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Thomas Flatt Division of Biology and Medicine, Department of Ecology and Evolutionary Biology, Brown University, Keywords canalization, constraint, epistasis, genetic architecture, genetic assimilation, genetic variance, genotype by environment interaction, genotype- phenotype, map, phenotypic plasticity, phenotypic variance, selection

Abstract Evolutionary genetics has recently made enormous progress in understanding how genetic variation maps into phenotypic variation. However, why some traits are phenotypically invariant despite appar-ent genetic and environmental changes has remained a major puzzle. In the 1940s, Conrad Hal Waddington coined the concept and term “canalization” to describe the robustness of phenotypes to perturbation; a similar concept was proposed by Waddington’s contemporary Ivan Ivanovich Schmal-hausen. This paper reviews what has been learned about canalization since Waddington. Canalization implies that a genotype’s phenotype remains relatively invariant when individuals of a particular genotype are exposed to different environments (environmental canalization) or when individuals of the same single- or multilocus genotype differ in their genetic background (genetic canalization). Con-sequently, genetic canalization can be viewed as a particular kind of epistasis, and environmental canalization and phenotypic plasticity are two aspects of the same phenomenon. Canalization results in the accumulation of phenotypically cryptic genetic variation, which can be released after a “decan-alizing” event. Thus, canalized genotypes maintain a cryptic potential for expressing particular phe-notypes, which are only uncovered under particular decanalizing environmental or genetic conditions. Selection may then act on this newly released genetic variation. The accumulation of cryptic genetic variation by canalization may therefore increase evolvability at the population level by leading to phenotypic diversification under decanalizing conditions. On the other hand, under canalizing con-ditions, a major part of the segregating genetic variation may remain phenotypically cryptic; canalization may therefore, at least temporarily, constrain phenotypic evolution. Mechanistically, canalization can be understood in terms of transmission patterns, such as epistasis, pleiotropy, and genotype by environment interactions, and in terms of genetic redundancy, modularity, and emergent properties of gene networks and biochemical pathways. While different forms of selection can favor canalization, the requirements for its evolution are typically rather restrictive. Although there are several methods to detect canalization, there are still serious problems with unambiguously demonstrating canalization, particularly its adaptive value. 288

HE STUDY of phenotypic variation is a typic variation may well be explained by clas- Tcentral theme in evolutionary biology. sical scenarios, such as selection or drift. Yet, Natural selection results from variation in fit- it remains a fascinating possibility that organ- ness among individuals; the response to selec- isms may have evolved specific mechanisms tion depends on the heritable determinants that make them insensitive to genetic and of the phenotype. Phenotypes are the prod- environmental change, thereby decreasing uct of developmental processes that depend their capacity for evolutionary change. Alter- both on the genotype and environment and natively, the molecular details of the func- their interaction. It is thus important to tional architecture of complex phenotypic understand how genetic variation maps to traits may lead to robustness as a nonadaptive phenotypic variation, and how this genotype- byproduct, an emergent property. This com- phenotype map is influenced by genetic and monly observed robustness of phenotypes environmental change. Although evolution- was named “canalization” by Waddington in ary biology is to a major degree concerned the 1940s to describe the mechanisms that with the study of variation, we have only a lim- cause the phenotype to be insensitive against ited understanding of the absence of varia- genetic and nongenetic perturbations and tion. Why are some traits phenotypically change (Waddington 1942; see also Schmal- invariant despite apparent genetic and envi- hausen 1949). ronmental changes? Why are some traits less Canalization is highly relevant for evolu- phenotypically variable in some taxa, but not tionary biology. For example, it implies that in others? phenotypes may be stable around their fitness One of the most intriguing observations in optimum despite genetic and environmental evolutionary genetics is that wild-type popu- change (e.g., Rendel 1967). By keeping phe- lations often harbor vast amounts of hidden notypic variation low, canalization may con- genetic variation, this variation being phe- strain phenotypic evolution (e.g., Charles- notypically expressed only in particular envi- worth et al. 1982; Maynard Smith et al. 1985) ronments or genetic backgrounds (e.g., Gib- and provide a microevolutionary mechanism son et al. 1999; Gibson and Dworkin 2004). for character stasis (e.g., Stearns 1994). Can- Thus, there seems to be a strong robustness alization also allows genetic variation that is of some phenotypes against genetic and non- phenotypically not expressed to accumulate. genetic change or perturbation. More gen- This cryptic variation can lead to the appear- erally, the amount and quality of phenotypic ance of new phenotypes when development variation can differ dramatically within and is “decanalized,” for instance by environmen- among populations. Some traits are highly tal stress, thereby allowing evolutionary invariant within species while simultaneously change (e.g., Rutherford and Lindquist being highly variable among closely related 1998). species; other characters seem to be highly Despite the long history of the canalization conserved among species or clades. Why is concept (Waddington 1942; Schmalhausen this the case? Similarly, the same trait may be 1949; also see Hall 1992; Gilbert 2000; Slack more variable in some taxa than in others. 2002), the evidence for canalization is limited Does robustness occur in all taxa and for all (e.g., Scharloo 1991; Gibson and G P Wagner traits to the same extent? Some of the differ- 2000; De Visser 2003). The evolutionary role ences in the amount and quality of pheno- of canalization has remained puzzling: the 289 concept is difficult to define, and its predic- One of the most fundamental problems of tions are often beyond the reach of experi- evolutionary biology is to better understand ments. Similarly, the molecular mechanisms the pathways that connect genotypes with governing canalization are poorly under- phenotypes, the genotype-phenotype map stood. Thus, not surprisingly, following the (e.g., Lewontin 1974a; Wright 1977; Houle classical work by Waddington, Schmalhausen, 1991, 2001; Schlichting and Pigliucci 1998). and others, empirical and theoretical Canalization is but one aspect of the geno- research on canalization declined, presum- type-phenotype map; the general problem is ably due to the lack of suitable theoretical and to understand the functional architecture of genetic methods to tackle the problem of complex phenotypes that depend upon mani- phenotypic robustness. However, recent fold interactions among underlying genes advances in molecular developmental genet- and the environment. A better understand- ics and theoretical biology have set the stage ing of canalization will contribute to a clearer for a comeback of the canalization concept conceptual picture of how genes, develop- among theoretical biologists (cf. Gibson and ment, and the environment interact to pro- G P Wagner 2000), evolutionary geneticists duce phenotypes. (cf. De Visser et al. 2003), and developmental biologists (cf. Gerhart and Kirschner 1997; The Phenomenon of Canalization Hartman et al. 2001). Thus, recent theoreti- the stability of the wild-type cal work (e.g., G P Wagner et al. 1997; Rice 1998; Kawecki 2000; Siegal and Bergman Waddington based the concept of canali- 2002; Bergman and Siegal 2003; Hermisson zation on the observation that genotypes dif- et al. 2003; Hermisson and G P Wagner 2004; fer in their phenotypic reactions to genetic Proulx and Phillips 2005), evolutionary and environmental change, and that wild- experiments (e.g., Stearns and Kawecki 1994; type phenotypes are phenotypically much less Stearns et al. 1995; Elena and Lenski 2001), variable than mutants or environmentally- and molecular studies (e.g., Gibson and Hog- induced phenotypes (Waddington 1942; Gib- ness 1996; Rutherford and Lindquist 1998; son et al. 1999). In a similar vein, Schmalhau- True and Lindquist 2000; A Wagner 2000b; sen (1949) argued that the “stability of the Queitsch et al. 2002; Gu et al. 2003; Sollars et morphogenetic system is destroyed (ren- al. 2003; True et al. 2004) have renewed inter- dered labile) due either to variation in envi- est in canalization. ronmental factors or to mutation” (Schmal- This paper provides a comprehensive hausen 1949/1986:79). Waddington (1957) review of what has been learned about the suggested that the reason for the difference evolutionary genetics of canalization since in variation between wild-type as opposed to Waddington (for recent, shorter reviews, see mutant or environmentally-induced pheno- Gibson and G P Wagner 2000; Meiklejohn types is that the wild-type has been exposed and Hartl 2002; De Visser et al. 2003; Gibson to many generations of stabilizing selection, and Dworkin 2004; also see the book edited whereas the mutant has not (see also Schmal- by Hall and Olson 2003). I ask six questions: hausen 1949). Several experiments seem to (1) How can canalization be defined? (2) support Waddington’s view by showing that What is the relationship between canaliza- stabilizing selection can reduce the variability tion, epistasis, and genotype by environment of environmentally-induced or mutant phe- interactions? (3) At the proximate level, notypes, and that phenotypic change becomes which molecular mechanisms may lead to progressively more difficult when the trait canalization? (4) At the ultimate level, how approaches the wild-type pattern (e.g., May- does canalization originate? Is it a sideprod- nard Smith and Sondhi 1960; Waddington uct or an emergent property of the genotype- 1960). This has been taken as evidence for phenotype map, or is it shaped by natural the adaptive canalization of the wild-type (cf. selection? (5) What are the consequences of Scharloo 1991). However, in most experi- canalization for evolutionary processes? (6) ments it is not clear whether stabilizing selec- How can canalization be measured? tion just decreased genetic variation or 290 whether it selected for canalizing mecha- phology, behavior, and life-history traits. For nisms itself. Furthermore, the wild-type con- instance, canalization may be observed at the cept is an ambiguous abstraction since natu- level of morphology, but not at the level of ral wild-type populations are often highly gene expression. The buffering would then genetically and phenotypically variable. See occur at some intermediate level between Scharloo (1991) for a comprehensive review gene expression and morphology. Further- of Waddington’s original view. more, to maintain a particular trait (e.g., body temperature in homeotherms) despite definition of canalization (e.g., thermal) perturbations, an organism Canalization is the reduced sensitivity of a may vary other traits (such as basal metabolic phenotype to changes or perturbations in the rate and dilation of blood vessels). Stability underlying genetic and nongenetic factors (homeostasis) at one level may depend upon that determine its expression (see also Mei- lability or sensitivity at another level. Thus, it klejohn and Hartl 2002; De Visser et al. 2003). seems that there exists an intrinsic paradox of canalization: buffering at one level (the phe- Canalization is a relative term, and can thus notype) may be coupled to higher variation only be defined as a matter of comparison. at another level (e.g., gene expression). Vari- Thus, a phenotype P is more canalized than ation in such buffering mechanisms can be another phenotype P* if P remains relatively heritable or nonheritable. invariant when the single- or multilocus ge- Homology, when two or more structures notype G, which determines P, is exposed to are alike because of shared ancestry, may be different environments (environmental ca- another interesting aspect of the biological nalization) or located in different genetic hierarchy related to canalization. Homology backgrounds (genetic