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PERSPECTIVES OPINION Opening Darwin’s black box: teaching evolution through developmental genetics Scott F. Gilbert When biologists are asked to discuss the evidence for evolution at public forums, they usually use well-established microevolutionary examples. Although these examples show the efficacy of evolution within species, they often leave audiences susceptable to the arguments of creationists who deny that evolution can create new structures and species. Recent studies from evolutionary developmental biology are beginning to provide case studies that specifically address these concerns. This perspective presents some of this new evidence and provides a framework in which to explain homology and phylogeny to such audiences. When asked to discuss evolution at public forums, an hour or so is often given to outline the main principles of evolution and to give examples. I think that, until recently, our best examples came from microevolutionary studies — looking at evolution within a species. However, although they are useful for explaining evolutionary processes, focusing exclusively on microevolutionary studies left openings for creationists to challenge whether species can be generated by evolutionary means. As well as providing new insights into evolution, evolutionary developmental biology has recently produced evidence that argues directly against the claims of creationists, by shedding light on macroevolutionary (above the species-level) processes. Here, I discuss some of this evidence and provide a framework for how we might wish to teach NATURE REVIEWS | GENETICS evolution. I discuss one possible way — there are certainly others. Evolution has generated our planet’s biodiversity, and over the past century scientists have become able to explain the mechanisms by which changes in animal body structure can be produced, inherited and selected. Genetics is crucial to this understanding. The MODERN SYNTHESIS initially explained evolution through the mathematics of population genetics. Although population genetics has remained the core of the modern synthesis, it has also come to include studies from ecology, biogeography, paleontology, and more recently cytogenetics and molecular genetics. Population genetics, itself a marriage of Mendelian particulate inheritance and Darwin’s natural selection, is able to explain how different alleles spread through the population, whereas cytogenetics and molecular genetics are able to explain the origins of these alleles through mutation and recombination. Until recently, the best examples of evolution in action came from microevolutionary studies. Such studies explained, for instance, how natural selection could cause moth colouration to become darker and change the beak shape in the Galapagos finches. For the Galapagos finches, selection by drought conditions allowed those individuals with certain beak shapes to survive, transmit their beak shape to their offspring and change the beak morphology that had been characteristic of the species1. This illustrates the main tenets of natural selection: variation within a species; overabundance of individuals within a species; differential survival of those most fit for the environmental circumstances; and the inheritance of those traits by the progeny of the survivours. The molecular version of the modern synthesis showed that evolution can even work over small timescales and therefore clearly showed how the principles of genetics and natural selection can, for example, explain the origins of pesticide-resistant insects and antibiotic-resistant bacteria2–4. The existence and efficacy of microevolution is widely accepted, even by most creationists5. But creationists are not concerned about antibiotic-sensitive bacteria becoming antibiotic-resistant or about the beak shape changes of Galapagos finch species. They have remained bacteria and finches, respectively, so nothing much has changed — no new species has been created. To creationists, the synthesis of evolution and genetics cannot explain how some fish became amphibians, how some reptiles became mammals, or how some apes became human (see the online links box for a link to material dealing specifically with arguments recently put forward by creationists). Behe6 named this inability to explain the creation of new taxa through genetics “Darwin’s black box”. When the box is opened, he expects evidence of the Deity to be found. However, inside Darwin’s black box resides merely another type of genetics — developmental genetics. If genetics is Darwin’s ‘missing evidence’7, then developmental genetics is needed to complete the picture given by molecular and population genetics. Fifty years ago, British biologist C. H. Waddington was trying to bring developmental biology into the modern synthesis8–10, and he noted that evolution had two principal components. There was the traditional component (that is, natural selection) that worked on adults that compete for reproductive success, and an embryological component, in which variation