Evo-devo beyond morphology: from genes to resource use

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1 Opinion Evo-devo beyond morphology: from genes to resource use Duncan J. Irschick 1,2*, R. Craig Albertson 1,2*, Patricia Brennan 1,2, Jeffrey Podos 1,2, Norman A. Johnson 1,2, Sheila Patek 1,2, and Elizabeth Dumont 1,2 1 Department of Biology, 221 Morrill Science Center, University of Massachusetts, Amherst, MA 01003, USA 2 Organismic and Evolutionary Biology Program, University of Massachusetts, 319 Morrill Science Center, University of Massachusetts, Amherst, MA 01003, USA How does genetic innovation translate into ecological innovation? Although evo-devo has successfully linked genes to morphology, the next stage is elucidating how genes predict resource use. This can be attained by broadening the focus of evo-devo from [genes!morphology], to [genes!morphology!functional ecology]. We suggest that the fields of evo-devo, functional morphology, and evolutionary ecology should be united under a common framework based on three predictions. The first is that morphological disparity should scale positively with functional complexity among different radiations. The second is that functional complexity should correlate negatively with the predictability of evolutionary divergence within lineages, and the third is that functional complexity should define the genetic architecture of adaptive radiations. These predictions could enable a broader understanding of how genetic variation is translated into variation in resource use. Evo-devo and the extended evolutionary synthesis How does phenotypic variation arise during the course of evolution? Adaptive evolution is enabled by two processes: (i) the generation of phenotypic variation and (ii) the culling of that variation via natural selection. The most synthetic attempt to address these questions occurred during the NeoDarwinian synthesis during the mid-20th century, which forged Mendelian genetics, Darwinian natural selection, and mathematical theory [1]. During recent decades, studies of genetics and evolution have converged in the emerging field of evolutionary developmental biology, or evo-devo [2], which probes the genetic and developmental bases of evolutionary variation. An ongoing extended evolutionary synthesis [3] considers how phenotypic and genotypic variation is generated and how it affects the potential for a lineage to evolve (i e., evolvability ) [4]. Comprehensive application of the extended evolutionary synthesis at the macroevolutionary level should incorporate organismal-level processes, mainly functional morphology and its influence on resource use (Box 1). Here, we offer a conceptual framework and road map for expanding the extended evolutionary synthesis from its current focus on relations between genes and Corresponding author: Irschick, D.J. (Irschick@bio.umass.edu) * These authors contributed equally to this work /$ see front matter ß 2013 Elsevier Ltd. All rights reserved. morphology (evo-devo) to one that incorporates the full range of levels relevant to evolutionary radiations, namely genes, morphology, function, and resource use. Although this formulation has been articulated at the intraspecific level [5,6], a synthesis at the macroevolutionary level has yet to be formalized. Multilevel studies provide insights into evolutionary processes Adaptive radiations are studied by evolutionary biologists because they can offer tractable systems in which to identify and study the proximate mechanisms of evolutionary change. Adaptive radiations have played a prominent role in the development of evolutionary theory [7], as exemplified by the many insights provided by examples such as Darwin s finches [8], East African cichlid fishes [9], Caribbean Anolis lizards [10], columbines of the genus Aquilegia [11], and Mediterranean Cistus [12]. Adaptive radiations are often rapid [13], although are not necessarily so, such as in plethodontid salamanders [14,15]. For many adaptive radiations, there is a close association among morphology, function, and resource use [13]. For example, Caribbean Anolis lizards vary notably in their hindlimb dimensions, variations that translate into the ability of different ecomorphs to occupy narrow versus broad perches in nature [16]. However, behavior and genetics also play a key role in dictating the shape of adaptive radiations. For example, in some cases, behavior and life-history variation drive the first phases of adaptive radiation, and variation in morphology can occur in secondary phases [17]. A logical next step in understanding adaptive radiation is to identify the genetic architecture underpinning diversification in key morphological traits [18]. Although developmental genetic research programs are beginning to examine certain key adaptive radiations (e.g., Darwin s finches, [18]), these programs focus mainly on the connection between genotype and morphology. Advances here will need to be matched by inquiry into how genetic interactions mediate resource use and clade-level patterns of divergence, as we address below. Toward a synthesis of evo-devo and adaptive radiation Although evo-devo and the study of adaptive radiations aim to explain organismal diversity, these two approaches have matured along independent lines, and focus on different levels. Evo-devo studies are concerned with the Trends in Ecology & Evolution, May 2013, Vol. 28, No