canalization): P is “resil- can occur between entities at different levels ient,” “robust,” or “insensitive” to genetic of the biological hierarchy; for instance some and/or environmental changes or perturba- organisms may show homologies at the tions. Canalization can therefore be recog- genetic level, but not necessarily at the mor- nized by observing that most genetic or envi- phological level (Laubichler 2000). Conse- ronmental changes leave the phenotypic quently, individuals from different species are expression of G, and thus the phenotype P, often composed of the same kind of (struc- invariant; the expression of G is changed such turally identical) building blocks (e.g., genes, r that specific phenotypic changes (P P*) are organs, and traits). Such homologous struc- induced only in some genetic backgrounds or tures may be a manifestation of canalization; environments (or combinations of genetic canalization may potentially explain the sta- backgrounds and environments). Conse- bility of homologues, since canalization may quently, a canalizing allele or genotype G limit or constrain variation in these structures reduces the phenotypic variation of a trait (e.g., G P Wagner 1996; G P Wagner and across a range of genetic backgrounds and Altenberg 1996; Laubichler 2000). A similar environments relative to a noncanalizing concept to homology is homoplasy, when two allele or genotype G*, and a canalized trait P or more structures are alike but not due to exhibits a restricted range of phenotypic vari- common ancestry. It is an interesting, yet ation across genetic backgrounds and envi- open empirical question whether and how ronments as compared to a noncanalized canalization affects homology and homoplasy trait P* (Meiklejohn and Hartl 2002). and whether, for example, homoplasy is more The proximate (molecular) mechanisms commonly observed in taxa with less cana- causing canalization, as well as the nature of lized traits. the perturbations, can be manifold. For Various authors have used different termi- example, the canalizing mechanisms can nologies when referring to canalization or potentially be located at any level of the bio- aspects of it (see Debat and David 2001; Mei- logical hierarchy, from gene expression, RNA klejohn and Hartl 2002). Autonomous de- stability, protein structure and folding, inter- velopment or autoregulation (Schmalhausen mediate metabolism and physiology to mor- 1938, 1949), homeostasis, homeorhesis or buf- 291 fering (Bernard 1865; Cannon 1932; Lerner ticular trait, are outcrossed with normal lab- 1954; Lewontin 1956; Zakharov 1992; Kauff- oratory strains, the trait is expressed only at man 1993; Hallgrı´msson et al. 2002), devel- very low levels, implying a return to the can- opmental stability (Thoday 1955; Palmer and alized state. Thus, the functional hsp83 locus Strobeck 1986, 1992), epigenetic stability (A is a gene with canalizing effects, masking the Wagner 1996), and robustness (e.g., Savageau effects of hidden genetic variation. Queitsch 1971; Kauffman 1993; Little et al. 1999; et al. (2002) confirmed the results of Ruth- De Visser et al. 2003) are essentially synony- erford and Lindquist (1998) by showing that mous with canalization. Sometimes canaliza- reducing the function of Hsp90 in various tion is taken to mean adaptive, evolved Arabidopsis genotypes increases genetic varia- robustness against heritable and nonherita- tion in morphological traits. ble perturbations, whereas terms such as buf- But does Hsp90 also buffer against environ- fering refer to any kind of mechanism, adap- mental, nongenetic perturbations such as tive or not, that will cause the phenotype to developmental noise? As work by Milton et al. be resilient against perturbations (cf. De- (2003) demonstrates, this does not seem to Visser et al. 2003). See Hall and Olson (2003) be the case (but see Queitsch et al. 2002): in for an excellent recent discussion of evolu- D. melanogaster, Hsp90 does not buffer against tionary developmental biology in general, nongenetic perturbations as measured by including various issues bearing on canaliza- fluctuating asymmetry in bristle traits. The tion discussed in this review. Here I use the Hsp90 system is undoubtedly the best current term canalization to mean any mechanism, example of a molecular canalizing mecha- structure, or process, adaptive or not, that will nism; it nicely illustrates how environmental reduce a phenotype’s sensitivity to perturba- cues (e.g., geldanamycin, temperature) or tions. mutation can lead to the release of hidden genetic variation. Intriguingly, Sollars et al. an example of canalization (2003) have shown that Hsp90 can act through epigenetic mechanisms, whereby a When the function of the Hsp90 protein, a reduced activity of Hsp90 causes a heritable chaperone and heat shock protein, encoded change in the chromatin state (see True et al. by the hsp83 locus in Drosophila melanogaster is 2004 for another example of epigenetic can- impaired by mutation or by the specific inhib- alization). itor geldanamycin, phenotypic variation The importance of heat shock proteins for increases both in laboratory and wild strains canalization has also been confirmed by Fares (Rutherford and Lindquist 1998). The et al. (2002): the authors show that overex- degree of increase and the nature of the phe- pression of the heat shock protein GroEL notypic variation depend on the genetic back- induces the recovery of fitness of Escherichia ground and environment (e.g., tempera- coli strains that have accumulated deleterious ture). A wide range of phenotypic effects is mutations. Yet, recent modeling work by Her- observed, including defects in bristles, eyes, misson and G P Wagner (2004) casts some halteres, legs, wings, the thorax, and abdo- doubt on whether Hsp90 can really be seen men. The authors showed that at least some as an example of canalization. Although can- of these phenotypes are produced by alleles alization is an attractive explanation for the that are not phenotypically expressed in the accumulation of cryptic genetic variation, presence of the functional Hsp90 protein. theory clearly shows that canalization is not This genetic variation is heritable, and the necessarily required to explain the buildup of pattern of heritability is unlikely to be due to cryptic variation. Accumulation of mutations de novo mutation but to genetic variance in at conditionally neutral loci for a sufficiently polygenic traits. Thus, impairment of Hsp90 long time can lead to the accumulation of uncovers previously silent genetic variation, cryptic variation even in the absence of can- which leads to an increase of phenotypic alization (Hermisson and G P Wagner 2004). variation (decanalization). When decanali- See Rutherford (2003) for a comprehensive zed lines, showing a high penetrance of a par- review of the role of protein chaperones such 292 as the Hsp family for canalization and buf- 1999; Gibson and Dworkin 2004), which are fering; see Scharloo (1991) for a review of only expressed when the genetic background some of the classic—but more ambiguous— changes—a common observation also made examples of canalization. by developmental geneticists working on spe- cific mutations in different genetic back- potential variation grounds. Similarly, in a mutation accumula- Canalization has been defined as a state of tion experiment, Davies et al. (1999) exposed reduced variability, that is, highly canalized a wild-type strain of Caenorhabditis elegans to a genotypes show a reduced potential to vary in mutagen and, from this strain, bred sixty response to genetic or environmental change independent lines toward homozygosity by as compared to some less canalized mutants selfing. In fitness assays of these lines, the or environmentally induced phenotypes (G P authors found that the frequency of delete- Wagner and Altenberg 1996; Gibson and van rious mutations is about 96%, and these Helden 1997; G P Wagner et al. 1997; Gibson mutations have fitness effects of less than 0.07%. Yet, whether these fitness effects are and G P Wagner 2000). Thus, the concept of reduced due to canalization, that is, they are “variability” (not to be mistaken by the term magnified in other environments or genetic “variation,” which refers to the actual level of backgrounds, remains to be determined. In change) is a way of expressing the observa- haploid mutant strains of yeast (Saccharomyces tion that highly canalized genotypes are cerevisiae), Thatcher et al. (1998) found that much more insensitive to mutations or envi- a major proportion of null mutations has ronmental changes than most other, less can- almost no effect on fitness because the func- alized genotypes (G P Wagner and Altenberg tion of a given knocked-out gene can be com- 1996; G P Wagner et al. 1997; Gibson and G P pensated by other genes. Thus, there is Wagner 2000). Thus, in comparison, geno- genetic variation due to null mutations with types with high variability (reduced state of potentially deleterious effects on fitness, but canalization) change their phenotype much this variation is cryptic and has no phenotypic more readily than genotypes with low vari- consequences for fitness. For a recent review ability (increased state of canalization) when on cryptic genetic variation see Gibson and faced by the same mutational or environmen- Dworkin (2004). tal change. Variability is itself under genetic The cryptic pool of genetic variation accu- control; some genotypes are more canalized mulated under canalization can be phenotyp- than others. For example, genetic polymor- ically expressed again if genetic or environ- phisms at the Ultrabithorax (Ubx) locus in Dro- mental change uncovers the silent genetic sophila melanogaster, a gene required for the variation (decanalization; e.g., Scharloo 1991; establishment of thoracic segment identity, Gibson and Hogness 1996; Rutherford and have been shown to be responsible for the Lindquist 1998; Gibson et al. 1999; Gibson differential insensitivity among Ubx genotypes and Dworkin 2004; Hermisson and G P Wag- against developmental perturbations by ether ner 2004), thereby increasing phenotypic vapor (Gibson and Hogness 1996). variation in the population. Decanalizing Canalization uncouples genetic variation conditions can be due to environmental per- from phenotypic variation among the cana- turbations that change environment-depen- lized genotypes in the population. This is dent gene expression or allele substitutions because, in contrast to weakly or noncanali- that render canalizing mechanisms nonfunc- zed genotypes, highly canalized genotypes tional. Whether a state of decanalization will can accumulate mutations or maintain exist- be reached is likely to depend on the degree ing alleles that are not phenotypically of canalization of a genotype or population expressed. Thus, part of the genetic variation as well as on the frequency and intensity of in the population can be maintained in a hid- the decanalizing conditions. Interestingly, den form. For example, wild-type genotypes recent theory shows that mutation accumu- often harbor substantial amounts of cryptic lation may eventually lead to a state of de- or hidden genetic variation (Gibson et al. canalization and indicates that a state of 293 high variability may be a generic property of ent phenotypic values: along a given contour mutant phenotypes. This is because under line, the underlying factors produce the same most forms of epistasis, mutation accumula- phenotypic value. The spacing between con- tion is expected to lead to an accelerating tours of equal phenotype indicates the slope increase of additive genetic variance (Hansen of the surface. At a given point, the slope and G P Wagner 2001). Consider the effect along the contour indicates the degree to of a single major mutation on the mutational which a given amount of variation in the variance of a particular trait in a given genetic underlying factors translates into phenotypic background. It has been found that the variation. The slope is therefore a measure of stronger the epistasis (i.e., the stronger the the degree of canalization. If the underlying effect of one locus on a substitution at factors contribute additively to the pheno- another locus), the stronger is the increase in type, then the landscape is a plane with no additive genetic variance under mutation curvature, the degree of canalization being accumulation (Hansen and G P Wagner everywhere the same. If the factors contribute 2001). Thus, mutations are decanalizing; the nonadditively (e.g., epistasis, genotype by more mutations that have already accumu- environment interactions), then the land- lated and the stronger the epistasis, the more scape is curved, and