Morphometrics and the role of the phenotype in studies of the evolution of developmental mechanisms

Developmental mechanisms are usually assumed to evolve by natural selection of the morphological traits they produce. Therefore, information on phenotypic traits is an important component of comparative studies of development. Morphometrics permits the rigorous quantitative analysis of variation in organismal size and shape, and is increasingly being used in developmental contexts. The new methods of morphometrics combine a geometric concept of shape with the procedures of multivariate statistics, and constitute a powerful and flexible set of tools for analyzing morphological variation. This paper briefly reviews these methods and provides examples of their application in studies of genetic variation and developmental modularity. The results of morphometric analyses can be readily interpreted in relation to the geometry and anatomical structure of the parts under study. Genetic studies of shape in the mouse mandible found two recurrent patterns in environmental and genetic variation from different origins, suggesting that the development system 'channels' the phenotypic expression of variation in similar ways. Moreover, by analyzing the correlations of left-right asymmetries of morphometric traits, it is possible to delimit the spatial extent of developmental modules. These methods complement the experimental approaches of developmental biology and genetics, and can be expected to be especially fruitful in combination with them.     

Studying morphological integration and modularity at multiple levels: concepts and analysis

Although most studies on integration and modularity have focused on variation among individuals within populations or species, this is not the only level of variation for which integration and modularity exist. Multiple levels of biological variation originate from distinct sources: genetic variation, phenotypic plasticity resulting from environmental heterogeneity, fluctuating asymmetry from random developmental variation and, at the interpopulation or interspecific levels, evolutionary change. The processes that produce variation at all these levels can impart integration or modularity on the covariance structure among morphological traits. In turn, studies of the patterns of integration and modularity can inform about the underlying processes. In particular, the methods of geometric morphometrics offer many advantages for such studies because they can characterize the patterns of morphological variation in great detail and maintain the anatomical context of the structures under study. This paper reviews biological concepts and analytical methods for characterizing patterns of variation and for comparing across levels. Because research comparing patterns across level has only just begun, there are relatively few results, generalizations are difficult and many biological and statistical questions remain unanswered. Nevertheless, it is clear that research using this approach can take advantage of an abundance of new possibilities that are so far largely unexplored.     

A developmental staging series for the lizard genus Anolis: a new system for the integration of evolution, development, and ecology

Vertebrate developmental biologists typically rely on a limited number of model organisms to understand the evolutionary bases of morphological change. Unfortunately, a typical model system for squamates (lizards and snakes) has not yet been developed leaving many fundamental questions about morphological evolution unaddressed. New model systems would ideally include clades, rather than single species, that are amenable to both laboratory studies of development and field-based analyses of ecology and evolution. Combining an understanding of development with an understanding of ecology and evolution within and between closely related species has the potential to create a seamless understanding of how genetic variation underlies ecologically and evolutionarily relevant variation within populations and between species. Here we briefly introduce a new model system for the integration of development, evolution, and ecology, the lizard genus Anolis, a diverse group of lizards whose ecology and evolution is well understood, and whose genome has recently been sequenced. We present a developmental staging series for Anolis lizards that can act as a baseline for later comparative and experimental studies within this genus.     

An interspecific QTL study of Drosophila wing size and shape variation to investigate the genetic basis of morphological differences

The Drosophila wing has been used as a model in studies of morphogenesis and evolution; the use of such models can contribute to our understanding of mechanisms that promote morphological divergence among populations and species. We mapped quantitative trait loci (QTL) affecting wing size and shape traits using highly inbred introgression lines between D. simulans and D. sechellia, two sibling species of the melanogaster subgroup. Eighteen QTL peaks that are associated with 12 wing traits were identified, including two principal components. The wings of D. simulans and D. sechellia significantly diverged in size; two of the QTL peaks could account for part of this interspecific divergence. Both of these putative QTLs were mapped at the same cytological regions as other QTLs for intraspecific wing size variation identified in D. melanogaster studies. In these regions, one or more loci could account for intra- and interspecific variation in the size of Drosophila wings. Three other QTL peaks were related to a pattern of interspecific variation in wing size and shape traits that is summarized by one principal component. In addition, we observed that female wings are significantly larger and longer than male wings and the second, fourth and fifth longitudinal veins are closer together at the distal wing area. This pattern was summarized by another principal component, for which one QTL was mapped.     

Do Correlation Patterns Reflect the Role of Development in Morphological Evolution?