2 Box 1. The extended evolutionary synthesis, and a role therein for functional morphology The Neo-Darwinian Synthesis assumed that phenotypic variation is generated in an unrestricted manner from genetic sources. It did not recognize other factors that can constrain the expression of adaptive phenotypic variation [47,48]. The recognition of this limitation of the Neo-Darwinian Synthesis, coupled with technological advances in genomics and molecular biology, has fueled the call for an extended evolutionary synthesis [3,49]. Phenotypic plasticity Phenotypic plasticity is the capacity of the phenotype of an organism to vary in distinct environments [3,50 52]. The ability of an individual to adjust its phenotype to circumstance may increase its fitness in changing and fluctuating environments, which suggests that developmental plasticity is adaptive and, therefore, itself subject to selection [51,52]. Phenotypic integration Phenotypic integration refers to the degree of covariation among groups of traits ( modules ). It is typically high among traits within modules and low between traits across modules [53]. Adaptive phenotypes likely emerge via the integration of multiple modules, which may occur when genetic variation that results in a particular pattern of trait covariation is favored by selection [54 57]. Evo-devo Evo-devo is helping to coalesce the extended evolutionary synthesis by unifying the concepts of phenotypic plasticity and integration. Both the plastic response of an organism (i.e., reaction norm) and its patterns of phenotypic integration are influenced by its developmental architecture. Two missions of evo-devo are to explain how variation is shaped by development, and the extent to which these processes act to bias the direction of evolutionary change [57]. Functional morphology Functional morphology is the study of the relation between form and function [58]. This field has an important role to play in the extended evolutionary synthesis by helping to unify its core concepts. For example, function is considered to be a key force influencing patterns of phenotypic integration [53]. It also represents the point of interface between an organism and its environment, and it is this interface that feeds back on the organism to shape (and reshape) its form over development. Adaptive radiation Adaptive radiation has been defined by Schulter ([13] p. 10) as the evolution of ecological and phenotypic diversity within a rapidly multiplying lineage. From this definition, the hallmarks of adaptive radiations are common ancestry, a correlation between phenotypic features and environmental conditions, the demonstration of utility of those phenotypic features in those environments, and rapid speciation [13]. By contrast, a nonadaptive radiation is the proliferation of species without substantial ecological differentiation. Columbines (genus Aquilegia) are an example of an adaptive radiation [59,60]. Differences in pollinators appear to have driven evolutionary changes in the spurs [11]. In another adaptive radiation in the plant genus Cistus, leaf characteristics (shape, labdanum content, and pubescence) correspond with climatic factors [12]. Genetic architecture Genetic architecture has been defined as the complex connection of genotype to phenotype through development [61] and, therefore, evo-devo falls squarely within the purview of this definition. As discussed in the main text, the genetic architecture will influence how traits will evolve. connection between genotype and phenotype, whereas investigations of adaptive radiation are concerned with how resource use is mediated by form and function. Integrating these research programs is timely, and will result in a more comprehensive understanding of evolutionary processes. Evo-devo studies have begun to account for complex morphology and function [19,20], and several studies have explored the genetic bases of morphological traits with functional significance (e.g., [21 23]). Although recent reviews have attempted to build a framework for linking genetic variation to variation in morphology, ecology, and function [5,6], these efforts largely examine variation at the intraspecific level. There is no synthetic framework integrating these different levels at the macroevolutionary level. Moreover, the above reviews focus on traits where the connection between form and resource use is straightforward. In these instances, understanding direct links from genes to morphology will suffice for understanding adaptive radiation [24]. However, it is not form per se that determines resource use, but rather the manner in which form is used in the context of behavior [25 27]. For example, patterns of trophic radiation in cichlids are similar