there are different decanalizing the mutations are (Hansen and degrees of canalization (as represented by the G P Wagner 2001). Thus, the results suggest unequal spacing of contour lines). If the sur- that a state of increased variability of mutant face curves, then the slope is likely to be lower phenotypes may be a generic property of epi- at some points along a contour than at others. static systems (Hansen and G P Wagner The points of minimal slope along a given 2001). As discussed by these authors, this contour line are the points of maximum can- finding does not falsify the concept of cana- alization for a given phenotype. In contrast, lization, but eliminates increased variability points of equal canalization along a contour of mutant phenotypes as indirect evidence line have the same partial derivative of the for the wild-type being canalized to reduce phenotype ␾ with respect to a given change the effects of genetic and environmental in an underlying factor. changes, as had been suggested by Wadding- This geometrical view makes two important ton (1942). Recent work supports this notion assumptions. First, the phenotypic landscape by showing that the release of genetic varia- is a function of several underlying factors that tion caused by mutation or environmental determine the expression of the phenotype. perturbation is a generic property of systems exhibiting epistasis or genotype by environ- Thus, based on the source of perturbation ment interactions (Hermisson and G P Wag- against which the phenotype is buffered, one ner 2004). can distinguish, for example, between genetic and environmental canalization (e.g., Wad- Genetic and Environmental dington 1942, 1957; Stearns and Kawecki Canalization 1994; G P Wagner et al. 1997). Second, non- additivity of the factors shaping the pheno- A useful way to illustrate canalization is to typic landscape implies different degrees of consider a phenotypic landscape (Rice 1998, canalization. Thus, canalization is best 2000, 2002; Wolf et al. 2001; Nijhout 2002). A phenotypic landscape is a surface that defines described in terms of nonadditive interac- the phenotype ␾ as a smooth function of tions, such as epistasis and genotype by envi- underlying factors, for example, genetic and ronment interactions. environmental factors (Rice 1998, 2000, 2002). Interactions among these factors rep- genetic canalization and epistasis resent the process of development and deter- Epistasis is the influence of one locus on mine the shape of the phenotypic landscape. the expression of genetic variance at another The height of the landscape is a measure of locus (Moreno 1994; Whitlock et al. 1995; some phenotypic trait, and the different con- Fenster et al. 1997; Wolf et al. 2000; Wade et tour lines of the landscape represent differ- al. 2001). A wealth of genetic observations 294 indicates that epistatic effects are extremely accumulation of phenotypically relevant common: genes may have specific effects on mutations (see Fontana and Schuster 1998; particular phenotypes, but these effects often De Visser et al. 2003). Interestingly, as shown strongly depend on other interacting genes by Dworkin et al. (2003), the gene for the epi- (Wade 2002). For example, wild-type geno- dermal growth factor receptor (Egfr) harbors syn- types often have large amounts of cryptic onymous substitutions that supply cryptic genetic variation, which is only phenotypi- genetic variation for photoreceptor determi- cally expressed in some but not in other nation. Remarkably, this variation, not genetic backgrounds (Gibson et al. 1999). observed in most environmental conditions Consequently, specific genetic effects on the or genetic backgrounds, shows the imprint of phenotype may be absent, weaker, or purifying selection (Dworkin et al. 2003). stronger, depending on the genetic back- Thus, the cryptic variation supplied by syn- ground. Genetic canalization means that onymous substitutions can, depending on the allele substitutions with a potential for phe- context, lead to the expression of phenotypic notypic change in some genetic backgrounds variation visible to selection (Dworkin et al. are not expressed in other genetic back- 2003). A more detailed review of the concept grounds. Thus, genetic canalization is an epi- of genetic canalization or robustness can be static phenomenon (buffering or canalizing found in De Visser et al. (2003). epistasis) (Nijhout and Paulsen 1997; G P Wagner et al. 1997; Rice 2000; Hansen and environmental canalization and G P Wagner 2001; Wade et al. 2001; De Visser phenotypic plasticity et al. 2003; Burch and Chao 2004); yet, as I Phenotypic plasticity is the sensitivity of the will discuss later, pleiotropy, the effect of a sin- gle gene or allele on two or more traits, is phenotype produced by a single genotype to another potentially important property of the variation in the environment (e.g., Stearns genotype relevant to canalization. 1989a; Roff 1997). In contrast, environmental By decreasing the effects of other loci, canalization is the insensitivity of a phenotype these epistatic effects allow an increase of to variation in the environment; in the broad genetic variation that is not phenotypically sense, environmental canalization refers to expressed. Consequently, genetic canaliza- any kind of robustness against nonheritable perturbations (Waddington 1942, 1957; Roff tion reduces the mutational variance VM, the phenotypic variance caused by the input of 1997; De Visser et al. 2003). Thus, environ- recurrent mutations (G P Wagner et al. mental canalization and phenotypic plasticity 1997). Thus, the inverse of the mutational describe different aspects of the same phe- variance can be taken as a measure of genetic nomenon: the dependency of the phenotype canalization or mutational robustness. Since on the environment (e.g., Stearns 1982; epistasis means that different genotypes react Palmer 1994; Roff 1994, 1997; Ancel and Fon- differently to allele substitutions, that is, more tana 2000; Rice 2000; Rutherford 2000; or less sensitively, its presence implies differ- De Visser et al. 2003; Proulx and Phillips ent degrees of genetic canalization. In the 2004; see Debat and David 2001 for a discus- broadest sense, genetic canalization refers to sion). This can also be seen by noting that the robustness against both heritable genetic phenotypic plasticity of one trait often causes as well as epigenetic perturbations (Sollars et environmental canalization for another trait. al. 2003). The environmental perturbations to which It is also noteworthy that genetic canaliza- a phenotype can be insensitive may either be tion can lead to genetic neutrality (A Wagner external environmental factors (e.g., tem- 1996; Nijhout and Paulsen 1997; Fontana and perature) or internal environmental factors Schuster 1998; Bornholdt and Sneppen 2000; (e.g., developmental noise); both kinds of De Visser et al. 2003): canalization allows the perturbation are nonheritable. In quantita- accumulation of phenotypically silent or neu- tive genetics, the phenotypic variation caused tral mutations, which can alter the genetic by variation in both external and internal, basis of a trait. This may set the stage for the nonheritable environmental factors is sub- 295 sumed in the environmental variance compo- errors in the development of an individual nent, VE. Thus, the simplest measure of envi- (developmental noise). Those errors may ronmental canalization is the inverse of VE. represent responses to random environmen- Environmental canalization, often called tal fluctuations immediately external to that development stability, has been defined by individual or nongenetic internal changes in some authors as phenotypic insensitivity to processes sensitive to chance fluctuations in microenvironmental perturbations (G P Wag- molecule numbers (McAdams and Arkin ner et al. 1997), whereas the concept of phe- 1997; Goss and Peccoud 1998). We do not yet notypic plasticity has usually been applied to know whether the patterns that have been phenotypic sensitivity to macroenvironments characterized as microenvironmental are in (Scheiner 1993). However, the concepts of fact due primarily to variation in the external environmental canalization and phenotypic environment or to nongenetic variation plasticity can be applied to variation in both within the organism, whether the perturba- micro- and macroenvironments (Via 1994; tions are external or internal (see Debat and Schlichting and Pigliucci 1998; Debat and David 2001; Kitcher 2001). Yet, at least part of David 2001). the “developmental noise” appears to be due to chance fluctuations in gene product levels macroenvironmental canalization (McAdams and Arkin 1997; Goss and Pec- Macroenvironments are shared by many coud 1998). For example, Ozbudak et al. individuals (e.g., climate conditions, habitat (2002) quantitatively examined whether and differences, food levels). Macroenvironmen- how molecular fluctuations within single cells tal phenotypic plasticity is phenotypic sensi- (biochemical noise) may explain the varia- tivity to macroenvironments. It is classically tion of gene expression levels between cells described with the concept of a reaction in a genetically identical population (pheno- norm, the set of all phenotypes produced by typic noise) in Bacillus subtilis. The authors a genotype across a set of environments (Wol- found that increased translational efficiency tereck 1909; Schmalhausen 1949; Falk 2001). is the predominant source of increased phe- By analogy, macroenvironmental canalization notypic noise, providing the first direct exper- is phenotypic insensitivity to macroenviron- imental evidence for a biochemical origin of ments. A genotype whose reaction norm is phenotypic noise. less steep than that of another genotype is Microenvironmental phenotypic plasticity macroenvironmentally more canalized, that is phenotypic sensitivity to microenviron- is, less plastic. Similarly, a reaction norm can exhibit a zone of canalization, a region of the ments. It can describe either nongenetic reaction norm that is relatively flat where interindividual or intraindividual phenotypic changes in the environment result in rela- variation. For instance, microenvironmental tively little phenotypic change. Variation in factors can cause phenotypic variation among the slopes of reaction norms implies different clonal individuals of the same genotype degrees of macroenvironmental canalization. within a single macroenvironment. Similarly, At present, we do not know much about the microenvironmental factors may cause ran- patterns of macroenvironmental canaliza- dom deviations from perfect symmetry in tion, for example, how frequently flat reac- individuals of bilaterally symmetric organ- tion norms or zones of canalization occur. If isms, that is, fluctuating asymmetry (e.g., we knew more about this, we could, for Palmer and Strobeck 1986, 1992; Debat et al. instance, address questions about which envi- 2000; Van Dongen and Lens 2000; Hoffmann ronmental ranges favor plasticity and which and Woods 2001; Polak and Starmer 2001). favor environmental canalization. This would By analogy, microenvironmental canalization tell us a lot about selection for optimal reac- is phenotypic insensitivity to microenviron- tion norms (Stearns and Koella 1986). ments. Micro- and macroenvironmental sensitivity microenvironmental canalization and insensitivity are not mutually exclusive: a Microenvironments are specific for a given phenotype can be plastic across macroenvi- individual since there are unpredictable ronments but microenvironmentally cana- 296 lized, or vice versa (e.g., Schlichting and Pig- include, for example, genetic redundancy, liucci 1998; Rutherford 2000). For instance, modularity of development, and system-level the developmental stability of the petal area properties of gene networks and biochemical of Phlox drummondii depends on the particu- pathways. Although canalization may be lar macroenvironment of low nutrients, low caused by several specific mechanisms, it is water, and herbivory (Schlichting and Pig- useful to retain canalization as a unitary con- liucci 1998), and changes in fluctuating asym- cept because it is likely to play a unitary role metry of wing length in D. melanogaster and D. in evolutionary processes. buzzatii depend on temperature (Imasheva et These mechanisms are often reflected as al. 1997). However, there is often no relation- genetic transmission patterns, such as epista- ship between micro- and macroenvironmen- sis, genotype by environment interactions, tal patterns of phenotypic variation (e.g., dominance, and pleiotropy, which I discuss Hoffmann and Woods 2001), suggesting that below. The