Correlation patterns have been widely used in evolutionary studies for exploring the role of development in channelling morphological evolution. The approach was firstly introduced by Olson and Miller in the 1950s, but it did not gain prominence until the 1980s, due to some extent to Gould and Lewontin’s (Proc R Soc Lond B 205:581–598, 1979) assertion of the importance of considering organisms as integrated entities, where the internal organization of a structure, and not only the selective regime acting upon it, would play a fundamental role in its evolution. Here we show that this approach, mainly focused on the study of small, quantitative shape changes of existing structures, does not deal with a fundamental aspect of developmental systems, that is, their intrinsic capacity of originating morphological novelties. We show that only when the physicochemical processes underlying morphogenesis and pattern formation are taken into account, would the causal role of development be fully incorporated into the evolutionary view.     

Rates of Evolution in Developmental Processes

The tempo and mode of morphological evolution are influencedby several factors, among which evolutionary transformationsin developmental processes are likely to be important. Comparingthe embryos of extant species in an explicit phylogenetic framwork allows the estimation of minimum average rates of evolutionin quantitative developmental parameters. It also allows delineationof the maximum time that complex qualitative transformationsin developmental mechanism take to evolve. This paper analyzesrates of quantitative and qualitative developmental evolutionusing examples drawn primarily from echinoderms. The resultsdemonstrate that rates of developmental evolution can be comparableto rates of morphological evolution. There is no indicationthat rates of evolution in development are lower for earlierstages, contrary to the prediction of "tree" models of epigeneticinteractions. In particular, rates of evolution in oogenesiscan exceed rates of evolution in adult body size. Rates of developmentalevolution can vary by up to two orders of magnitude within aclade. Whether such large scale variation in evolutionary ratesof developmental processes is a general phenomenon can onlybe answered by further study.     

Genes into geometry: imaging for mouse development in 3D

Mammalian development is a sophisticated program coordinated by a complex set of genetic and physiological factors. Alterations in anatomy or morphology provide intrinsic measures of progress in or deviations from this program. Emerging three-dimensional imaging methods now allow for more sophisticated morphological assessment than ever before, enabling comprehensive phenotyping, visualization of anatomical context and patterns, automated and quantitative morphological analysis, as well as improved understanding of the developmental time course. Furthermore, these imaging tools are becoming increasingly available and will consequently play a prominent role in elucidating the factors that direct and influence mammalian development.     

Embryonic development of the fossorial gymnophthalmid lizards Nothobachia ablephara and Calyptommatus sinebrachiatus

The evolutionary history of the lizard family Gymnophthalmidae is characterized by several independent events of morphological modifications to a snake-like body plan, such as limb reduction, body elongation, loss of external ear openings, and modifications in skull bones, as adaptive responses to a burrowing and fossorial lifestyle. The origins of such morphological modifications from an ancestral lizard-like condition can be traced back to evolutionary changes in the developmental processes that coordinate the building of the organism. Thus, the characterization of the embryonic development of gymnophthalmid lizards is an essential step because it lays the foundation for future studies aiming to understand the exact nature of these changes and the developmental mechanisms that could have been responsible for the evolution of a serpentiform (snake-like) from a lacertiform (lizard-like) body form. Here we describe the post-ovipositional embryonic development of the fossorial species Nothobachia ablephara and Calyptommatus sinebrachiatus, presenting a detailed staging system for each one, with special focus on the development of the reduced limbs, and comparing their development to that of other lizard species. The data provided by the staging series are essential for future experimental studies addressing the genetic basis of the evolutionary and developmental variation of the Gymnophthalmidae.     

New Components of Drosophila Leg Development Identified through Genome Wide Association Studies

The adult Drosophila melanogaster body develops from imaginal discs, groups of cells set-aside during embryogenesis and expanded in number during larval stages. Specification and development of Drosophila imaginal discs have been studied for many years as models of morphogenesis. These studies are often based on mutations with large developmental effects, mutations that are often lethal in embryos when homozygous. Such forward genetic screens can be limited by factors such as early lethality and genetic redundancy. To identify additional genes and genetic pathways involved in leg imaginal disc development, we employed a Genome Wide Association Study utilizing the natural genetic variation in leg proportionality found in the Drosophila Genetic Reference Panel fly lines. In addition to identifying genes already known to be involved in leg development, we identified several genes involved in pathways that had not previously been linked with leg development. Several of the genes appear to be involved in signaling activities, while others have no known roles at this time. Many of these uncharacterized genes are conserved in mammals, so we can now begin to place these genes into developmental contexts. Interestingly, we identified five genes which, when their function is reduced by RNAi, cause an antenna-to-leg transformation. Our results demonstrate the utility of this approach, integrating the tools of quantitative and molecular genetics to study developmental processes, and provide new insights into the pathways and networks involved in Drosophila leg development.     

