JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 291:213–225 (2001)

© 2001 WILEY-LISS, INC.

Elements of Butterfly Wing PatternsH.F. NIJHOUT*Department of Biology, Evolution, Ecology and Organismal Biology Group,Duke University, Durham, NC 27708-0338

ABSTRACT The color patterns on the wings of butterflies are unique among animal colorpatterns in that the elements that make up the overall pattern are individuated. Unlike the spotsand stripes of vertebrate color patterns, the elements of butterfly wing patterns have identitiesthat can be traced from species to species, and typically across genera and families. Because ofthis identity it is possible to recognize homologies among pattern elements and to study theirevolution and diversification. Individuated pattern elements evolved from non-individuated pre-cursors by compartmentalization of the wing into areas that became developmentally autonomouswith respect to color pattern formation. Developmental compartmentalization led to the evolutionof serially repeated elements and the emergence of serial homology. In these compartments, serialhomologues were able to acquire site-specific developmental regulation and this, in turn, allowedthem to diverge morphologically. Compartmentalization of the wing also reduced the developmen-tal correlation among pattern elements. The release from this developmental constraint, we be-lieve, enabled the great evolutionary radiation of butterfly wing patterns. During pattern evolution,the same set of individual pattern elements is arranged in novel ways to produce species-specificpatterns, including such adaptations as mimicry and camouflage. J. Exp. Zool. (Mol. Dev. Evol.)291:213–225, 2001. © 2001 Wiley-Liss, Inc.

Butterfly color patterns are very different fromthose of leopards and zebras. The color patternsof leopards and zebras are made up of spots andstripes that are placed either randomly or evenlyand whose number and position differ from indi-vidual to individual. These coat patterns have thesame characteristics of randomness and individualvariability as the ridge patterns of human finger-prints. In butterflies, by contrast, the same spotor stripe occurs in exactly the same location inall individuals of a species. More importantly, agiven spot or stripe can be traced from species tospecies within a genus and often from genus togenus within a family. The elements that makeup the wing pattern of butterflies are an anatomi-cal system that is as organized and diverse as thevertebrate skeleton and the body segmentationand tagmatization of arthropods. It is a systemin which there is homology and in which prob-lems of developmental and evolutionary origin,adaptation, and diversification can be analyzed.

A butterfly color pattern element is an individu-ated character in the way that a bone is an indi-viduated character but a leopard spot is not. Acolor pattern element is, however, a character ofa peculiar sort. A pattern element is not an objectthat has a substance and that can be isolated, asone can dissect out a bone or an imaginal disk;rather, a pattern element is the product of a lo-

calized event of pigment synthesis that resultsfrom a spatially patterned activation of an enzy-matic pathway. Why then is the resultant patchof pigment a character? And why is a spot on abutterfly wing an individuated character that canbe given a name and whose identity can be tracedacross phylogenetic space, whereas a leopard spotis not? The answers to these questions bear onthe issue of how characters originate in develop-ment and evolution, and how characters becomeindividuated. In the following sections I will firstoutline the general structure and properties of theelements of butterfly wing patterns and then dis-cuss what we have learned about their develop-mental and evolutionary origins. We will see thatthe principles that underlie color pattern devel-opment, evolution, and individuation are generalones that apply also to more conventional mor-phological characters.

From The Character Concept in Evolutionary Biology, G.P. Wagner,editor. Reprinted with permission. © 2000 by Academic Press, SanDiego.

Grant sponsor: National Science Foundation.*Correspondence to: H. Fred Nijhout, Dept. of Biology, Duke Uni-

versity, Durham, NC, 27708-0325. E-mail: hfn@duke.eduReceived 18 May 2000; Accepted 6 November 2000

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STRUCTURE OF WING PATTERNSThe main organizing principle of butterfly color

patterns is the symmetry system. In its simplestform, a symmetry system consists of a pair of pig-ment bands that run roughly parallel to each otherfrom the anterior to the posterior margin of thewing and approximately normal to the longitudi-nal veins of the wing. It is called a symmetry sys-tem because the pigment distribution in each bandof the pair mirrors that of the other (Nijhout, ’91).The field between the bands of each symmetrysystem typically differs in pigmentation (usuallydarker) from the general background of the wing.Three such systems of bands make up the basicwing pattern of butterflies: the basal symmetrysystem, the central symmetry system, and the bor-der symmetry system. In addition there may beone or two narrow bands that run closely parallelto the distal wing margin, the submarginal bands(Fig. 1A).