most general statement we can micro- and macroenvironmental canalization make about the causes of canalization is that are independent. Thus, a single canalizing canalization often is the result of an interac- mechanism will not confer robustness to all tion between the perturbation (e.g., muta- kinds of environmental perturbation; many tion, environment) and the genotype (De- different mechanisms exist by which pheno- Visser et al. 2003). In fact, as recently shown types maintain insensitivity to environmental by Hermisson and G P Wagner (2004), the change (Ancel Meyers and Bull 2002; De- accumulation of cryptic genetic variation, Visser et al. 2003; Milton et al. 2003). one of the hallmarks of canalization, is a generic property of systems showing epistasis genetic canalization of reaction and genotype by environment interactions. norms Genetic canalization may not only suppress epistasis the phenotypic expression of genetic varia- As I have argued above, canalization is an tion of a trait in a given environment but also epistatic phenomenon (Dobzhansky 1971; the expression of genetic variation for phe- Gibson 1996; Fenster et al. 1997; G P Wagner notypic plasticity. That is, different genotypes et al. 1997, 1998; Rutherford and Lindquist can produce the same or a similar phenotype 1998; Hansen and G P Wagner 2001; Hart- across environments, resulting in the same or man et al. 2001; Wade et al. 2001; De Visser a similar level of phenotypic plasticity among et al. 2003; Burch and Chao 2004; Hermisson genotypes (Wijngaarden et al. 2002). Some and G P Wagner 2004). authors have used patterns of variation A well-understood example concerns diaz- among genotypes across environments to inon insecticide resistance in blowflies, Luci- infer genetic canalization (Lewontin 1974b; lia cuprina (e.g., Clarke and McKenzie 1987; Stearns 1994). Whether a given bundle of Davies et al. 1996; overview by Clarke 1997). reaction norms is relatively canalized or not Resistance to diazinon is essentially conferred is decided by comparison between popula- by the Rop-1 locus and is correlated with tions. In a given population, reaction norms increased levels of fluctuating asymmetry in may be called on average more or less genet- several bristle traits. Consequently, resistant ically canalized when compared to another genotypes have reduced fitness. After resis- population of reaction norms. tance had become widespread, however, resis- tant genotypes were no longer selectively dis- The Molecular Mechanisms of advantaged in the absence of the insecticide Canalization and had similar fitness as susceptible wild- Several molecular mechanisms, which are type genotypes. It was found that selection on not mutually exclusive, may contribute to can- the genetic background of resistant geno- alization (Wilkins 1997; Gibson and G P Wag- types had favored an allele whose spread led ner 2000; Hartman et al. 2001; De Visser et to increased levels of environmental canali- al. 2003; Burch and Chao 2004). They zation (Davies et al. 1996). This allele is an 297 allele of the Scalloped wings (Scl) locus, a provides an elegant experimental test for homolog of the Drosophila gene Notch. Thus, genetic canalization by epistasis in E. coli. The both fluctuating asymmetry as well as envi- authors examined genetic interactions ronmental canalization are likely to result between random insertion mutations and from an interaction between Rop-1 and Scl. other mutations by transducing each of Another example comes from Gibson et al. twelve insertion mutations into two genetic (1999) who showed that introgression of backgrounds, one ancestral and the other homeotic Antennapedia mutations, affecting derived. The derived background evolved for the antenna-to-leg transformation in D. 10,000 generations in a laboratory environ- melanogaster, into different wild-type genetic ment. Elena and Lenski (2001) hypothesized backgrounds substantially increases pheno- that the derived background may have typic variation, ranging from complete sup- evolved to be better buffered against harmful pression (i.e., canalization) to complete mutations. In their experiment, there was no antennal leg formation. Furthermore, using compelling evidence for adaptive genetic a composite interval mapping method, the canalization: while some mutations were less authors could demonstrate that one allele harmful in the derived background (canali- (E(Ubx)3L) accounts for three-quarters of the zation), others showed the opposite pattern, phenotypic variance for haltere-to-wing mar- and there was on average no clear trend in gin transformation in Ubx mutant flies. This either direction. Although the authors iden- allele, depending on the genetic background, tified clear cases of genetic canalization strongly enhances the transformation in Ubx depending on the epistatic interaction pres- flies, but has no obvious effects on wild-type ent, their data neither demonstrate adaptive haltere development. canalization nor offer a clear rejection of the In Caenorhabditis elegans, at least ten genes hypothesis that canalization evolves by selec- have been identified that produce mutant tion (Elena and Lenski 2001). vulval phenotypes only in combination with mutations in other genes (Ferguson and functional redundancy Horvitz 1989; Sternberg and Han 1998; Fay and Han 2000). Whereas wild-type C. elegans Functional redundancy refers to a special hermaphrodites have a single vulva, the mul- kind of epistasis among genes with similar tivulva phenotypes of certain mutants have function, causing a mutation at one of the three or more vulva-like structures resulting loci to have little or no phenotypic effect from defects in two functionally redundant (Lerner 1954; Tautz 1992; Nowak et al. 1997; pathways. Evidence suggests that the multi- Wilkins 1997; A Wagner 1999; Rutherford vulval phenotypes result from interactions 2000; Hartman et al. 2001; De Visser et al. between cell-cycle/transcriptional controllers 2003). Thus, functional redundancy in the (Rb transcriptional regulatory complex) and strict sense means that two or several genes regulators of vulval development, such as (or pathways) are performing the same (or a members of the RTK/Ras/Map kinase path- similar) function; this redundancy is caused way (e.g., Fay and Han 2000). This case exem- by the presence of paralogous genes resulting plifies synthetic lethality, an epistatic phe- from gene duplication among members of nomenon (see Hartman et al. 2001): two the same gene family (see Tautz 1992; Wilkins mutations exhibit synthetic lethality if either 1997; McAdams and Arkin 1999; Dover 2000; of the single mutations (with the full function Hartman et al. 2001; De Visser et al. 2003). of the other gene) is viable but mutations in Unfortunately, the distinction between cana- both genes are not. The majority of synthetic lization and redundancy is often not clear in lethal relationships occurs among loci acting the literature. For example, some authors in the same pathway or process (intrinsic buf- also refer to redundancy when functionally fering), but some occur among loci in bio- unrelated genes are involved in the epistatic chemically distinct pathways or processes interaction (Wilkins 1997; De Visser et al. (extrinsic buffering; Hartman et al. 2001). 2003). Although interactions among unre- A recent study by Elena and Lenski (2001) lated genes may be important for phenotypic 298 robustness (e.g., De Visser et al. 2003), func- argued that pleiotropy thereby contributes to tional redundancy as operationally defined canalization: some traits may become so by Wilkins (1997) is not different from the embedded in development that they lose general definition of genetic canalization by their evolvability (Stearns 1994; Hansen and epistasis. Thus, the term functional redun- Houle 2004). While pleiotropy may impose a dancy should be reserved for robustness due constraint for the evolvability of the focal to the presence of paralogs; also see the above trait, it may also increase the mutational tar- discussion of synthetic lethality. get size and thus improve the evolvability of Functional redundancy seems to be wide- the trait (Hansen and Houle 2004). While spread. For instance, in budding yeast, S. cer- pleiotropy is a common mode of gene action evisiae, the inactivation of single genes has lit- and often emerges as a natural property of tle phenotypic effect whereas the knockout of networks of transcriptional regulation (Gib- all paralogs produces a strong mutant effect son 1996), the relevance of pleiotropy for (Kataoka et al. 1984). In the case of paralogs canalization remains unclear. of the yeast ras genes, the CLN (cyclin) genes, and the two genes for 3-hydroxy-3-methyl- dominance glutaryl-coenzyme A, only multiple knockouts Dominance can be viewed as another man- have an effect, suggesting that in all three ifestation of canalization (e.g., Proulx and cases paralogs serve as functional backups Phillips 2004 and references therein). It was (Basson et al. 1986; Hadwiger et al. 1989). noticed early on that heterozygotes for However, a recent study on the genome of mutant and wild-type alleles almost invariably S. cerevisiae shows that redundancy may not show the wild-type rather than the mutant be important for canalization (A Wagner phenotype; only dominant allele substitu- 2000b). If gene duplications are predomi- tions are expressed. Thus, dominance refers nantly responsible for robustness, one would to the concealment of the presence of one expect a correlation between the similarity of allele by the strong phenotypic effects of two duplicated genes, both in terms of another allele (Sagaret 1826; Mendel 1866). sequence and temporal expression patterns Dominance may therefore contribute to can- and the effects of mutations in one of these alization by masking the effects of deleterious genes. However, A Wagner (2000b) did not recessive mutations. As has been argued by find such an association, suggesting that Wright (1934), recessiveness and dominance redundancy due to gene duplication contrib- are likely to be intrinsic properties of metab- utes little to mutational robustness. Yet, Gu et olism. Probably most deleterious alleles cause al. (2003) demonstrate that null mutations in a reduction in enzyme activity. However, if the duplicate genes have a 20% higher probabil- wild-type allele is more active than required, ity of exhibiting only weakly deleterious fit- the rate of reaction is substrate rather than ness effects as compared to knockouts in sin- enzyme limited: deleterious mutations appear gle-copy genes. then as recessive or nearly recessive. For instance, as shown by metabolic control the- pleiotropy ory, the many gene-controlled steps in a path- Pleiotropy, the multiple effects of a single way imply that a 50% reduction in the activity gene or allele on two or more traits, is of any one step is often likely to have only another potentially important genotypic minimal effects on the amount of the final property bearing on canalization. Since traits product (Wright 1934; Kacser and Burns are often linked by pleiotropic gene action, 1981; Charlesworth 1998). pleiotropy may compromise the indepen- dence of traits so that it becomes impossible genotype by environment to change a trait without disturbing other interactions traits at the same time (Hansen and Houle Genotype by environment interactions are 2004). Such pleiotropy, therefore, may limit another mechanism for canalization, concep- the evolvability of the focal trait, and it can be tually similar to that of epistasis (e.g., Brodie 299