was created and constrained. “The changes that produced new body plans…”, wrote Waddington11, “…were inheritable changes VOLUME 4 | SEPTEMBER 2003 | 7 3 5 PERSPECTIVES in the patterns of embryonic development. Changes in genotypes only have ostensible effects in evolution if they bring with them alterations in the epigenetic processes by which phenotypes come into being; and the kinds of change possible in the adult form of an animal are limited to the possible alterations in the epigenetic system by which it is produced.” Both the selective and the developmental sides of evolution are important. In 1977, the two sides of evolution began to be bridged. Nobel Laureate François Jacob proposed the idea that evolution was bricolage — tinkering, not engineering12. Moreover, Jacob claimed that evolutionary biologists should look at evolution not only in adults, but also during development, because evolution works not only on adults but also on the ‘recipes’ for adults. Therefore, to study large changes in evolution, biologists needed to look for changes in the regulatory genes that make the embryo, not just in the structural genes that provide fitness within populations. The last quarter of a century has borne out this idea. It took a while for developmental genetics to reach maturity, but it can now be added to the mix of population genetics and molecular genetics to explain evolution. Moreover, when developmental genetics is added, macroevolution can be explained much more easily. The microevolutionary processes of mutation and recombination can be analysed to determine how specific genetic changes have created new types of organisms by altering their development. So, when confronted with the classic evolutionary question of how the arthropod body plan arose, Hughes and Kaufman began their study by stating, “To answer the question by invoking natural selection is correct but insufficient. The fangs of a centipede … and the claws of a lobster accord these organisms a fitness advantage. However, the crux of this mystery is this: From what developmental genetic changes did these novelties arise in the first place?” (REF. 13). Here, I propose a way of teaching evolution that highlights developmental genetics. As such, it confronts the creationist challenges directly and shows how changes in gene expression can cause a marked evolutionary change. Both population genetics and developmental genetics, as well as contributions from other fields such as palaentology and biogeography, are required for any theory of evolution, but I propose that students will learn the principles of evolution most easily from examples of development. I illustrate my argument with examples, most of which come from vertebrates and insects, and so I apologize in advance to the allies of all other clades. 736 | SEPTEMBER 2003 | VOLUME 4 Teaching phylogeny and homology In 1859, Darwin wrote “It is generally acknowledged that all organic beings have been formed on two great laws — Unity of Type and Conditions of Existence” (REF. 14).While natural selection explained adaptation to the “offices of existence”, embryonic homologies explained “unity of type” (REF. 14). Together, they would produce the idea of ‘descent with modification’. Using this concept, Darwin could explain the similarities of animal form through descent from a common ancestor and the differences by natural selection in different environments. When introducing evolution to students, biologists usually concentrate on natural selection, the main mechanism of Darwinian evolution. This is important. It shows that there is variation within a species; that most animals die before reproducing; that the mortality can be selective; and that the progeny resulting from such selection are more fit for their environment, having inherited the genes that made their parents fit. But this is only half of the story. The other half concerns the unity of type and the question of homology, that is, the fact that animals are joined together into groups with similar features. “…I propose that students will learn the principles of evolution most easily from examples of development.” To show this unity of type and how animals are variations on a relatively small set of themes, Darwin followed von Baer’s principles and looked to embryonic and larval stages, because the earlier stages of animal development could show homologies that are obscured in the adult. In On the Origin of Species, Darwin celebrated the case of the barnacle, the larvae of which showed that it is a shrimp-like arthropod14, and in Descent of Man15, he gloried in Alexander Kowalevsky’s discovery that the tunicate — previously classified as a shell-less mollusk — is actually a chordate. It has a NOTOCHORD and pharyngeal slits that come from the same cell layers as those of fish and chickens. So, the two great domains of the animal kingdom — invertebrates and vertebrates — were united through larval homologies. “Thus, if we may rely on embryology, ever the safest guide in classification, it seems that we have at last gained a clue to the source whence