Along the midline of the border and central sym-metry systems there is often a distinctive set ofpigmented marks. Those within the border sym-metry system are often highly elaborated into eye-spots and are called the border ocelli (Fig. 1A).Along the midline of the central symmetry sys-tem there is typically a single large mark calledthe discal spot, a name that derives from the factthat this spot always occurs at the apex of the so-called discal cell.1 In some moths the discal spotis elaborated into a large eyespot.

In most cases each pigment band is interruptedwherever it crosses a wing vein so that it looks

Fig. 1. The nymphalid groundplan is made up of pairedpigment bands, forming a set of three symmetry systems,the border symmetry system, central symmetry system andbasal symmetry system. The discal spot (d), and the borderocelli (bo) are found along the midlines of the central andborder symmetry systems, respectively. Panel (A) emphasizesthe vertical structure of the groundplan and its constitutive

symmetry systems, while panel (B) emphasizes the role ofwing veins which break up the bands of the symmetry sys-tems into semi independent pattern elements. One exampleof how real patterns are derived from this groundplan byselective expression, displacement and morphological modifi-cation of these elements is illustrated in Fig. 9.

1A “cell” in this case is an area bounded by wing veins. The spatialpattern of veins on insect wings is relatively constant for all speciesand general within a taxonomic family, although it differs greatlyamong families. All butterflies therefore have the same number ofwing cells, although the shape of these cells varies with the overallshape of the wing. In about half the species the discal cell is closedoff by a set of crossveins, and in those cases the discal spot coincidesprecisely with the position of these crossveins.

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like a linear series of short segments. The align-ment of such a series is typically imperfect, sothat the whole band has the appearance of a geo-logic fault zone with each segment of the bandhaving slipped proximally or distally along a wingvein. By breaking up the bands of the symmetrysystems into series of short segments, the wingveins provide the second organizing principle forbutterfly wing patterns. The wing veins behaveas boundaries that compartmentalize the wingsurface with respect to color pattern formation.This compartmentalization breaks the three sym-metry systems up into parallel series of isolatedand, as we shall see, semi-independent patternelements. The result of this interaction betweensymmetry systems and wing veins is illustratedin Figure 1B. This figure emphasizes the disloca-tion of the pigment bands and presents the so-called Nymphalid Groundplan: the set of patternelements out of which butterflies build their wingpatterns (Nijhout, ’91). The groundplan thus con-sists of eight parallel series of pattern elements:one for each band of each of the three symmetrysystems, a row of border ocelli, and a set of sub-marginal bands. Each wing cell has a representa-tive of each of these pattern elements, so theoverall groundplan can be viewed as a serial rep-etition of the same set of pattern elements.

PATTERN ELEMENTS AS CHARACTERSColor pattern evolution and diversification oc-

curs by the selective modification of the patternelements illustrated in Figure 1B. During evolu-tion, each pattern element can change indepen-dently from the other elements in each of itsmodalities, of color, shape, size, and position (seeFig. 9 for an example). The degree of independenceof the pattern elements is most easily demon-strated by examining their correlated variationamong individuals. There is always a smallamount of individual variation in the size, shape,and position of pattern elements, which comesabout through individual variation in genotypesor environment experienced during development,and by an appropriate experimental and breed-ing design it is possible to partition the relativecontribution of genetic and environmental varia-tion to variation in the phenotype (Paulsen, ’94;Falconer and Mackay, ’96; Lynch and Walsh, ’98).If two pattern elements covary in size or positionfrom individual to individual, it could indicate thatthe two share developmental determinants for theprocesses that determine size or position. If twopattern elements do not covary (that is, if the sec-