2000; Hermisson and G P Wagner 2004). The necessarily be identical. For example, canaliz- presence of these interactions implies that ing systems such as Hsp90 probably do not different genotypes differ in their degree of generally control both genetic and microen- plasticity/environmental canalization. Many vironmental canalization (Milton et al. 2003). experiments have established that pheno- Yet, there are several studies showing that typic plasticity—and thus, as I argue herein, genetic and environmental canalization are environmental canalization—is a heritable, correlated and may share the same develop- evolvable trait (e.g., Waddington 1960; Schei- mental basis (A Wagner and Stadler 1999; ner and Lyman 1991; see Scheiner 1993 and Ancel and Fontana 2000; Burch and Chao Roff 1997 for reviews). Recent studies on the 2004). genetics of phenotypic plasticity suggest that environment-dependent gene regulation plays modularity a prevailing role in determining a genotype’s A modular unit of the phenotype is a com- sensitivity to variation in the environment plex of traits, which collectively serve a pri- (Pigliucci 1996; Schlichting and Pigliucci mary functional role. Modules are tightly 1998). For instance, environment-dependent integrated by strong pleiotropic effects and alterations of DNA transcription and RNA are more or less independent from other translation rates are known from the re- such units (G P Wagner 1996; G P Wagner sponse of flowering plants to light, induced and Altenberg 1996). Modules can be seen at heat-shock proteins in plants, responses of different levels, from base pairs or amino cyanobacteria to sulfur limitation, and tem- acids to cellular structures or developmental perature-induced sex determination in liz- processes (Hartwell et al. 1999; Dover 2000). ards (Pigliucci 1996). Similarly, Wu (1998) has demonstrated that a few regulatory loci, For a recent discussion of modularity see also differing from QTLs (quantitative trait loci) Gass and Bolker (2003). for trait values within environments, are Modularity of development may contribute responsible for the environment-dependent to canalization (Stearns 1989b; Maynard control over structural gene expression in Smith 1998; Hartwell et al. 1999; Stern 2000). Populus trees. This also suggests that epistasis Changes in the organism in one of its parts and genotype by environment interactions should not compromise other achievements: are not mutually exclusive: the environment independent functions should be coded inde- interacts with genes that determine the mag- pendently so that the change of one function nitude of the response to the environment, does not interfere with other optimized func- which in turn interact with genes that affect tions (G P Wagner and Altenberg 1996). the expression of the trait (epistasis model of Modularity can be a way to achieve this inde- phenotypic plasticity; e.g., Scheiner and pendence of functions. The significance of Lyman 1991). modularity for canalization is that perturbing As shown by several authors, the genetic one module does not necessarily perturb the basis of micro- and macroenvironmental plas- development of the whole organism: the ticity/canalization is likely to be different embedding of particular functions into dis- (Wu 1998; Debat and David 2001). For tinct modules allows for phenotypic change instance, Rutherford and Lindquist (1998) by altering connections among the modules have shown that Hsp90 buffers both against while the core function of a given module genetic and macroenvironmental perturba- remains unchanged (G P Wagner and Alten- tions (e.g., temperature; see also Queitsch et berg 1996; Hartwell 1999; Stern 2000; Schank al. 2002). However, the buffering ability of the and Wimsatt 2001). First, if deleterious muta- hsp83 locus does not affect microenvironmen- tions are highly pleiotropic (but see Stern tal variation, as measured by the stochastic 2000), then these mutations are likely to have effects producing fluctuating asymmetry a negative effect on many traits. If gene net- (Rutherford 2000; Milton et al. 2003). This works have a modular structure, however, implies that the mechanisms for the buffering then genetic change in one of the modules of different kinds of perturbation may not does not necessarily influence the others 300

(Bonner 1988). Thus, by restricting pleiot- chemical pathways can lead to robustness ropy, modularity allows some modules to con- against perturbations. Such networks and tinue to function when others change pathways exhibit emergent properties such as (Schank and Wimsatt 2001). Second, random integration of signals across time scales, gen- mutations of a given phenotypic effect are eration of distinct outputs that depend on likely to be more deleterious in complex input strength and duration, and feedback organisms consisting of many traits as com- loops (e.g., Bhalla and Iyengar 1999; Mc- pared to simpler organisms with less traits Adams and Arkin 1999). The dynamic stabil- (Fisher 1930; Orr 2000). Fisher (1930) sug- ity of these networks often results from con- gested that mutations of small phenotypic nections among the components of the effect are more likely to be favorable than system, such as functional redundancy and mutations with large effects. In a topological feedback regulation. model, he showed that the probability of There are several good examples for the approaching (or deviating from) the fitness robustness of gene networks and biochemical optimum is higher (or smaller) if a mutation pathways, both from theoretical and empiri- has a small phenotypic effect than if it has a cal work (De Visser et al. 2003). For instance, large phenotypic effect. In the latter case, a enzymes of intermediary metabolism often mutation is more likely to go beyond the opti- show saturation kinetics, that is, flux is a con- mum or to deviate from it more strongly than cave or (sigmoid) function of enzyme activity if the mutation has only a small phenotypic (e.g., of the Michaelis-Menten form). If selec- effect. For the same intuitive reason, random tion favors increased enzyme activity to max- mutations of a given phenotypic effect are imize flux, a point of diminishing returns may more likely to disrupt a complex than a sim- be reached where any change in flux results ple organism (Orr 2000). It would therefore in a disproportionately small change in fit- be evolutionarily advantageous to reduce the ness. Consequently, mutations that affect number of independent traits by bundling activity will often result in only minor changes them into modules (Orr 2000). in fitness (Hartl et al. 1985). Some of the best examples of modularity As shown by Gibson (1996), threshold- concern promoter regions of genes (Dover dependent transcriptional regulation, the 2000; Stern 2000). For instance, recent stud- cooperative binding of several transcriptional ies of gene regulation, particularly in Drosoph- regulators to a gene promoter, can also lead ila, have demonstrated that the cis-regulatory to canalization. Minor variations in individual sequences of many genes are organized into parameters can be balanced by variation in independent modules (e.g., Arnone and other parameters, which allows for the same Davidson 1997; Stern 2000). These modules threshold to be set by different genotypes are constructed from multiple binding sites (Gibson 1996). In a similar vein, A Wagner for individual transcription factors, and the (1996) modeled sets of transcriptional regu- same module can have several binding sites lators that form a gene expression network. for the same transcription factor. Thus, the He demonstrated that the network’s stability binding sites are redundant both in number against deleterious mutations increases and function. While there are many examples under stabilizing selection so that the fraction of modularity at various levels of biological of mutations causing changes in gene expres- organization (Hartwell et al. 1999), it is not sion patterns is substantially reduced, regard- yet clear how relevant modules are for cana- less of the network structure and the mode of lization. This situation may change with the reproduction. advent of new tools for studying modularity Barkai and Leibler (1997) showed that sim- (Magwene 2001). ple biochemical networks of bacterial che- motaxis are robust against perturbations due emergent properties of gene to the network’s connectivity. Similarly, the- networks and biochemical pathways ory developed by Frank (1999) shows that There is increasing evidence that system- the more highly connected a Boolean regu- level properties of gene networks and bio- latory network, the smaller the effect of muta- 301 tional perturbation. Lenski et al. (1999) con- type, and this effect does not seem to require structed digital organisms and showed that that the mutations are conditional on the organisms with a more complex genetic environment. architecture are more robust than simple ones with respect to the average effects of The Evolution of Canalization mutations. adaptive and nonadaptive origins of As found by Little et al. (1999), the behav- canalization ior of the phage Lambda gene regulatory cir- The evolution of canalization is difficult to cuit exhibits robustness of transcriptional address because there are many alternatives regulation. The phage can switch between that are difficult to distinguish empirically. In two states: lysogeny (integration of the viral principle, canalization could result from genome into the host’s genome without adaptive evolution (e.g., Schmalhausen 1949; destroying the host cell) and lysis (viral rep- Waddington 1957; Layzer 1980; Gavrilets and lication and destruction of the host cell). This switch involves a promoter lying between two Hastings 1994; G P Wagner et al. 1997; Rice autoregulatory genes, which have different 1998; Kawecki 2000) or as an emergent prop- affinities for different modular binding sites erty of complex gene and trait networks (e.g., in the promoter. The authors experimentally Kauffman 1993; Barkai and Leibler 1997). altered the normal binding pattern such that Thus, following De Visser et al. (2003), we the binding sites for the two regulatory pro- may distinguish three origins of canalization: teins had identical sequences. Despite these (1) adaptive; (2) intrinsic; and (3) congruent. changes, the mutations in the two regulatory An adaptive origin implies that canalization sites showed the same qualitative in vivo pat- evolves because the insensitivity to perturba- tern of gene expression as the wild-type. Sim- tions increases fitness; an intrinsic origin ilarly, using a synthetic gene circuit in E. coli, implies that canalization arises as a nonadap- Becskei and Serrano (2000) demonstrated tive correlated byproduct of functional trait that autoregulatory negative feedback loops architecture; a congruent origin implies that can dramatically reduce variation in gene genetic canalization evolves as a correlated expression. Wilke et al. (2001) demonstrated response to selection for environmental can- that populations of digital organisms that alization. have evolved under high mutation rates tend As I have outlined above, there may not be to be more robust to mutation as compared a single specific mechanism for canalization. to populations that have evolved in identical On the other hand, if specific single mecha- environments at low mutation rates. At high nisms do exist, the evolution of canalization mutation rates, mutationally more robust will be much simpler to address—both theo- populations prevailed in competition with retically and empirically—than if there are less robust populations. multiple mechanisms for canalization, some Interestingly, Siegal and Bergman (2002) of them having evolved for reasons other than showed that developmental processes, mod- canalization. Thus, there are several impor- eled as interaction networks of transcrip- tant questions to which we do not yet have tional regulators, can produce canalization, clear answers. Here I concentrate on the case even without selection towards an optimum. in which canalization evolves by natural selec- Finally, using both computer simulations of tion, as assumed by Waddington and in most gene networks and an analysis of gene-knock- theoretical treatments of the evolution of can- out data from S. cerevisiae, Bergman and Sie- alization (e.g., Gavrilets and Hastings 1994; gel (2003) presented evidence that suggests G P Wagner et al. 1997; Kawecki 2000). that most genes release phenotypic variation when their function is compromised (but see, models for the evolution of for example, Thatcher et al. 1998). Further- canalization more, the availability of such loss-of-function It is thought that canalization holds phe- mutations can—at least theoretically—accel- notypes close to their fitness optimum. Con- erate adaptation to a new optimum pheno- sequently, it is expected that stabilizing selec- 302 tion favors the evolution of both genetic and static modifier loci that reduce the pheno- environmental canalization (e.g., Schmalhau- typic expression of the structural loci. Simple sen 1949; Waddington 1957; Rendel 1967; models therefore rely on the somewhat Layzer 1980; Stearns and Kawecki 1994; unrealistic assumption that only one or a few A Wagner 1996; G P Wagner et al. 1997; Rice genes or alleles have strong epistatic effects 1998). whereas most genes or alleles have only addi- First, and most generally, canalization can tive genetic effects (G P Wagner et al. 1997). in principle evolve whenever there is a non- Consequently, more general theoretical mod- linear relationship between traits or between els of canalization have been developed in loci (e.g., A Wagner 1996; Rice 1998, 2000; which loci both have direct effects on the trait see the phenotypic landscape model above). and