to those observed in other fish lineages [28], and the major axis of craniofacial divergence in New World bats is similar to that in other mammals [29,30]. The mechanics of adaptive radiation: a conceptual framework Adaptive radiations are diverse in their complexity, with some radiations comprising only a few species on a single island, or set of islands, and others exhibiting an array of forms across continents [13]. Here, we offer a schematic of this variation, exemplified by four vertebrate radiations, in terms of the relations between genes, morphology, function, and resource use (Figure 1). Some adaptive radiations are defined by variation primarily on one or a few resource axes, with concomitant change in similarly few morphological and functional traits. Other radiations are defined by divergence in multiple morphological traits, resulting in multiple axes of resource use and greater diversity in functional outcomes. Radiation-specific patterns can be detected empirically by examining variation in their morphological fullness, in which some radiations vary only along a single axis, whereas others vary in multiple dimensions [e.g., principal component (PC) 1,, etc.; Figure 1]. One simple way to quantify fullness is with respect to variation in morphological trait space that can be explained by the first size-adjusted PC axis relative to subsequent axes, which is high for some radiations (e.g., Caribbean anoles [31]), intermediate for others (bats [32]), and relatively low for others (cichlid fishes [28]). Specifically, cichlid fish jaws present different ecomorphological groupings, each describing a distinct aspect of shape variation [30], whereas in Anolis limbs, most of the variation can be explained by one axis [31]. Although these examples are not directly comparable in a statistical sense, given that they use different types of measure and consider different numbers of trait, they represent instances where morphological variation in key traits associated with an adaptive radiation is analyzed, and thus provide a useful comparison within the context of a predictive framework. We note that other metrics of disparity exist that may be useful in comparing the different morphological characteristics among taxa. Foote disparity is useful for multivariate 268

3 Func on Cichlid jaws Bat skulls Link to resource use Genotype Morphology Resource use Finch beaks Genotype Morphology Func on Resource use Morphology Anolis limbs Func onal complexity TRENDS in Ecology & Evolution Figure 1. Morphological traits exhibit variation in functional complexity (e.g., number of moving elements or degrees of freedom). For example, the number of independently moving elements in the mammalian skull is relatively small, because most elements are fused together, whereas the teleost head has many independently moving elements, which translates into more degrees of freedom in terms of jaw kinetics. Adaptive radiations are arrayed along the x-axis in terms of the functional complexity of the radiating phenotype. As functional complexity increases, the relation between form and function should also become more complex (e.g., greater potential for many-to-one mapping of form to function). When functional complexity is high, trait function (e.g., running, crushing, or biting) will be a more accurate predictor of resource use than will form. When functional complexity is low, the relation between form and function will be more linear, and morphology can serve as a reasonable proxy for resource use. The y-axis defines the continuum between morphology and function with respect to predicting resource use. Our model predicts that phenotypes with greater functional complexity underlie radiations that exhibit greater morphological disparity, but have a less well-defined primary axis of divergence (as illustrated by the large ellipse for the jaws of cichlid fish). By contrast, those phenotypes with lower functional complexity will show less morphological disparity, but will exhibit a more well-defined ecomorphological axis of divergence (as illustrated by the narrow ellipse for Anolis limbs). Within the context of evo-devo, the connection between genes, morphology, and resource use will be more straightforward in systems where the relation between form and function is simple (green-shaded area). Conversely, in systems where the relation between form and function is inherently complex, simple genotype phenotype mapping will not be as successful in explaining evolutionary processes (blue-shaded area). For these lineages, an understanding of the relations between genotype, form, and function, including the remodeling of form throughout the life of an organism, will be required to provide a mechanistic basis for evolutionary radiation. traits [33], and can be used to complement or verify fullness as determined by PC analysis (PCA) (e.g., [30]). We suggest that these differences among radiations are due to differences in functional complexity. We define functional complexity as the number of moving elements within a trait complex, which together allow