Crossroads, milestones and landmarks in insect development and evolution: implications for systematics

Our understanding of insect development and evolution has increased greatly due to recent advances in the comparative developmental approach. Modern developmental biology techniques such as in situ hybridization and molecular analysis of developmentally important genes and gene families have greatly facilitated these advances. The role of the comparative developmental approach in insect systematics is explored in this paper and we suggest two important applications of the approach to insect systematics--character dissection and morphological landmarking. Existing morphological characters can be dissected into their genetic and molecular components in some cases and this will lead to more and richer character information in systematic studies. Character landmarking will he essential to systematic studies for clarifying structures such as shapes or convergences, which are previously hard to analyze anatomical regions. Both approaches will aid greatly in expanding our understanding of homology in particular, and insect development in general.     

EVOLUTION AND MORPHOGENETIC RULES: THE SHAPE OF THE VERTEBRATE LIMB IN ONTOGENY AND PHYLOGENY

The notion of a “developmental constraint” has become a catchphrase for a collection of poorly defined notions about how ontogeny affects phylogeny. In this paper, we shall attempt to define this idea more precisely by examining the vertebrate limb from three viewpoints. First, theoretical models of morphogenesis suggest several generalizations about how limb geometry is laid down during development. Comparative studies and experimental manipulations of developing limbs independently confirm these generalizations, which amount to a set of “construction rules” for determining how the major features of limb architecture are established in ontogeny. Armed with these rules, we can inquire how limb morphology can be varied during evolution and suggest a more precise operational definition of “developmental constraints” on morphological evolution.     

Developmental and genetic origins of murine long bone length variation

If we wish to understand whether development influences the rate or direction of morphological evolution, we must first understand the developmental bases of morphological variation within species. However, quantitative variation in adult morphology is the product of molecular and cellular processes unfolding from embryonic development through juvenile growth to maturity. The Atchley-Hall model provides a useful framework for dissecting complex morphologies into their component parts as a way of determining which developmental processes contribute to variation in adult form. We have examined differences in postnatal allometry and the patterns of genetic correlation between age-specific traits for ten recombinant inbred strains of mice generated from an intercross of LG/J and SM/J. Long bone length is closely tied to body size, but variation in adult morphology is more closely tied to differences in growth rate between 3 and 5 weeks of age. These analyses show that variation generated during early development is overridden by variation generated later in life. To more precisely determine the cellular processes generating this variation we then examined the cellular dynamics of long bone growth plates at the time of maximum elongation rate differences in the parent strains. Our analyses revealed that variation in long bone length is the result of faster elongation rates of the LG/J stain. The developmental bases for these differences in growth rate involve the rate of cell division and chondrocyte hypertrophy in the growth plate.     

DISPARITY: MORPHOLOGICAL PATTERN AND DEVELOPMENTAL CONTEXT

Abstract:  The distribution of organic forms is clumpy at any scale from populations to the highest taxonomic categories, and whether considered within clades or within ecosystems. The fossil record provides little support for expectations that the morphological gaps between species or groups of species have increased through time as it might if the gaps were created by extinction of a more homogeneous distribution of morphologies. As the quantitative assessments of morphology have replaced counts of higher taxa as a metric of morphological disparity, numerous studies have demonstrated the rapid construction of morphospace early in evolutionary radiations, and have emphasized the difference between taxonomic measures of morphological diversity and quantitative assessments of disparity. Other studies have evaluated changing patterns of disparity across mass extinctions, ecomorphological patterns and the patterns of convergence within ecological communities, while the development of theoretical morphology has greatly aided efforts to understand why some forms do not occur. A parallel, and until recently, largely separate research effort in evolutionary developmental biology has established that the developmental toolkit underlying the remarkable breadth of metazoan form is largely identical among Bilateria, and many components are shared among all metazoa. Underlying this concern with disparity is a question about temporal variation in the production of morphological innovations, a debate over the relative significance of the generation of new morphologies vs. differential probabilities of their successful introduction, and the relative importance of constraint, convergence and contingency in the evolution of form.     

Embryonic and larval staging of summer flounder (Paralichthys dentatus)

Early development of flatfishes such as the summer flounder Paralichthys dentatus (Pleuronectiformes) has not been extensively documented, largely because of a dearth of material; however, the recent expansion of flatfish aquaculture has made embryos of P. dentatus readily available for developmental studies. We divide development of P. dentatus embryos and larvae into two main periods, pre- and posthatching, and assign stages within each of those primary divisions. Stages from fertilization to hatching loosely follow the general teleost staging scheme suggested by Shardo ([1995] J Morphol 225:125-167); stages from hatching through metamorphosis are aligned with the series used for Japanese flounder, P. olivaceus (Minami [1982] Nippon Suisan Gakkaishi 48:1581-1588; Fukuhara [1986] Nippon Suisan Gakkaishi 52:81-91). Although length, width, and age may serve as approximate indicators of developmental progression in summer flounder, these characteristics are too variable to form the sole basis of a staging table. Therefore, we define stages by morphological criteria drawn from the development of the jaw apparatus and digestive system, eye migration, and notochord tip flexion. Examination of these morphological features in hatched larvae allows accurate and consistent assessment of developmental stage despite variation in timing and size. The staging scheme for flounder embryonic and larval development presented here should facilitate both experimental and comparative research on summer flounder and other flatfish species.     