modify the phenotypic expression of other Second, traits are often connected to fitness loci (G P Wagner et al. 1997; Rice 1998). nonlinearily. If the relationship between fit- ness and a trait is convex, then reducing the selection for genetic canalization phenotypic variance of the trait will increase Stabilizing selection reduces genetic vari- fitness. If the relationship is concave, how- ance by driving the segregating loci underly- ever, then increasing the variance will ing the trait to fixation. However, even under increase fitness (e.g., Stearns 2000). This stabilizing selection, deviations from the fit- principle, discovered by the Swiss mathema- ness optimum may be frequent because, if tician Daniel Bernoulli in 1738 (Stearns epistasis and pleiotropy are common, selec- 2000), is important in economics and has tion cannot eliminate all genetic variance for been applied to evolutionary biology by Ste- the trait. Genetic variance that causes individ- phens and Krebs (1986) and Real and Ellner (1992). As shown by theoretical work, cana- uals in the population to deviate from their lization can evolve when fitness functions are fitness optimum is thus thought to be the convex (i.e., stabilizing selection; Lande driving force for the evolution of genetic can- 1980; Layzer 1980; Rice 1998; Kawecki 2000). alization: the more genetic variance, the Third, convexity of the fitness function is not stronger the selection for canalization (G P necessarily synonymous with selection for Wagner et al. 1997). All factors contributing canalization: both randomly and periodically to genetic variance—large population size, fluctuating linear as well as Gaussian selection high mutation rate, and a large number of regimes can select for genetic canalization loci—favor the evolution of genetic canaliza- (Kawecki 2000). Fourth, directional selection tion. Under mutation-selection balance, selec- is thought to select against canalization (Tho- tion cannot completely eliminate deleterious day 1958; Layzer 1980; G P Wagner et al. alleles because they occur continuously and 1997): directional selection can favor modi- recurrently, and canalization may therefore fiers that increase the phenotypic expression be selected. However, the conditions under of genetic variation. Fifth, new work shows which genetic canalization can evolve under that the maximum strength of selection for mutation-selection are rather restrictive (G P environmental or genetic canalization is gen- Wagner et al. 1997). The strength of stabiliz- erally a function of the fitness load imposed ing selection may have counteracting effects. by the environmental or genetic perturbation On the one hand, increasing the strength of (Hermisson et al. 2002; Proulx and Phillips stabilizing selection favors selection for genetic 2004). canalization. On the other hand, as the The evolution of canalization has usually strength of selection increases more and been modeled assuming canalizing modifi- more (i.e., selection becomes purifying), sta- ers. Canalizing modifiers are alleles that bilizing selection eliminates genetic variance reduce the phenotypic effect of one or several to a point where canalization cannot evolve other alleles (G P Wagner et al. 1997). Models anymore (G P Wagner et al. 1997). Further- of canalization often assume that a trait is more, if one assumes that alleles with canaliz- affected by two kinds of loci: structural loci ing effects have direct (pleiotropic) effects that have a direct effect on the trait and epi- on the trait, any direct effect will be dele- 303 terious and selected against whereas canaliz- natural populations (e.g., Turelli 1988) and ing effects will be selected for. Under strong may be a driving force for the evolution of stabilizing selection, genetic canalization may canalization. Thus, mechanisms for the main- not evolve because selection for canalizing tenance of genetic variation other than muta- effects cannot override selection against tion-selection balance may facilitate selection direct effects (G P Wagner et al. 1997). Thus, for genetic canalization. Third, in the model stabilizing selection may favor genetic cana- by G P Wagner et al. (1997), environmental lization only at low to moderate strengths of canalization (see below) evolves rather easily selection (G P Wagner et al. 1997). Interest- as compared to genetic canalization (also see ingly, recent work by Hermisson et al. (2003) Proulx and Phillips 2004). There are several shows that stabilizing selection does not typi- lines of evidence suggesting that patterns of cally lead to genetic canalization of the trait. genetic and environmental canalization may In fact, the conditions for the evolution of be correlated (Ancel and Fontana 2000; genetic canalization of traits are even more Burch and Chao 2004): traits that are under restrictive than the work by G P Wagner et al. stronger stabilizing selection seem to be both (1997) suggests. Instead, canalization may environmentally and genetically more cana- evolve at the gene level; such genic canaliza- lized than traits under weaker selection tion refers to the buffering of mutational (Stearns and Kawecki 1994; Stearns et al. effects of certain subunits or groups of genes 1995). If this is the case, then the evolution (Hermisson et al. 2003). Only genes with of genetic canalization could be a correlated high mutation rates will primarily be buf- response to the evolution of environmental fered, however, and the genic buffering may canalization (G P Wagner et al. 1997). For often occur at the expense of decanalization instance, A Wagner and Stadler (1999) dem- of other genes (Hermisson et al. 2003). Also onstrated that the secondary structure of a see De Visser et al. (2003) for a discussion of viral RNA genome is more stable than a ran- the effects of mutation rates, modes of repro- domly chosen sequence that folds into the duction, strength of selection, and popula- same structure, suggesting that the virus has tion size on the evolution of genetic canali- evolved to a state of increased mutational sta- zation. bility. In this case, the mutational stability Three points are particularly important (genetic canalization) may be a side effect of here. First, selection for genetic canalization selection for increased thermodynamic sta- is usually weak, that is, selection coefficients bility (environmental canalization). Such a are small (G P Wagner et al. 1997; Kawecki mechanism has also been shown to be at work 2000; Proulx and Phillips 2004). However, as in a model of the evolutionary dynamics of shown by Proulx and Phillips (2004), the RNA secondary structure (Ancel and Fon- strength of selection for genetic canalization tana 2000). may depend on the size of the genetic net- Theoreticians have also studied the evolu- work: selection for canalization is typically tion of particular mechanisms of genetic can- stronger in larger genetic networks because alization, for example, genetic redundancy. large networks exhibit a higher mutational One problem with the theory of the evolution load than small networks. Second, different of genetic redundancy is that genes encoding assumptions about the maintenance of functional proteins must be under selection genetic variance may considerably change the pressure: if a gene is truly redundant, then it theoretical predictions made by G P Wagner is not protected against the accumulation of et al. (1997). Forces other than mutation- deleterious mutations (Nowak et al. 1997). selection balance can account for the main- Consequently, redundancy may not be evo- tenance of genetic polymorphism (e.g., Roff lutionarily stable. However, as shown by 1997), such as environmental heterogeneity Nowak et al. (1997), evolutionarily stable (Felsenstein 1976; Hedrick 1986), pleiotropy redundancy can evolve by selection in several (Rose 1982), epistasis (Gimelfarb 1989), and simple models. The models assume that heterosis. These processes can maintain rela- genes are initially neither redundant nor plei- tively large amounts of genetic variance in otropic: each gene performs a single unique 304 function. Then, spontaneously arising muta- selection for canalization against microenvi- tions that lead to a functional overlap can be ronmental perturbations. Their analyses sug- favored by selection. These mutations can gest that stabilizing selection will favor the become fixed, and redundancy can become evolution of canalization against develop- evolutionarily stable if random mutations mental noise. Under stabilizing selection, occurring in the genome are more likely to reduced microenvironmental variation is destroy all functions of a gene rather than to always associated with increased mean fitness: destroy just one function while leaving others the stronger the stabilizing selection and the unaffected. The final genetic configurations larger the reduction of microenvironmental often consist of complex genetic networks, variation through the canalizing effects of a exhibiting both redundancy and pleiotropy: modifier, the stronger the selection for can- each function is performed by several genes, alization (G P Wagner et al. 1997). Microen- and each gene performs several functions vironmental variation is never reduced to (Nowak et al. 1997). Similarly, A Wagner zero, however, suggesting that there is a limit (1999) has found that selection can maintain to microenvironmental canalization (G P as well as increase redundancy among genes Wagner et al. 1997). This may be due to can- in a population provided that mutations gen- alizing modifiers having negative direct (plei- erate sufficient variation in redundancy and otropic) effects on the trait (G P Wagner et that populations are large. Selection acts al. 1997). Furthermore, empirical evidence through the decreased number of offspring suggests that it is possible to select for with deleterious mutations that individuals increased microenvironmental canalization. with redundant genes generate (A Wagner For example, it is possible to select for 1999). As shown by A Wagner (2000a), how- decreased fluctuating asymmetry in scutellar ever, the average effects of deleterious muta- bristle number in D. melanogaster (see Schar- loo 1988, 1991). tions on fitness do not greatly influence the Several examples suggest that macroenvi- evolution of functional overlap and vice versa: ronmental canalization (i.e., reduced plastic- the extent of functional overlap maintained ity) can be selected. For instance, Wadding- in a population is independent both of the ton (1960) applied a family selection regime mutation rate and the average effects of muta- to select on the facet number of the Bar tions. Thus, overlapping gene functions may mutant in D. melanogaster, in which the off- not be maintained because they confer a spring of pair-matings were divided between higher mean fitness by reducing the impact two treatments, one at 18ЊC and one at 25ЊC. of deleterious mutations. This implies that The facet number is greater at lower than at the maintenance of functional overlap may higher temperatures. Selection was made on not be an adaptive case of canalization. the basis of differences among family means. Waddington’s experiment yielded fast pro- selection for environmental gress: in the unselected Bar stock, the facet canalization number at 18ЊC was initially three times Generally, since environmental canaliza- higher than at 25ЊC. However, after six gen- tion confers insensitivity to environmental erations of selection, the difference had been change, it will only be selected for if the opti- reduced to about 10%. At this point, the phe- mum phenotype is the same across environ- notypes at both temperatures were interme- ments. In contrast, if the optimal phenotype diate between those seen at the start of the depends critically on the particular environ- experiment. ment, phenotypic plasticity should be favored Similarly, Scheiner and Lyman (1991) by selection (De Visser et al. 2003). In con- selected on phenotypic plasticity of thorax trast to genetic perturbations, environmental size in response to temperature in D. melano- perturbations often cause strong selection for gaster. In the lines selected for decreased plas- canalization (e.g., Proulx and Phillips 2004). ticity, a selection limit of zero plasticity was Models by Gavrilets and Hastings (1994) reached, but additive genetic variance for and G P Wagner et al. (1997) have addressed plasticity still existed. The authors succeeded 305