ond varies in the opposite direction from the firstas often as it varies in the same direction, or ifthe first element varies and the second does not),then the two either share no variable developmen-tal determinants that affect the characteristic inquestion, or the variation in one direction imposedby one set of determinants is balanced exactly byvariation in the opposite direction imposed by an-other set of determinants. There are some inter-esting correlation patterns among the elementsof the nymphalid groundplan in Precis coenia. Thesize of pattern elements that belong to a homolo-gous series (i.e., are members of the same sym-metry system band) covaries significantly whereasthe sizes of nonhomologous elements are un-correlated. The position of pattern elements withinthe same wing cells are moderately correlated(even if they belong to different symmetry sys-tems), but the positions of elements in differentwing cells are not (Paulsen and Nijhout, ’93;Paulsen, ’94). Moreover, the correlated variationamong pattern elements diminishes with distancebetween them (elements physically farther aparton the wing have less correlated variation thanelements closer together) as well as with the de-gree to which they have diverged from each othermorphologically (homologous elements that differin properties such as pigmentation or complexitycovary less than homologous elements that do notdiffer in these properties).

Finally, studies on the genetics of wing patterndiversity in Heliconius have revealed a hierarchyof genetic regulation of pattern element charac-teristics. Some genes affect the color, position, orsize of single pattern elements, while others af-fect the properties of an entire rank of serial ho-mologues. Several genes are known that affect thepattern on one wing surface only (i.e., either forewing or hind wing), while others affect the pat-tern in homologous regions on both fore and hindwing. Finally, some genes affect the properties ofmany unrelated pattern elements at once (Nijhoutet al., ’90; Nijhout ’91). There appear to exist ge-netic mechanisms that control the properties ofpattern elements individually, in homologous se-ries, or in ensembles over large areas of the wing.

With independent control of the various proper-ties (presence/absence, size, shape, color, position)of the individual members of the eight series ofpattern elements, the number of possible permu-tations that can be used to build up the overallwing pattern is enormous. One of the conse-quences of this great permutational complexity isthat no two species of butterflies have exactly the

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same overall pattern, although within a speciesthe color pattern is so characteristic and constantthat it can be used for species identification. In-sofar as pattern elements and their properties aregenetically and developmentally independent, theyare compartmented and free to respond indepen-dently to natural selection.

The function of color patterns is visual commu-nication. Accordingly, butterfly wing patterns haveundergone extensive adaptive evolution for suchfunctions as sexual signaling, aposematism, cam-ouflage, and mimicry. In building up the wing pat-tern, some pattern elements are eliminated whileothers are brought into mutual alignment and,their color and shape is adjusted to achieve a par-ticular overall visual effect. The extraordinary di-versity of patterns, and the fact that extremelyaccurate Batesian, Muellerian, and dead leaf mim-icry can evolve, suggests the operation of a highlyversatile developmental mechanism that placesand shapes the pattern elements in a precise yetflexible way. The evolution of such a flexible de-velopmental mechanism and the way in which itproduces a highly evolvable color pattern will bethe subject of the remainder of this article.

EVOLUTION OF A PATTERNINGMECHANISM

Origin of symmetry systemsIn order to understand the evolution of the

highly compartmented Nymphalid groundplan, weneed to first understand the nature and origin ofsymmetry systems. Symmetry systems are prob-ably the most widespread of all color patterns, notonly in butterflies and moths but also in fish andmammals. The general nature of symmetry sys-tems in moths and other animals was first recog-nized by Henke (’33). He noted that the bands ofa symmetry system sometimes merge togetherand fuse to form a U-shaped pattern (Fig. 2A,B),or a variety of closed patterns (Fig. 2C,D). A sym-metry system then appears to be nothing morethan a closed loop figure, or a circle, that has beentruncated. An eyespot is therefore a symmetry sys-tem, as is any pattern that has a differentiationof color or structure that runs from center to pe-riphery. Symmetry systems can therefore be ra-dial, as in the case of eyespots (or Fig. 2C), orbilateral, when there is extensive truncation (asin Fig. 2A).