the Vertebrata were derived.” (REF. 15). But homology can be a tricky concept. If used alone (and some creationists imply that it is), it risks forming a circular argument wherein structures are considered homologous because of common origin, and these animals are said to have a common origin because they have homologous structures. An independent assessment of ancestry and origins is needed. Although we have this independent assessment now, it was not avilable to Darwin. Without this independent assessment, the study of macroevolution became embroiled in disputes about whether similar structures were the result of common ancestry or convergence. The frustration about whether similarities came from common ancestry (homology) or common environments (HOMOPLASY) caused biologists of the first three decades of the twentieth century to leave this area of research and begin the microevolutionary studies that related evolution to genetic variation within species (for example, see REFS 16,17).“The geneticist…”, said William Bateson in 1922, “…is the successor of the morphologist.” (REF. 18). I find that I can teach the relationships of homologies and evolution best by looking at one of the other passengers on the HMS Beagle. The Tierra del Fuegan, York Minster, was a bit older than Darwin, but like Darwin, he had been trained in theology (FIG. 1a). York Minster had been abducted by Captain Fitzroy on the first voyage of the HMS Beagle to the southern tip of South America, and Fitzroy had him educated in London as a missionary. Both he and Darwin completed their theological training in 1830. Newly baptized, this Tierra del Fuegan was now being repatriated to spread the Gospel in his homeland19. Imagine his education. He would have been told of different religions: Judaism, Catholicism, Russian and Greek Orthodoxy, Lutheranism and, of course, Anglicanism. There are differences and similarities between these religions: they share similar, but different Bibles; sing similar, but different hymns; and worship similar, but different Gods. How was he to make sense of them? Do these similar religions have a common origin or are they separate acts of religious faith? No-one can go back in time. How would he determine whether the religions come from a common ancestor and if the similarities are due to this common origin? First, there would be material evidence. The Tierra del Fuegan would have the evidence of archaeology. Biblical archeology had uncovered artefacts showing shared histories and transitions. He could be shown concrete evidence of religious history (such as Herod’s palace), linking Judaism and Christianity. Just as archeology can reveal the history and www.nature.com/reviews/genetics PERSPECTIVES a b West/East Greek Orthodox Christian Judaism (Roman empire variants) c Papal authority Greek Orthodox Christian Russian Orthodox Lutheran Judaism (Roman empire variants) Russian Orthodox Lutheran Anglican Anglican Catholic Catholic Reform Judaism Reform Judaism Conservative Judaism Conservative Judaism Orthodox Judaism Orthodox Judaism Figure 1 | York Minster and two rival cladograms of western religion. a | York Minster, a Tierra del Fuegan, aboard the HMS Beagle. b,c | Cladograms that could be constructed on the basis of the theological analogues of material evidence, homology and vestigial apparatus. In b, non-allegiance to the papacy is a derived chatacter that evolved twice in the history of the Christian clade, once in Eastern Orthodoxy, and then independently in Protestantism. In c, papal authority is considered a derived trait that evolved before the split between the East and West churches. For simplicity, Islam and many other religions have been omitted from the diagram. genealogy between religions, so paleontology reveals the history and transitions between groups of animals. Second, York Minster could use vestigial apparatus and homologies to show the genealogical connection between faiths. He might argue that Christian Bibles have lists of Semitic kings that would not be there were it not for their inclusion in an earlier religion. The fact that so many strange stories, such as that of Onan, Samson, Ahab and Jezebel, occur in the Hebrew Bible, the Protestant Bibles and the Catholic Bible would indicate that there is a common origin. His shipmate Darwin would similarly argue that such strange things as the aortic arches would not be present in mammals and amphibians were they not in the earlier ancestors from which they arose. The aortic arches would not be ‘invented’ twice, especially when most of the mammalian aortic arches degenerate. On the basis of his knowledge of homologies, vestigial apparatus and material artefacts, York Minster could draw a cladogram of religions. One such cladogram is shown in FIG. 1b. It should be noted that the first separation is between the Jewish and Christian ‘clades’. Then there is a division in the Christian ‘clade’ between the Eastern and Western Churches. Then there is a split in the Western Church between Catholicism and the Protestant denominations. This cladogram views the