in decreasing plasticity so that flies raised at frequencies at locus i and ai is the effect of 19ЊC were on average the same size as flies the two alleles segregating at locus i. There raised at 25ЊC. This pattern was irreversible are two ways to decrease genetic variance: (i) since it was not possible to select for increased either by driving pi or qi to zero or to one, plasticity again. Although a selection limit was thus eliminating genetic variance; or (ii) by reached, there were still substantial amounts decreasing the effects of the segregating of additive genetic variance as measured by alleles, ai. Canalizing selection does the latter; narrow-sense heritabilities based on sib cor- it reduces the magnitude of allelic effects relations after 16 generations of selection. (e.g., Stearns and Kawecki 1994; G P Wagner The authors explain this apparent contradic- et al. 1997). tion by suggesting that the plasticity of the Selection that does not alter the mean of a trait is determined by loci separate from those trait but reduces its variance is commonly determining the mean of the trait. called stabilizing selection. Subsequently, it While Waddington (1960) and Scheiner has been suggested that canalizing selection and Lyman (1991) managed to select for refers to a special kind of stabilizing selection increased macroenvironmental canalization (Schmalhausen 1949; Waddington 1957; (i.e., less steep reaction norms), a recent Stearns and Kawecki 1994; G P Wagner et al. selection experiment failed to change the 1997). However, canalization can evolve not shape and the slope of the reaction norms for only by stabilizing selection but also by fluc- eyespot size in the butterfly Bicyclus anynana tuating linear or Gaussian selection (Kawecki (Wijngaarden et al. 2002). The authors only 2000). Selection thus qualifies as canalizing managed to change the elevation of a bundle selection, no matter what the fitness function of reaction norms. This may suggest that the looks like, if it favors the suppression of the bundle is canalized so as to exhibit the same phenotypic expression of genetic variation. overall shape and slope by some unknown In principle, selection can therefore reduce mechanism, for instance by hormonal regu- phenotypic variation by reducing genetic var- lation. Alternatively, the shape and slope of iance, its phenotypic expression, or both. In the reaction norms may not be controlled by reality, it is most likely that the two types of the same loci as the reaction norm’s eleva- selection are not completely independent tion. Thus, the result may simply be and that they occur simultaneously (Stearns explained by a lack of standing genetic vari- and Kawecki 1994). For most episodes of ation for the shape and slope of the reaction selection in natural or experimental popula- norms. tions, we do not know whether phenotypic variation decreased because alleles were fixed canalizing selection or eliminated or whether the phenotypic The classic view of selection assumes that effects of segregating alleles were masked by selection eliminates additive genetic variance selection for canalizing mechanisms. by reducing the number of segregating alleles, that is, by driving their frequencies to The Evolutionary Significance of zero or one. In contrast, canalizing selection Canalization (Waddington 1957) selects for genetic mech- canalization and fitness sensitivity anisms that suppress the phenotypic expres- sion of genetic variance. It thereby retains the Any developmental mechanism that cana- genetic potential for phenotypic change. The lizes a trait by keeping it close to its fitness difference between the two forms of selection optimum should be favored by selection can be illustrated by considering the additive (Rendel 1967). Consequently, traits that are genetic variance of a trait determined by n under strong stabilizing selection should be loci in linkage equilibrium: more canalized than traits under weak selec- n tion (but see G P Wagner et al. 1997). This is מ 2 ס VA 2ap(1͚ iip) i exactly what Stearns and Kawecki (1994) and Stearns et al. (1995) found in experiments on 1סi מ ס where pi and qi 1 pi represent the allele D. melanogaster. The lines they used differed 306 in the position of a P-element insert or in duced in reliable, functional trait combina- genetic background; flies within lines were tions despite genetic recombination. Genetic genetically nearly identical. Genetic canali- canalization may be one way of stabilizing zation was measured as the inverse of the the development of sexual organisms facing coefficient of variation among lines; microen- the problem of recombination (e.g., Stearns vironmental canalization was measured as the 1994; G P Wagner and Altenberg 1996). We inverse of the coefficient of variation among do not know enough yet to check the validity replicates within lines. For both genetic and of this hypothesis. However, theory suggests environmental canalization, the canalization that there are similarities between the selec- of traits increased with their impact on fitness tion regimes that favor genetic canalization and did not depend on genetic differences and the conditions that favor low recombi- among lines. The genetic and environmental nation rates (see discussion in Kawecki 2000). coefficients of variation were positively cor- Both genetic canalization (e.g., G P Wagner related. This suggests that (i) there is differ- et al. 1997) and low recombination rates ential genetic and environmental canaliza- (e.g., Charlesworth 1993) can be favored by tion of fitness components and (ii) the same stabilizing selection. developmental mechanisms may buffer the phenotype against genetic and microenviron- canalization and constraint mental perturbation. However, these results Canalization may constrain phenotypic and the underlying assumptions were ques- evolution, that is, prevent (or slow down) the tioned by Houle (1998), who reanalyzed data attainment of fitness optima by an evolving on genetic variances in D. melanogaster from population (Maynard Smith et al. 1985). the literature. He found evidence that the Since the response to selection is propor- mutational variance of traits was likely to be tional to the amount of additive genetic vari- influenced by the mutational target size (i.e., ance expressed in the phenotype, canaliza- traits with a complex functional architecture tion can potentially reduce a trait’s capacity are larger targets for mutation) and by the for evolution by reducing its level of timing of trait expression (i.e., traits expressed variation. Although mutations con- expressed later in life inherit variation from tinually occur in the genes affecting all traits, earlier stages). Canalization did not explain some traits are invariant within clades. differences in the genetic variances of traits Stearns (1994) suggested that canalization (Houle 1998), however. Thus, Houle (1998) may be responsible for this invariance by concluded that canalization is unlikely to play decoupling variation in traits from variation a dominant role in determining variation in in genes. Under this view, canalization may mutational and standing genetic variances lead to macroevolutionary patterns (e.g., (see also Hermisson et al. 2003). traits fixed within lineages) that suggest phy- logenetic and developmental constraints. canalization and recombinational Thus, short-term canalization may temporar- load ily constrain phenotypic evolution, and long- Canalization may reduce the problem of term canalization may lead to character stasis recombinational load. Whereas in asexual (Charlesworth et al. 1982; Stearns 1982; May- organisms mutations enter the population nard Smith et al. 1985; Scharloo 1987; Brad- sequentially, progeny of sexual organisms shaw 1991; Stearns 1994). face the problem of genetic recombination (e.g., Barton and Charlesworth 1998). Recom- canalization and evolvability bination can induce a genetic load if the trait Canalization, followed by decanalization, is under stabilizing selection and the mean may increase evolvability, the ability of a phenotype has approached the fitness opti- population to respond to selection. The mum. Under recombination, newly produced evolvability of a trait is proportional to the phenotypes will often deviate from the opti- amount of heritable genetic variance, a result mum. However, phenotypes have to be pro- following from Fisher’s fundamental theorem 307 of selection (Houle 1992). Canalization can There are several experimental examples of increase evolvability because it allows the genetic assimilation (Scharloo 1991; Ruther- accumulation of genetic variation not “seen” ford and Lindquist 1998; True et al. 2004). by selection. The cryptic variation allowed to For instance, Waddington (1953) has studied build up by canalization can be released genetic assimilation of the cross-veinless phe- under decanalizing conditions, and selection notype (cvl), a defect in wing venation pat- on the trait can change the frequency of the tern in D. melanogaster. Waddington started a previously cryptic alleles (Levin 1970; Agur selection experiment with a wild-type base and Kerszberg 1987). Experimental examples population in which no cvl flies appeared from D. melanogaster (Rutherford and Lind- when raised at 25ЊC. However, when pupae quist 1998; see above) and budding yeast, S. were given a heat shock of 4 hours at 40ЊCat cerevisiae, show that canalization can increase 17 to 23 hours after puparium formation, evolvability. In budding yeast, a modified pro- some proportion of the flies showed the cvl -is an epigenetic mod- phenotype. Waddington created two selec ,[םtein, the prion [PSI ifier of the fidelity of translation termination. tion lines by treating pupae in every genera- It provides the means to uncover hidden tion with heat shock and by selecting flies as genetic variation for growth rates and to pro- parents for the next generation which either duce new heritable growth rate phenotypes showed the cvl phenotype after heat shock across diverse environments (True and Lind- (“upward” selection line) or not (“downward” quist 2000; True et al. 2004). However, it is selection line). The frequency of the cvl phe- questionable whether these systems have notype increased in the upward treatment adaptively evolved to a state of increased and decreased in the downward treatment. evolvability (Partridge and Barton 2000). After 13 generations, the difference in the percentage of the cvl phenotype between the canalization and genetic assimilation two treatments had become about 60%. Fur- thermore, Waddington examined in every A particular way in which canalization may generation a large number of flies from increase evolvability is genetic assimilation. untreated pupae of the upward selection Genetic assimilation is defined as the process regime. Among these untreated control flies, by which a phenotype originally produced no cvl individuals were found until genera- only in response to some environmental tion 14. At generation 16 the frequency of cvl change becomes stably expressed indepen- had raised to 1–2%, and Waddington set up dently of the evoking environmental effect paired matings between such cvl individuals. (Waddington 1953; Pigliucci and Murren From these flies, he created four selection 2003). It requires that: (i) a population is lines and obtained a high proportion of cvl originally genetically polymorphic for a par- phenotypes at 25ЊC without any heat shock. ticular polygenic threshold trait, but that the Thus, Waddington had “genetically assimi- alleles for this phenotype are not expressed lated” an environmentally induced pheno- because of canalization; (ii) an environmen- type. tal stimulus or allele substitution decanalizes Another example comes from Gibson and the expression of these alleles; and (iii) selec- Hogness (1996), who repeated an experi- tion alters the frequency of these alleles to the ment of Waddington’s (1956) by selecting for point where the phenotype appears in the differential sensitivity to the induction of absence of the environmental stimulus or bithorax phenocopies by ether vapor (see allele substitution (Stern 1958; Waddington also Ho et al. 1983a,b,c). Bithorax phenocop- 1961; Scharloo 1991; Hall 1999; Gilbert ies resemble bithorax mutants, which have two 2000). pairs of wings. Using molecular genetics, they Genetic assimilation may lead to rapid phe- demonstrated that the differential pheno- notypic divergence and may—at least theo- typic expression of the ether-induced retically—facilitate adaptation to novel envi- bithorax phenotype can be mostly attributed ronments (Levin 1970; Eshel and Matessi to genetic polymorphism at the Ubx (Ultrabi- 1998) and speciation (Pa´l and Miklo´s 1999). thorax) locus. Loss of expression of Ubx pro- 308 tein in the third thoracic imaginal discs cor- age effect of the mutations—a consequence related with increased sensitivity to ether. of the Haldane-Muller