The development and evolution of symmetrysystems can perhaps be best understood by firstlooking at some simple color patterns of verte-

brates. Zebra stripes, for instance, are symmetrysystems, although this is not always easy to de-tect. Some races of Burchell’s zebra (Equusburchelli variety antiquorum from Namibia) havegray so-called shadow stripes in the white areasbetween their black stripes and some individualshave yet narrower and paler gray stripes betweenthe shadow stripes and the black stripes (Fig. 3A).I’ll call these the primary and secondary shadowstripes, respectively. Shadow stripes are expressedonly where the main black stripes are very far

Fig. 2. Various simple modifications of symmetry systemsfound in the moths which give clues to the nature of symme-try systems. (A) Normal symmetry bands. (B) Symmetrybands fused together at one end. (C) Symmetry bands fusedat both ends to form a circle. (D) Multiply closed pattern.For real examples of such patterns see Henke (’33) andNijhout (’91).

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apart, so it looks like the patterning system isattempting to fit additional stripes in where theseparation between existing stripes becomes large.A more spectacular intercalation of stripes can beseen in lionfish (various species of Pterois; Fig.3B), where species differ in the number of levelsof intercalation.

Intercalation of elements is fairly common inpattern-forming systems that are growing or thatoccur on curved surfaces where the spacing be-tween existing elements must necessarily expand.Brain corals are a good example. Here the spac-ing of polyps gradually increases as the coral headgrows and the surface of the colony expands; newpolyps then form where the distance between ex-isting polyps exceed some value. An overall evenspacing is maintained by intercalation when thegap between existing structures exceeds a criti-cal value. Pattern intercalation of the sort we see

in brain corals and zebra stripes actually tells ussomething important about the mechanism of pat-tern formation. In order to understand what isgoing on we need to recognize that there are twoalternative ways in which the space between twodiverging stripes can be filled: by branching of oneof the stripes, or by intercalation of a stripe un-connected to either of the flanking stripes. Thedifference between these two patterns lies in thetiming of the processes of pattern formation rela-tive to growth. Understanding the mechanism bywhich repeated spatial patterns arise during de-velopment helps explain why this is so. It is wellestablished that in development and physiologythe formation of evenly spaced repeated patternsrequires a mechanism that involves lateral inhi-bition (Bard, ’77, ’81; Meinhardt, ’82; Murray, ’89;Oster, ’88; Held, ’92). In lateral inhibition the pres-ence of a given structure inhibits the formationof similar structures in its immediate vicinity. Thisinhibition diminishes with distance, so the nextstructure can only form where the inhibition fallsbelow a particular threshold. Whether a newstructure will actually form outside the area ofinhibition depends on the presence of a mecha-nism that produces the conditions for the initia-tion of that structure. Typically this would be thesame mechanism that gave rise to the initialstructure, and if this initiation mechanism is con-stitutive and spatially widespread then the con-ditions exist for the generation of a relativelyevenly spaced repeated pattern.

Depending on the nature of the inhibitorymechanism and the way it interacts with the ini-tiation mechanism, the pattern that is formed canbe either evenly spaced points (such as bristles ofinsects, or the spots on cheetahs), or evenly spacedstripes (as the stripes of zebras), the spacing be-tween the elements being determined by thestrength of the inhibition. Suppose then that weare considering two neighboring stripes and sup-pose that the field on which they occur is expand-ing. Then there are three possible outcomes,depending on the stage of pattern formation: (1)if pattern formation is fully complete, then thestripes and interstripe region simply becomebroader; (2) if pattern formation is still ongoingand the stripes are not yet fixed, then the pat-tern reorganizes itself so that three stripes canbe formed instead of two, and if growth is moreextensive in one region of the field than in an-other so that the stripes diverge in a wedge-shaped pattern, then one stripe will produce abranch that occupies the widening interband re-

Fig. 3. (A) Striping pattern on a Burchell’s zebra fromNamibia (Equus burchelli var antiquorum) with intercalatedshadow stripes. (B) Color pattern of the lionfish, Pteroisvolitans, illustrating several levels of intercalated stripes.Fig.3. (A) Striping pattern on a Burchell’s zebra from Namibia(Equus burchelli var antiquorum) with intercalated shadowstripes. (B) Color pattern of the lionfish, Pterois volitans, il-lustrating several levels of intercalated stripes.