liturgy and Bible translation as fundamental characteristics. However, another cladogram can be made. This alternative (FIG. 1c) views the papacy as a derived trait (not seen in Jewish, Orthodox or Protestant lineages). So, together, Protestantism and Orthodoxy split from NATURE REVIEWS | GENETICS Catholicism, and only later do they branch off into Eastern and Western derivatives. Instead of the East/West distinction being primary, the second cladogram is made on the papacy/non-papacy distinction. How could York Minster decide which model is correct? How do we know that the model in FIG.1b is correct and the model in FIG.1c is wrong? Here, York Minster had a huge advantage over Charles Darwin.There were textual records for the histories of the churches.York Minster might say that the best evidence is neither from material artefacts nor from arguments about homologies and vestigial apparatus (as the lack of allegiance to the papacy could result either from common ancestry or from two independent events), but from textual evidence.In religions,this would be the set of documents that show the splitting of one religion into two or more new religions.Henry VIII’s 1534 Act of Supremacy and Martin Luther’s 95 Theses (1517) would be such documents,showing the separation of Protestant Christianity from Catholicism and not fromOrthodoxy. Darwin and his colleagues could draw branched-chain evolutionary trees of the animal kingdom that were based on embryonic homologies, adult homologies, vestigial apparatuses and the fossil record. But, until recently, there were many versions of these trees, because there was no independent assessment of homology except for the fossil record. And here is where macroevolution, the study of phylogeny — of evolution above the species-level — encountered a problem. It did not have such documentation. Except for the fossil record, which was much worse for Darwin than it is for us today, there was no independent assessment for common origins. Not until the arrival of molecular genetics would evolution have a powerful way of getting around the homology question. It wasn’t until the late 1970s that we could access the historical records of genes, and they turned out to be a treasure trove beyond measure. By finding rare and shared molecular similarities, the genealogical relationships between higher taxa can be found. Later phylogenetic reconstructions also included LIKELIHOOD AND BAYESIAN ESTIMATION TESTS for deciding which paths of evolution were most probable. Evolutionary problems that had been intractable to Darwin and early evolutionary biologists have now been solved. The phylogeny of insects, for example, was fraught with problems of homoplasy16. However, a rare gene rearrangement shared between insects and crustaceans has now shown the common ancestry of these two groups20, refuting the alternative hypothesis that insects were derived from a myriapod-like ancestor. Similarities in 18S ribosomal RNA sequences, coupled with the same duplication of certain Hox genes in particular phyla, linked a set of invertebrates into the ecdysozoan clade and established a particular set of genealogical relationships among the invertebrates21,22. Similarly, we have a record telling us that the whale and hippopotamus are closely related and that both arose from an ancestor that is in common with the cow and deer23. The phylogeny of animals has become less a matter of interpreting visual evidence and more a matter of reading the documentation. By looking at the cladograms of religion, and by showing how molecular evidence fills VOLUME 4 | SEPTEMBER 2003 | 7 3 7 PERSPECTIVES the gaps in our assessment of homology, we can explain evolution to the public, and we can also directly counter creationists’ claims that evolutionary biology is based on circular arguments about homology relationships. Evolutionary developmental biology The ability to find and sequence genes has done more than provide evolutionary biologists with an independent assessment of animal relationships. It has enabled developmental biologists to look at the causal mechanisms wherein small changes in genes can generate large morphological differences. In 1977, three publications paved the way for evolutionary developmental biology. These publications were the aforementioned article Evolution and tinkering12, Stephen J. Gould’s book Ontogeny and Phylogeny 24 and Maxam and Gilbert’s technique for DNA sequencing 25. In Ontogeny and Phylogeny, Gould showed how the German biologist Ernst Haeckel had misrepresented the field of evolutionary embryology and made it into an unscientific and racist doctrine. Indeed, the first half of this book exorcises Haeckel’s ghost so that some other model of evolution and development could be put in place of Haeckel’s Biogenetic Law, wherein ‘ontogeny recapitulates phylogeny.’ Jacob’s paper proposed a new model that could be tested, and the paper on DNA sequencing established a method that could test it. The first discoveries in evolutionary developmental genetics showed the remarkable homology of genetic instructions. Indeed, the developmental instructions for forming several analogous organs were shown to be homologous. For example, the fly eye and mouse eye have very little in common as to their origin or structure. However, Walter Gehring’s group showed that the instructions to form both the mouse and the fly eye are based on a set of homologous genes, such as Pax6 (REF. 26). The instructions are so similar that fly IMAGINAL DISCS will form an eye (a Drosophila eye) under the instruction of the rodent Pax6 gene27. Not only is eye development dependent on the Pax6 gene throughout the animal kingdom, but the genes that interact with Pax6 to form the photoreceptor cells also seem to be evolutionarily conserved28,29. Therefore, whereas it was previously thought that eyes formed independently many different times30, we now find that each type of eye is but a variation on a theme. Moreover, many of the genes that specify the formation of the heart, the nervous system and the anterioposterior body axis also seem to be the same throughout the animal kingdom, despite the enormous diversity of these structures31,32. So, whereas creationists are fond of saying that Darwin admitted that he had no idea how an organ as precise and as complicated as an eye could evolve, we now have the basic Chicken hindlimb Duck hindlimb BMP Gremlin Apoptosis Newborn Figure 2 | Regulation of chicken limb apoptosis by BMPs. Autopods of chicken feet (top) and duck feet (bottom) at similar stages. The in situ hybridizations show that while bone morphogenetic proteins (BMPs) are expressed in both the chicken and duck hindlimb webbing, the duck hindlimb also shows expression of gremlin in the webbing (arrows). Gremlin is an inhibitor of BMPs. The pattern of cell death (shown by neutral red dye accumulation) becomes distinctly different in the two types of webbing. Reproduced with permission from REF. 33 © Sinauer Associates (2003) and REF. 41 © The Company of Biologists (1999). 738 | SEPTEMBER 2003 | VOLUME 4 scheme. First, the genes that generated the photoreceptors of the primitive eyes of flatworms and other invertebrates are now used in the formation of the complex eye types. The entire optic system did not have to be invented de novo. Rather, there was descent with modification. Second, the retina and lens are in their respective positions because the lens helps build the retina and the retina helps build the lens26,33. This type of reciprocal induction is found whenever complex organs are being made. The blood vessels are able to get into the RENAL GLOMERULI because the developing cells of the renal glomeruli induce blood-vessel formation in the mesoderm next to them34,35. Third, complex systems with new properties can be generated by the interactions of simpler systems. Using computer programs that show the salient features of evolution — replication, variation and differential fitness — Lenski and colleagues36 showed that mutation and selection could evolve complex functions out of simpler ones, and that, in some instances, mutations that were originally deleterious when they first emerged became steppingstones in the evolution of more complex programmes. Importantly, there were several cases in which the amalgamation of simpler programmes into a more complex programme involved no intermediates. Development is complicated and interconnected; it is not irreducibly complex. However, although the first wave of evolutionary developmental biology uncovered the remarkable similarities among very diverse organisms, it was obvious that there had to be profound differences in the ways these genes were being used. For example, although we specify our anterior-posterior axis using Hox genes that are similar to those of fruit flies, we do not activate them by the same paths, nor do they have the same targets. Similarly, we might use Pax6 to specify our eye-forming regions, but our visual system does not form from imaginal discs. So there must be important differences as well as important similarities. If we are talking about descent with modification, we expect both underlying similarities and secondary differences. Can these differences in developmental genetics explain morphological differences? During the past five years, we have seen that there are at least five ways in which changes in developmental regulatory genes can mediate evolutionarily important morphological changes. Mutating the regulatory genes. One way to produce evolutionary change is to mutate regulatory genes. This seems to have been the case with the Ultrabithorax (Ubx) gene, which has undergone a mutation in the insect clade. www.nature.com/reviews/genetics PERSPECTIVES As a result of this mutation, the Ubx protein of insects (but not of other arthropod groups) now represses Distal-less expression37,38. This means that insects will only have thoracic legs. Whereas other arthropods — millipedes, centipedes and crustaceans — are known for their many legs, insects have but six. As Jacob predicted, mutations in regulatory genes can give them new properties. Altering the genetic targets of regulatory