principle (G P Wagner This shows that there is heritable genetic vari- et al. 1997). Furthermore, the frequent obser- ation for the propensity to exhibit ether- vation that mutants or environmentally induced bithorax phenotypes, which can be induced phenotypes are more variable than selected upon. Selection will then lead even- the wild-type does not imply that the wild-type tually to the loss of Ubx expression, resulting is adapted to be more canalized (Hansen and in a high proportion of bithorax phenotypes, G P Wagner 2001). Similarly, whether canali- which are stably expressed in the absence of zation has a constraining effect, by reducing ether. the response to selection, will depend on the Although Waddington (1953) suggested stability of the canalizing mechanisms and on that changes in canalization may account for the frequency and magnitude of decanalizing assimiliation, he did not propose an explicit perturbations. For instance, directional selec- mechanistic model. However, most assimila- tion on a trait can favor decanalization (e.g., tion experiments are consistent with thresh- Layzer 1980). However, theory suggests that old models of trait expression (Scharloo even strong directional selection is likely to 1991; Hall 1999); these models assume that be rather slow in decanalizing, and short the trait under consideration is a polygenic periods of directional selection are unlikely threshold trait. These assumptions have been to decanalize a well-canalized trait (G P Wag- confirmed in several experiments (Ruther- ner et al. 1997). The temporary breakdown ford and Lindquist 1998). In these models, a of canalizing mechanisms may result in alter- decanalizing perturbation can move the nating phases of canalization and decanali- population outside of a zone of canalization zation, leading to punctuated phenotypic in which the variation is masked. Conse- dynamics (e.g., Agur and Kerszberg 1987). quently, previously hidden genetic variation for the trait becomes exposed, making selec- Detecting and Measuring tion more effective. Eventually, selection will Canalization shift the trait into a new zone of canalization standard methods where the new phenotype becomes stably expressed in the absence of the enabling There are essentially two approaches to stimulus. detect and measure canalization (Gibson and G P Wagner 2000; De Visser et al. 2003; also conflicting evolutionary roles of see Gibson and Dworkin 2004 for methods of canalization? measuring cryptic genetic variation), both Canalization may have important implica- involving genetic/environmental perturba- tions for both micro- and macroevolution: tion of a reference population. Genetic per- optimal phenotypic design, rapid phenotypic turbation can be achieved, for instance, by divergence and speciation, stasis and con- P-element insertion (Stearns and Kawecki straints. However, the relative importance of 1994), mutation accumulation, or introgres- canalization for these different phenomena is sion of mutations into isogenic lines (Moreno not yet clear (De Visser et al. 2003). For 1994; Gibson et al. 1999). Environmental per- instance, whether genetic canalization is turbation can be achieved, for example, by adaptive, that is, leads to an increase in the heat shock, treatment with specific inhibitors mean fitness of a population, crucially of gene action (geldanamycin in the case of depends on the mechanism maintaining the hsp83 locus), or chemicals such as ether genetic variation (G P Wagner et al. 1997). vapor (Scharloo 1991; examples above). Theory shows that in the case of mutation- First, genetic canalization can be detected selection balance, for example, there is no fit- most clearly by comparing mutational vari- ness benefit from genetic canalization ances among different lines in a mutation because under mutation-selection balance accumulation experiment. Starting with an the mean fitness is independent of the aver- isogenic line, the phenotypic variance is 309 entirely due to environmental effects. Over With such methods it is possible to determine time, however, sublines founded from the iso- the number, location, and magnitude of genic line will accumulate mutations so that effects of quantitative trait loci (QTLs). QTL the variation among sublines as well as the mapping has helped to define genotype by average within-line phenotypic variance will environment interactions and can also be increase over time. If the rate of increase of used to define epistatic effects (Cheverud and variation in one trait is smaller than in Routman 1995). Thus, the method in prin- another, the trait is canalized (Gibson and ciple allows one to identify genes involved in G P Wagner 2000). However, this approach buffering by identifying QTLs for the trait, has several drawbacks including large mea- which is canalized. Then, one can ask surement errors, a bias due to the use of iso- whether these loci act additively or whether genic lines, and other confounding factors they interact epistatically. For instance, QTL affecting variation (see Gibson and G P Wag- mapping has been successfully applied in ner 2000). mice where QTLs affecting fluctuating asym- Second, one can check for canalization by metry in skull morphology have been identi- measuring the change in phenotypic variance fied (Leamy et al. 1998). Similarly, recombi- among lines that are perturbed and not per- nation mapping protocols have been used to turbed (Scharloo 1991 for some classic exam- identify Notch as a candidate gene responsible ples). For instance, one can compare two sets for the reduction of fluctuating asymmetry in of inbred sublines originating from two dif- blowflies (Davies et al. 1996). ferent lines. Then one compares the two sets of sublines when perturbed and not per- problems with detecting turbed. When unperturbed, the sublines in canalization: a cautionary note both sets are likely to exhibit some variation This review has discussed and highlighted in mean phenotype. If the two sets are now the potential evolutionary importance of can- perturbed, the overall mean phenotype is alization and cryptic variation. Canalization likely to change in both sets. If the variation can be defined as reduced sensitivity to in mean phenotypes among sublines in a genetic or environmental perturbation, and given set changes less or not at all as com- its presence can be demonstrated by showing pared to the other set, then this set is more a release of cryptic variation caused by a canalized than the other. Such an approach decanalizing perturbation. Thus, it is rather has been used for instance to examine the straightforward to show that canalization effects of introgression of mutations into well- occurs as a consequence of epistasis or geno- defined genetic backgrounds (e.g., Moreno type by environment interactions (Elena and 1994; Gibson and van Helden 1997; Elena Lenski 2001), and several examples of cana- and Lenski 2001). Phenotypic variances of lization now exist (Scharloo 1991 for a review; the same trait or of different traits can then Rutherford and Lindquist 1998 for an exam- be compared by using coefficients of varia- ple). Furthermore, the recent development tion, that is, variances standardized by the of measurement-theoretical representations mean or the square of the mean (e.g., Houle of epistasis will facilitate the analysis and 1992; Stearns et al. 1995; Houle 1998). interpretation of experimental tests of genetic canalization (Hansen and G P Wag- molecular methods ner 2001). The recent advent of molecular tools Yet, despite the potentially important evo- makes it now possible to address questions lutionary implications of canalization, the about the molecular mechanisms of canali- concept remains problematic. Recently, Her- zation directly (e.g., Gibson and Dworkin misson and G P Wagner (2004) have chal- 2004). For instance, the genetic control of lenged the concept by demonstrating that canalization by short chromosome segments cryptic variation can build up even in the can be studied by using DNA-based markers absence of canalization of the wild-type. As a (Routman and Cheverud 1994; Jansen 1995). consequence, classic experiments showing 310 the release of cryptic variation may not pro- ing event. On the other hand, canalization vide sufficent evidence that the hidden vari- may, at least temporarily, constrain pheno- ation was held in check by canalization. Even typic evolution. (9) Several molecular mech- if specific canalizing patterns do exist and anisms may lead to canalization, among them cause the buildup of cryptic variation, show- genetic redundancy, modularity, and system- ing that canalization is adaptive is extremely level properties of gene networks and bio- difficult. Most, if not all, experiments (Schar- chemical pathways. These mechanisms are loo 1991) do not clearly address whether (or not mutually exclusive; they are often fail to demonstrate that) canalization is adap- reflected in properties such as epistasis, geno- tive. Thus, devising novel approaches to dem- type by environment interactions, and plei- onstrate unambiguous cases of canalization otropy. Generally, it can be stated that canali- will be of crucial importance (Hansen and zation is an interactive and context-dependent G P Wagner 2001; Hermisson and G P Wag- phenomenon. (10) Different forms of selec- ner 2004). Yet, the phenomenon of cryptic tion can favor canalization, but the require- variation seems to be a real and generic prop- ments for the evolution of canalization are erty of populations with epistasis and geno- usually rather restrictive. (11) Canalizing type by environment interactions (Gibson selection favors genetic mechanisms that and Dworkin 2004; Hermisson and G P Wag- result in the suppression of genetic variation ner 2004). Future work needs to address at the level of the phenotype. (12) Although whether the phenomenon of cryptic varia- there are several methods to detect canaliza- tion, its buildup and release, is evolutionarily tion, there are still serious problems with important, independent of whether it is unambiguously demonstrating canalization, caused by canalizing mechanisms or not (Gib- particularly adaptive canalization. son and Dworkin 2004; Hermisson and G P Despite the recent progress reviewed here, Wagner 2004). there is not yet a consensus in the field on some of the most fundamental problems. Summary and Conclusions Two major unresolved problems are about I have emphasized several points in this (i) how canalization should be measured, and review. (1) Canalization is the relative phe- (ii) whether canalization is an adaptive phe- notypic insensitivity of a genotype to genetic nomenon or rather an intrinsic property of or environmental change. A canalized geno- gene networks and biochemical pathways. We type produces the same (or a similar) phe- have many patterns and hypotheses but little notype in different genetic backgrounds solid data. Unraveling the developmental and (genetic canalization) or in different environ- evolutionary basis of canalization will be an ments (environmental canalization). (2) important task for evolutionary developmen- Concepts such as homeostasis and buffering tal biology and evolutionary genetics. Most are essentially synonymous with canalization. importantly, however, unambiguously dem- (3) Genetic canalization is a special kind of onstrating that canalization is more than a epistasis. (4) Environmental canalization and beautiful idea remains a major challenge for phenotypic plasticity are two aspects of the future research. same phenomenon. (5) Genetic canalization acknowledgments and phenotypic plasticity are not mutually exclusive. (6) Canalization and phenotypic I am very grateful to Steve Stearns for encouragement plasticity can be generalized using the notion and inspiration and to Nicole Vouilloz for all her of variability, that is, the capacity to respond insight, help, and constructive critique. I also thank Christian Braendle, Dieter Ebert, Joachim Hermisson, to genetic and environmental change. They Tad Kawecki, Benedikt Schmidt, Steve Stearns, Gu¨nter can be modeled using phenotypic land- Wagner, and two anonymous referees for helpful com- scapes. (7) Genetic canalization results in the ments on the manuscript. Santiago Elena, Richard accumulation of phenotypically cryptic Lenski, and Claus Wilke kindly provided me with pre- genetic variation. (8) On the one hand, can- prints of their work. My work was supported by the Swiss alization may increase evolvability and lead to Nationalfonds (grant to T J Kawecki), the Roche phenotypic diversification after a decanaliz- Research Foundation, and the Swiss Study Foundation. 311

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