Hsp90 canalizes developmental perturbation

Stochastic processes are intrinsic phenomena that perturb developmental processes. However, the canalization process restricts the magnitude of perturbation and hence the magnitude of morphological variation during development. Heat-shock protein 90 (Hsp90) chaperones are a class of proteins stabilizing a network of 'client' proteins that are involved in diverse signal transduction pathways affecting development. Here it is reported that a reduction of Hsp90 gene dose creates canalization perturbations that affect many aspects of Arabidopsis development and results in a plethora of morphological alterations. Hence, Hsp90 restricts stochastic phenomena by minimizing perturbations, thereby canalizing development. It is also shown that morphogenesis is determined by three mutually inter-related parameters: genotype, environment, and time. Hsp90 is involved in the interaction of these three parameters which ultimately affect developmental processes. The amount of phenotypic variation upon the reduction of Hsp90 function could be perceived as adaptive and could have an impact on the evolutionary process.     

Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis

In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis.     

Making teeth to order: conserved genes reveal an ancient molecular pattern in paddlefish (Actinopterygii)

Ray-finned fishes (Actinopterygii) are the dominant vertebrate group today (+30 000 species, predominantly teleosts), with great morphological diversity, including their dentitions. How dental morphological variation evolved is best addressed by considering a range of taxa across actinopterygian phylogeny; here we examine the dentition of Polyodon spathula (American paddlefish), assigned to the basal group Acipenseriformes. Although teeth are present and functional in young individuals of Polyodon, they are completely absent in adults. Our current understanding of developmental genes operating in the dentition is primarily restricted to teleosts; we show that shh and bmp4, as highly conserved epithelial and mesenchymal genes for gnathostome tooth development, are similarly expressed at Polyodon tooth loci, thus extending this conserved developmental pattern within the Actinopterygii. These genes map spatio-temporal tooth initiation in Polyodon larvae and provide new data in both oral and pharyngeal tooth sites. Variation in cellular intensity of shh maps timing of tooth morphogenesis, revealing a second odontogenic wave as alternate sites within tooth rows, a dental pattern also present in more derived actinopterygians. Developmental timing for each tooth field in Polyodon follows a gradient, from rostral to caudal and ventral to dorsal, repeated during subsequent loss of teeth. The transitory Polyodon dentition is modified by cessation of tooth addition and loss. As such, Polyodon represents a basal actinopterygian model for the evolution of developmental novelty: initial conservation, followed by tooth loss, accommodating the adult trophic modification to filter-feeding.     

Plant Proteus: brown algal morphological plasticity and underlying developmental mechanisms

Brown algae are multicellular photosynthetic marine organisms, ubiquitous on rocky intertidal shores at cold and temperate latitudes. Nevertheless, little is known about many aspects of their biology, particularly their development. Given their phylogenetic distance (1.6 billion years) from other plant organisms (land plants, and green and red algae), brown algae harbor a high, as-yet undiscovered diversity of biological mechanisms governing their development. They also show great morphological plasticity, responding to specific environmental constraints, such as sea currents, reduced light availability, grazer attacks, desiccation and UV exposure. Here, we show that brown algal morphogenesis is rather simple and flexible, and review recent genomic data on the cellular and molecular mechanisms known to date that can possibly account for this developmental strategy.     

Modularity, comparative embryology and evo-devo: Developmental dissection of evolving body plans

Modules can be defined as quasi-autonomous units that are connected loosely with each other within a system. A need for the concept of modularity has emerged as we deal with evolving organisms in evolutionary developmental research, especially because it is unknown how genes are associated with anatomical patterns. One of the strategies to link genotypes with phenotypes could be to relate developmental modules with morphological ones. To do this, it is fundamental to grasp the context in which certain anatomical units and developmental processes are associated with each other specifically. By identifying morphological modularities as units recognized by some categories of general homology as established by comparative anatomy, it becomes possible to identify developmental modules whose genetic components exhibit coextensive expressions. This permits us to distinguish the evolutionary modification in which the identical morphological module simply alters its shape for adaptation, without being decoupled from the functioning gene network (‘coupled modularities’), from the evolution of novelty that involves a heterotopic shift between the anatomical and developmental modules. Using this formulation, it becomes possible, within the realm of Geoffroy's homologous networks, to reduce morphological homologies to developmental mechanistic terms by dissociating certain classes of modules that are often associated with actual shapes and functions.