proteins. In many instances, large changes in evolution can be made by maintaining the same patterns of regulatory protein expression but altering their downstream targets. This can be seen in the difference between four-winged butterflies and two-winged fruit flies and houseflies. The butterfly hindwing differs from the Drosophila HALTERE, but both structures are generated through the action of the Ubx protein on the larval imaginal discs of the third thoracic segment. However, Ubx regulates different genes in the butterfly imaginal discs from the ones in the Drosophila imaginal discs. So the DIPTERANS have diverged from other four-winged insects through changes in the Ubx-responsive genes of the thoracic imaginal discs39, 40. Altering the spatial expression of regulatory genes. A mutation in the actual protein or its receptor is not needed to effect evolutionary change. A change in the expression pattern of a regulatory protein can also be vitally important. For example, changes in the spatial expression of regulatory genes can explain how the duck got its webbed feet41. Here, the expression of bone morphogenetic protein 4 (BMP4) in the interdigital spaces signals these ‘webbing’ cells to undergo apoptosis. Duck and chicken hindlimbs have the same pattern of BMP4 gene expression, both embryos expressing BMP4 in the interdigital cells of the foot. Where they differ is in the expression of a BMP inhibitor protein, Gremlin (FIG. 2). Gremlin expression is seen in both the chicken and duck hindlimbs, where it is found around the digits. In the duck, however, and not in the chicken, the Gremlin gene is also expressed in the interdigital cells. The Gremlin protein made there prevents BMP from signaling for cell death in the webbing, and the result is a webbed foot. This can be experimentally tested by adding Gremlin-containing beads into the interdigital regions of the embryonic chicken hindlimb. When this is done, the chicken foot resembles that of a duck (FIG. 3). This is bricolage. The protein hasn’t changed, it is just being expressed at a different place. Changing the pattern of regulatory gene expression has also been shown to correlate with the type of vertebra formed by the SOMITES and the type of appendage formed by crustaceans42–44. It has also been correlated with the formation of feathers from scales. Feathers have long been proposed as an evolutionary novelty, and creationists say that feathers cannot be generated from any other structure. However, recent papers present evidence that differences in the expression of Sonic hedgehog (Shh) and BMP proteins separate the feather from the scale45,46. Moreover, when the expression of Shh or BMP2 is altered, the feather pattern changes. The results corre- a b Figure 3 | Inhibition of cell death by inhibiting BMP. a| Control chicken hindlimbs have extensive apoptosis in the space between the digits, leading to the absence of webbing. b | When beads soaked with Gremlin protein, an inhibitor of bone morphogenetic protein (BMP), are placed into the interdigital mesoderm, the webbing persists and generates a duck-like foot. Reproduced with permission from REF. 41 © The Company of Biologists (1999). spond exceptionally well to a proposed mechanism of feather production from archosaurian scales. Changing the pattern of regulatory gene expression has also been correlated with the loss of limbs in snakes47. Changes in the temporal expression of regulatory genes. In addition to getting evolutionary change by altering the spatial expression of genes, remarkable changes can be obtained by altering the temporal expression of genes. Heterochronic (temporal) changes in Wnt5 gene expression are responsible for the changes between direct and indirect developing sea urchins48. Heterochronic changes also seem to Glossary BAYESIAN IMAGINAL DISCS NOTOCHORD A branch of statistics that focuses on the posterior probability of hypotheses. The posterior probability is proportional to the product of the prior probability and the likelihood. Thickenings of the epidermis in larval insects. These structures produce the adult wings, legs, eyes, antennae, mouth and genitalia during metamorphosis. A rod of mesodermal cells in the dorsal midline beneath the neural tube. It is the major characteristic of chordates. INSTAR POLYPHENISM The period between larval insect molts. Phenotypic variation not attributable to genetic differences: the product of environmental stimuli on particular genotypes. One example is seasonal polyphenism, whereby individuals with the same genotype manifest different phenotypes owing to temperature. DIPTERANS Insects with two wings, such as flies and mosquitos. ECLOSION The emergence of the adult insect from its pupal case. HALTERE Balancers. Club-like tissue arising from the imaginal disc in the third segment of dipterans. HOMOPLASY Similarity whereby structures have similar form or function but not the same ancestral origin. Homoplasies often result from convergent evolution whereby different organisms in the same environment produce similar adaptions. HYPOPHYSEAL