animals to perform ecologically relevant functional tasks, and which can evolve independently so as to produce new morphological variants. Although links between form and function are often discussed in the context of generalized tasks (e.g., limbs and locomotion), it is more useful to focus on how morphology influences the ability to perform specific tasks (e.g., the role of the beak in crushing seeds or the role of the limb in fleeing from predators, [34,35]). An emphasis on specific performance outcomes of morphological traits makes it feasible to assess how different groups of animal vary in functional complexity. To illustrate, in mammals, most bony elements that comprise the skull are fused. There are two primary moving parts, namely the tongue and the lower jaw; the upper jaw is fixed to the braincase and the tongue and the lower jaw move relative to it, movement that can be modulated to influence the rate of feeding or bite strength [36]. Diversity in feeding mechanics among mammals is achieved through a limited array of morphological divergence. By contrast, in teleosts, the jawbones are not fused, and can translate and pivot along a variety of axes, creating potential for a broader range of movements and, thus, a broader suite of possibilities in evolution. Although the underlying jaw anatomy of both fishes and mammals is complex, each featuring multiple kinds of muscle, soft tissue, bone, or other tissue, from a functional perspective, the complexity of the jaw is higher in teleost fishes. We note that the above is only one estimate of functional complexity, and different criteria may be needed for different functional traits. For example, locomotion in most animals occurs through movement of limbs or other appendages, and animals have tremendous flexibility in overcoming morphological constraints to achieve high levels of locomotor performance. Therefore, one cannot equate morphological and functional complexity, and the latter does not derive from the former in every case. Our main assertion is that adaptive radiations are most accurately defined by adaptive variation in function, and this should hold true across systems. 269

4 The mechanics of adaptive radiation: predictive framework We make three predictions about how adaptive radiations should manifest across different clades. First, morphological disparity, or the degree to which integrated morphological traits interact to perform a function, should scale positively with functional diversity. This prediction can be conceptualized with reference to the relative area (in all dimensions) of the ellipses illustrated in Figure 1. As the number of moving elements of a trait increases, the number of possible combinations of trait values is also likely to increase, which should lead to a greater potential for morphological disparity. This prediction implies that, as functional complexity increases, it is trait function, rather than morphology, that will most accurately predict resource use and, thus, trajectories of natural selection. Trait function should have greater predictive power than trait morphology, because as the relation between morphology and function becomes less stringent, the role of behavior in shaping trait function becomes more prominent [27]. Returning to cichlid fishes, the evolutionary success of this clade is credited, in part, to a marked radiation in feeding architecture, and how this radiation has enabled different species to consume different kinds of prey. However, because fish jaws function through the interaction of multiple morphological elements that are integrated in a complex and dynamic manner, the mapping of form to function in fishes is not linear. Thus, species with different jaw types can consume the same prey (i.e., many to one mapping, [37,38]), whereas species with similar jaw types can consume different prey (one to many mapping). This nonlinear relation between form and function increases the potential of cichlids to diverge in many directions, and makes it unlikely that the ability of cichlids to evolve (evolvability) can be understood by examining variation in one or few morphological traits in isolation. When functional complexity is low, the relation between form and function will be more direct, and morphology can serve as a proxy for resource use [24]. Second, we predict that functional complexity should correlate negatively with the tendencies of different clades to evolve in specific, repeatable patterns, such as when invading multiple, parallel novel environments (e.g., lakes or islands). That is, an adaptive radiation with limited functional complexity should manifest in similar phenotype distributions across multiple replicates, whereas radiations with high functional complexity should manifest in distinct, somewhat random distributions, contingent on the specific ecological pathways encountered in each replicate. We presume that, in evolutionary radiations with more potential pathways for phenotype evolution, historical contingency can result in different pathways despite similar initial conditions [39]. Thus, there is a many-to-one mapping of different combinations of morphological traits being used for the same function. Inherent constraints on how morphology can evolve, and how morphological variation translates into functional variation, could fuel either outcome, depending on the nature of the constraints. In plethodontid salamanders, a gular pump is needed for respiration and, as such, the skeleton could not become highly specialized for projection of the tongue [14,15,40]. These and other constraints resulted in extensive homoplasy in salamanders [40], signifying evolution along certain routes with little or no variation between them. For this and other highly predictable systems, it seems likely that constraints would channel evolution in the same directions repeatedly, whereas for less predictable systems, constraints could also channel evolutionary change differently in independent radiations. If correct, this relation would have consequences for how evolutionary divergence and convergence occur. Both East African cichlid fishes and Caribbean Anolis lizards show substantial convergent evolution, yet the degree to which these adaptive radiations are repeatable (across replicates) differs. Whereas African rift-lake cichlids appear to have diverged morphologically in a similar manner among the three large lakes in the region [28], there is a fair degree of variation in this trend. For example, species that specialize on scraping tough filamentous algae from rocks in Lakes Malawi and Tanganyika have evolved distinct trophic anatomies to accomplish this task. This pattern can be attributed to the tendency of cichlids to converge on function via different anatomical configurations. By contrast, anatomical convergence among Caribbean anoles is repeatable, which is due to the relatively simple mapping of form to function to resource use, in response to the occupation of parallel microhabitats on different islands [10]. One must also consider potential confounding factors when comparing different radiations, such as the role of behavior in modifying relations between morphology and function, and differences among different regions in environmental variables, such as food or vegetation, among other possibilities. Our third prediction is that variation in functional and morphological complexity will define the genetic architecture of adaptive radiation across different clades. This prediction refers to the complexity of the pathways connecting genotype to resource use in Figure 1. For cases in which functional complexity is low, a relatively small number of genes with direct effects on the phenotype should mold phenotypes to match resource use. In such cases it may be possible, at a macroevolutionary level, to match genes directly to resource use by simply matching them to variation in the phenotype (green-shaded region, Figure 1). For example, relatively simple anatomical shifts in beak depth, width, and length in Galapagos finches are directly linked to resource use (seed size), and correspondingly simple genetic shifts may account for these anatomical changes [18,41]. By comparison, if functional complexity is high, then the genetics of an adaptive radiation are also likely to be complex. The genetic architecture of such radiations should be characterized by a greater number of genes and genetic interactions. Although adaptive shifts in jaw traits have been traced to variation in particular genes in cichlids [21,22], traits investigated thus far are part of relatively simple functional systems (i.e., lever mechanics), where we would expect the genetic basis to be similarly simple. A recent genetic mapping study in cichlids demonstrated that more complicated functional systems (i.e., four-bar linkages) are under the control of more complicated genetic systems characterized by nonadditive allelic effects and epistasis [42]. We also expect 270

5 that the developmental timing will be different for traits with different levels of functional complexity. There is greater potential for functionally simple traits to be established via early patterning mechanisms (e.g., outgrowth of limb or jaw primordia), whereas later developmental remodeling should figure more prominently in the shaping of functionally complex systems that involve the integration and coordination of multiple elements that appear at different stages of development. Although bone patterning and remodeling involve different cell types and developmental mechanisms (neural crest development versus osteoclastogenesis, respectively), the same signaling pathways can orchestrate both (e.g., fibroblast growth factor [FGF] signaling, [43]). Thus, it will be interesting to show whether the same molecular signaling pathways are involved in the evolutionary divergence of both functionally simple and complex systems, and how conserved these are across taxa. Because different forms can often perform the same function in complex systems, it is likely that parallel adaptive peaks can be reached along different genetic avenues. A more direct link between the genotype