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gion; (3) finally, if the first set of stripes is fullydetermined but the interstripe region is still ca-pable of pattern formation, then a new stripe canform halfway between the existing stripes, butunconnected to them. The second mechanism isbelieved to be responsible for stripe branching inzebras, and the third mechanism for the forma-tion of shadow stripes (Bard, ’77, ’81). Becauseshadow stripes are determined after the mainstripes, and not simultaneously with them, it ispossible for different factors to influence the de-velopmental details of each. This can account forthe fact that the two types of stripes differ in pig-mentation, the shadow stripes being gray ratherthan black.

In the lionfish Pterois volitans, the primarystripes and shadow stripes differ not only in pig-mentation but also in structure (Fig. 3B). Not onlyare the shadow stripes paler than the mainstripes, but the main stripes have a graded pig-ment distribution, darker near the periphery andlighter near the center of the stripe. This grada-tion is not seen in the secondary shadow stripes,although it is occasionally evident in the primaryshadow stripes, wherever these are particularlybroad. This graded pigment distribution makeseach stripe self-symmetrical with regard to colorpattern.

We have now arrived at the point where we canconceptualize two kinds of symmetry systems. Apair of stripes and their intervening shadow stripeis a symmetry system, and each band is itself asymmetry system. We have already seen that inthe first case the symmetry arises from the lat-eral inhibition that existing stripes exert on theformation of new stripes. For this to work the ex-isting stripes need to be developmentally fixed,or determined, before the shadow stripe is in-duced. In the second case, the self-symmetry of astripe tells us something about the shape of thegradient that induces pigment synthesis. This gra-dient must also be self-symmetrical, which meansthat its graph must be an elongated hump. Thetwo edges of a stripe differentiate in the same waybecause they respond to the same gradient value,which is different than the value at the centralcore of the stripe. For the purposes of the presentargument I’ll assume that the value at the mar-gins is lower than at the core, but it is also pos-sible that the difference is one of timing if, forinstance, determination proceeds like a grass firefrom a line-shaped central core outward. In thelatter case, the dark outer margins represent theactive fire, while the paler core represents the

burnt grass left behind; lower and higher valuesof the gradient can be thought of as shorter orlonger times since ignition. As we’ll see later inthis article, the grass fire model cannot easily ex-plain the next stages in development of a symme-try system, and a simple self-symmetrical spatialgradient is a more useful conceptual model for un-derstanding what happens next.

The symmetry systems of the nymphalid ground-plan appear to have evolved from simple self-sym-metrical pattern elements (Nijhout, ’94). It ispossible to trace the steps by which this evolu-tion occurred by examining the symmetry systemsand their precursors in various groups of moths.Moths in the suborders most closely related to thebutterflies have color patterns based on symme-try systems, but moths in the more basal subor-ders of the Lepidoptera do not. In the latter,patterns are typically made up of fairly irregularspots or blotch-like figures, placed in species-spe-cific but irregular patterns on the wing surface.Often these figures are monochrome (like zebrastripes), but when they are not, their pigmenta-tion exhibits self-symmetry (like lionfish stripes),so that they look like irregularly rounded concen-tric figures or rings (Figs. 2B and 4). In some spe-cies there is much intraspecific variation in thesize and shape of these pattern elements. In indi-viduals where two neighboring elements are en-larged, they simply fuse smoothly at their pointof contact to form a single closed figure (Figs. 4and 5). The fact that two self-symmetrical patternscan fuse smoothly to form a single closed patternindicates that the two are produced by identical de-velopmental processes. When these composite fig-ures enlarge so that they extend to the anterior andposterior wing margins, the pattern that results re-sembles a pair of irregular bands and constituteswhat we recognize as a symmetry system. Sym-metry systems are constructed by the fusion ofthe outer pigment rings of a row of adjoining cir-cular elements (Henke, ’33; Nijhout, ’91).