Pituitary. NATURE REVIEWS | GENETICS LIKELIHOOD ESTIMATION TEST A statistical method that calculates the probability of the observed data under varying hypotheses to estimate model parameters that best explain the observed data and determine the relative strengths of alternative hypotheses. MODERN SYNTHESIS Neo-Darwinism. The theory that natural selection, acting on randomly generated variation, is the major cause of evolution. REACTION NORMS A situation in a population in which the genotype provides a graded response to environmental conditions. NEURAL CREST CELLS RENAL GLOMERULI A migratory cell population that arises at the lateral edges of the neural plate, and which differentiates into numerous cell types including skull and facial bone, pigment cells, adrenal medullary cell, and the neurons and glia of the sensory and autonomic nervous systems. A cluster of capillaries in the kidney cortex. SOMITES Blocks of mesoderm along the vertebrate body axis that further differentiate into dermal skin, bone and muscle. VOLUME 4 | SEPTEMBER 2003 | 7 3 9 PERSPECTIVES Population genetics Developmental genetics Variation within populations Variation between populations Genes in adults competing for reproductive success Genes in embryonic and larval organisms building structures Survival of the fittest Arrival of the fittest Natural selection Phylogeny A new evolutionary synthesis explaining biodiversity Figure 4 | A newly emerging evolutionary synthesis. The classic approach to evolution has been that of population genetics. It emphasized variations within a species that allowed certain adult individuals to reproduce more frequently. In this way, it could explain natural selection. The developmental approach looks at variation between populations, and it emphasizes the regulatory genes that are responsible for organ formation. Its explanations are more apt to explain evolutionary novelty and constraint. Together, population genetics and developmental genetics make a more complete genetic approach to evolution. be responsible for another evolutionary novelty, the vertebrate jaw — one of the problems that puzzled evolutionary embryologists in the early 1900s. In the lamprey, the naso-hypophyseal plate is retained and prevents the migration of NEURAL CREST CELLS rostrally. In jawed vertebrates, this plate separates very early in development into the nasal and HYPOPHYSEAL neural tissues, thereby making a path for the rostral migration of neural crest cells. This allows the formation of a new structure, the jaw. As Kuratani and colleagues conclude, “The clue to solve this problem, therefore, will not be obtained by comparative anatomy of the adult structures, but rather by discrimination of conserved and newly acquired patterns of gene expression… Molecular developmental biology has taken the initial steps into this old question of comparative zoology, but it has already suggested new directions in which a solution may lie.” (REF. 49). Selection of variants that are caused by developmental plasticity. In many organisms, the genotype does not directly predict the phenotype. Rather, the genotype provides a range of potential phenotypes and the exact phenotype will be induced by the environment. Usually, the environmental stimulus is sensed by the neuroendocrine system and the hor- 740 | SEPTEMBER 2003 | VOLUME 4 mones control the expression of particular genes9,50. This is known as developmental plasticity and it is the basis for REACTION NORMS and POLYPHENISMS. At different points in their ranges one extreme of this plasticity might become genetically fixed51–54. This ability of a developmentally plastic trait to become genetically fixed has been documented numerous times in the laboratory and could provide a rapid means for the origin of new species55,56. A new evolutionary synthesis The developmental genetic approach to evolution complements the traditional population genetic approach. They are both needed. The mechanisms of genetic change in regulatory genes are the same as those of structural genes, and once these evolutionary novelties are generated they must still be selected. The traditional differences between the population genetic and developmental genetic approaches to evolution are summarized in FIG. 4 . These differences are beginning to blur as both population geneticists and developmental geneticists start studying allelic variations of developmental regulatory genes within populations57. I would emphasize the importance of including examples from the developmental genetics approach when discussing evolution in public. In addition to being easier to understand than the population genetic approach to teaching evolution, the developmental approach might have other advantages. First, the developmental genetic approach uses examples that everyone can understand from visual experience. Second, evolution at the level of developmental regulatory genes might be the main source of variation. In humans (that animal for which variations have been catalogued most carefully), genes are heterozygous at more functional cis-regulatory sites (>16,000) than at exon sites (<13,000). Ordinary small-scale mutations contribute to large variations in transcription rates across the genome and so to human variation57. Third, these examples address the questions of evolutionary novelty that creationists say cannot be explained by evolution — how insects have fewer legs than centipedes, how snakes lost their legs, how birds got feathers and ducks got their webbed feet. We are therefore able to show where creationists are wrong and how their ideas about homology and morphological novelties are out of date. If the processes of evolution were viewed solely from the population genetic perspective, it would appear very difficult to explain the origins of feathers, teeth and eyes. They seem to be (in the words of the creationists) “irreducibly complex”6. However, when the perspectives of embryonic induction and developmental genetics are added, these novelties become explainable, at least in outline, if not yet in detail. Such information is crucial if we are to counter the distortions of science by creationists58. J. B. S. Haldane, the editor of the volume in which Waddington published the abovementioned paper, commented on this idea using a wonderfully apt developmental metaphor,“To sum up, then, a number of workers are groping from their own different standpoints towards a new synthesis, while producing facts which do not fit too well into the currently accepted synthesis. The current instar of the evolution theory may be defined by such books as those of Huxley, Simpson, Dobzhansky, Mayr, and Stebbins. We are certainly not ready for a new moult, but signs of new organs are perhaps visible.” (REF. 59). Today, exactly half a century later, we can report that the organs have formed and the INSTAR has ECLOSED. Evolutionary developmental biology has become a normative part of evolutionary biology and should provide wonderful and informative new ways of teaching evolution to biology students and to the general public. Scott F. 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I wish to thank the Education Committee of the SDB for the opportunity to write it and to Kenneth Miller and Sean Carroll for their helpful comments. Funding was from the National Science Foundation and from Swarthmore College, Pennsylvania. Online links DATABASES The following terms in this article are linked online to: FlyBase: http://flybase.bio.indiana.edu Distal-less | Ubx LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink Pax6 Swiss-Prot: http://www.expasy.ch BMP2 | BMP4 | Gremlin | Sonic hedgehog FURTHER INFORMATION Evolution, development, and creationism — a supplement: http://zygote.swarthmore.edu/Darwin Access to this interactive links box is free online. OPINION Vertebrate gene predictions and the problem of large genes Jun Wang, ShengTing Li, Yong Zhang, HongKun Zheng, Zhao Xu, Jia Ye, Jun Yu and Gane Ka-Shu Wong To find unknown protein-coding genes, annotation pipelines use a combination of ab initio gene prediction and similarity to experimentally confirmed genes or proteins. Here, we show that although the ab initio predictions have an intrinsically high false-positive rate, they also have a consistently low false-negative rate. The incorporation of similarity information is meant to reduce the false-positive rate, but in doing so it increases the false-negative rate. The crucial variable is gene size (including introns) — genes of the most extreme sizes, especially very large genes, are most likely to be incorrectly predicted. We live in the halcyon days of large-scale DNA sequencing. Each release of a sequenced genome is accompanied by a list of genes, many of which are computer predictions. Experimental confirmation in the form of sequenced transcripts of full-length cDNAs is extensive for mice1,2, less so for humans3 and non-existent for pufferfish4. For invertebrate genomes, cDNAs are less important because the genes are smaller and easier to predict. Nevertheless, the many fruitfly 5 and nematode6 cDNAs that were produced after their genomes were sequenced have been invaluable in finding residual errors in the definition of exon boundaries. As it is difficult to get cDNAs that are expressed transiently, or at low levels, in specific tissues and at specific developmental stages, predicting genes will remain an integral part of DNA sequence analysis for the foreseeable future. Therefore, it is imperative for the biologists who use gene-prediction programs to understand what they can and cannot do. Although some features of these programs are better than is commonly thought, others are worse. It is tempting to dismiss the programs as being inherently unreliable (see BOX 1 for a note on fluctuations in gene number in the human genome), but, in fact, they fail for specific reasons that can be understood with minimal jargon and without delving into algorithmic minutiae. ANNOTATION PIPELINES have been comprehensively reviewed7. Every pipeline incorporates information from known genes. No pipeline ever substitutes a predicted gene for a known VOLUME 4 | SEPTEMBER 2003 | 7 4 1