and function will be necessary to explain lineage diversity (blue-shaded region, Figure 1). Testing predictions: a road map Our first two predictions are related because they each address how functional complexity influences phenotypic variation. They can both be tested by empirically comparing the magnitude of phenotypic variance among different radiations (e.g., relative contribution of ) to levels of functional complexity. However, methods to compare data from different taxa and traits are not yet developed and are needed for such comparisons to proceed. Studies of the evolutionary origin of traits, which requires accounting of homology and homoplasy [38], might thus provide a valuable tool. Our third prediction will be the most challenging, but the tools are in place. These represent an extension of genotype to phenotype mapping, including both genetic mapping [quantitative trait locus analysis (QTL) or association mapping] and candidate gene approaches. According to our prediction, the number of genes or number and types of genetic interaction (e.g., pleiotropy, epistasis, or gene environment) should be greater in adaptive radiations with greater functional complexity. Although this may seem intuitive, it has implications for how genetic mapping experiments can be optimized for the study of functionally complex systems. We suggest extending the phenotypic assay to include functionally explicit measures. This can be done both directly, by generating kinematic measures to be mapped, or indirectly by using engineering principles to derive trait values (e.g., lever mechanics or four-bar linkage systems, [21]). Another potentially fruitful approach would be to map genetically patterns of phenotypic integration (e.g., [44]), because these are determined, in part, by functional processes. Combining morphological and functional traits into a common mapping experiment would enable investigators to assess the extent to which anatomical and functional QTL overlap. We expect that there would be large overlap between anatomical and functional QTL for traits with simple functions, and relatively little overlap between these two types of QTL for traits with complex, emergent functions. Although QTL analyses have been limited in their ability to identify the causative genes involved in adaptive divergences, especially for complex traits, the advent of population genomics in a comparative context has been useful in helping to refine QTL intervals to the level of signal genes (e.g. [22,45]). With these advances, the specific genes associated with complex functions should be within our grasp. Elucidation of genotype to function links could also benefit by reference to laboratory model organisms. Measuring phenotypic integration or locomotor and feeding kinematics in mutant mice or zebrafish, for example, could yield valuable information about how simple genetic changes (e.g., point mutations) translate to variation in function. We might expect that a mutation that acts early in development to modulate limb length will have a direct effect on performance, providing a clear link between genotype, phenotype, and function. Alternatively, a mutation that influences bone mineral density and bone metabolism may have a negligible effect on morphology in a static environment, but a profound effect on performance across a range of environments (e.g. [46,43]). Thus, the analysis of organismal function within the context of different types of mutation (i.e., patterning versus remodeling) across a range of environments can provide insights into the connection between genes, morphology, function, and the environment. Because genetic and developmental mechanisms across metazoans are conserved, laboratory studies can be brought to the field by screening natural populations for polymorphisms in candidate genes that alter function and performance in experimental models. Concluding remarks A framework that considers relations among genes, morphology, function, and resource use, which we present here, can be used to assemble and guide integrated research programs in functional morphology, evolutionary ecology, and evo-devo. The aim of this article was to propose a set of hypotheses that can form the basis for further studies. We advocate strengthened collaborations among molecular geneticists, developmental biologists, functional morphologists, and evolutionary ecologists, with the goal of addressing the genetics of adaptive radiations by considering all links from genes to resource use. Although other reviews have attempted to build a framework linking genes, morphology, ecology, and even function, they have focused on the intraspecific level, or among several closely related species. We believe that our expansion to macroevolutionary studies is novel, given that one cannot easily predict processes above the species level from those that occur within species [5,6]. We have focused here on several well-studied adaptive radiations, but our ideas are applicable to many other systems and taxa. 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