In the course of pattern evolution in the moths,the arrangement of the elements that producedsymmetry systems became increasingly regular.They came to be arranged in several parallel se-ries, running from the anterior to the posteriormargins of the wing. Some species of moths haveas many as six such parallel symmetry systems,presumably derived from six parallel rows of cen-ters of origin. In many moths, particularly in theArctiidae and Geometridae, the bands of an adja-cent symmetry system can fuse along their length(Fig. 5), indicating again that the different sys-

ELEMENTS OF BUTTERFLY WING PATTERNS 219

tems are produced by identical developmental pro-cesses (Nijhout, ’94).

The butterflies typically have three symmetrysystems, although in many species the basal sym-metry system is missing or represented only byits distal-most band. The bands of each symme-try system usually have a distinctive pigmenta-tion and morphology. The central elements of theborder symmetry system (called the border ocelli;Fig. 1B) are almost always distinct and highly dif-ferentiated, in contrast to the situation in mothswhere these elements are usually absent or onlyfaintly expressed. There are no cases in which thebands of adjacent symmetry systems fuse so asto make the central fields of those systems con-tiguous, as we see in certain moths (Fig. 5). Itappears then that as the butterflies diverged fromthe moths, each of their symmetry systems ac-quired the ability to express a distinctive pigmen-tation and morphology (Fig. 6), and becamesufficiently differentiated from each other that fu-sion between adjacent systems is no longer pos-sible. The most complex evolution has occurredin the border symmetry system. Not only havethe central elements of this system undergonecomplex elaboration and differentiation (Nijhout,

’91), but even the two bands have diverged fromeach other in pigmentation and morphology sothat in many species it is difficult to see that thetwo are sister bands (or homologous members) ofthe same symmetry system.

Hierarchy of symmetry systemsWe have seen that there are two ways of mak-

ing sets of stripes that look like symmetry sys-tems: (1) by the intercalation of stripes betweenexisting ones (Figs. 3 and 7A), and (2) by the ex-pansion and truncation of radially symmetricalpatterns (Figs. 2 and 5). In the first case, exem-plified in this article by zebra and Pterois colorpatterns, it is necessary that the primary elements(the main pigment stripes) first be irreversiblydetermined so that the process that produces theintercalated element (the shadow stripe) can nolonger alter the two flanking primary elements.In this case symmetry is imposed by the tempo-ral sequence in which bands are determined. Inthe second case, symmetry is a preexisting condi-tion that is the consequence of the concentric or-ganization of a pattern element. We can think ofthe concentric rings of such a pattern as corre-sponding to isoclines or contours of the determi-

Fig. 4. Fusion among rounded pattern elements in mothsof the genus Zygaena (Lepidoptera: Zygaenidae). These vari-ous patterns of fusion are found as interspecific diversity as

well as intraspecific variation, and indicate that the roundedelements of the pattern are produced by identical processes(after Nijhout, ’94).

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nation gradient. Spot-like patterns would be pro-duced by humped or circular mound-like gradi-ents and stripes or bands by elongated moundsor ridge-shaped gradients.

An added complication to this system is that insome butterflies and moths the bands of a sym-metry system are themselves composed of a sym-metrical arrangement of pigments (Nijhout, ’91).In order for a band to be self-symmetrical in pig-mentation, a band cannot simply be a thresholdor contour on a gradient but must be a ridge ofsome sort. The simplest way to obtain this wouldbe if the band is initially formed at a thresholdon a gradient but then that site becomes thesource or high point of a new gradient (Fig. 7B),very much in the way the sequence of gradientsin early embryonic development of Drosophila isset up (Lawrence, ’92). A symmetry system thusbegins as a single self-symmetrical band and eachof its edges then becomes the organizing centerof a new symmetry system. The edge of a band,initially merely an aspect of a structure (the band),can thus acquire an identity independent from thestructure of which it was a part.

Individuation in symmetrical structuresThe hierarchy of symmetry systems outlined in

this article poses an interesting problem in char-acter evolution. One can question whether themembers of a pair of symmetrical structures aredifferent characters. Two appendages of a sym-metrical pair or the petals of a radially symmetri-cal flower, for instance, are certainly different

Fig. 5. Fusion among bands of symmetry systems in mothsof the family Arctiidae. Bands belonging to the same symme-try system can fuse, as can bands belonging to adjacent sym-metry systems. Such fusions indicate that identical processesproduce each of the symmetry systems (for photos of speci-mens illustrating these kinds of fusion patterns see Nijhout,’91, ’94).

Fig. 6. Origin of symmetry systems form random spot-ting patterns (A). Primitive moths have patterns that origi-nate by fusion of irregularly arranged centers of origin (B,

see also Fig. 4). In the ancestors of the butterflies these cen-ters became arranged in three regular rows, producing threesymmetry systems (after Nijhout, ’94).

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structures; they have independent developmentalorigins, and if the development of one is perturbedthe other is unaffected. But insofar as they areperfect mirror images of each other it is difficultto see them as different characters because ex-actly the same information went into their manu-facture and exactly the same information can beextracted from their analysis. Symmetrical struc-tures may have developmental independence, butthey have no individuality, if by individuality wemean the possession of characteristics that dis-tinguish one from the other. In bilateral symme-try (but not in radial symmetry) the two are atleast mirror images of each other, so they are notperfectly identical and are at least recognizableas left or right instantiations of the character, butthat does not qualify them as different charac-ters. Only if the two for some reason diverged intheir morphology, like the cutter and crusher clawsof a lobster, do they become different characters,and then only in those features in which they di-

verged. The causes of divergence can be manifold.A simple change in absolute size, if it is accompa-nied by allometric changes, can lead to theappearance of many differences between twostructures that, had they been the same size,would have been absolutely identical in all details.What is needed in such a case is some develop-mental event that influences the two members ofthe pair in a qualitatively or quantitatively dif-ferent way.

If the members of a symmetrical pair developin a spatially asymmetrical environment then dur-ing its ontogeny each will experience unique in-teractions not shared with its sister structure.Differences in physical interactions and differ-ences in local patterns of gene expression willcause each member to develop unique character-istics. This is the case during the development ofsymmetry systems, since they develop on a wingwith distinct proximo-distal and antero-posteriordifferentiation. This is why the proximal and dis-tal bands of a symmetry system can differ inshape, pigmentation, and distance from their com-mon center. Individuation of a pigment band re-quires that it be subject to some developmentalinfluences that are not shared with other bands,so that it develops unique characteristics by whichit can be distinguished from all other pigmentbands.

Presumably any symmetrical structure can bethe locus for the multiplication and subsequentdifferentiation of parts. Differentiation of an ini-tially homogeneous structure, whether a zygote,an insect segment, or a symmetry system, intomany different parts must necessarily occur by amechanism that somehow generates differencesin different regions of that structure. Those dif-ferences can be imposed from the outside by a spa-tially heterogeneous environment, or they can begenerated from within by processes that break upthe initial homogeneity. Recent findings about thecontrol of embryonic determination show that thishappens primarily by means of concentration gra-dients of gene products that control the differen-tial expression of genes. Different thresholds thengive rise to expression of new genes in some ar-eas and not in others. The new gene products thendiffuse and produce new gradients, and a succes-sion of such gradient and threshold events gradu-ally subdivides a field into ever smaller and morespecialized regions each of which can become rec-ognized as a different part or character.

In order for such a process of successive subdi-vision of a developmental field to produce discrete

Fig. 7. Mechanisms for the formation and multiplicationof symmetry systems. Pigment bands are formed at severalthreshold levels of a symmetrical gradient of pigment deter-mination, here depicted as a roughly bell-shaped curve. (A)and (B), Intercalation of a new symmetry system (or stripe)when two existing symmetry systems (or stripes) move apart.(C) and (D), each threshold becomes the origin of a new sym-metrical gradient for pigment determination.

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structures, there must be boundaries of some sortso that the effects of region-specific gradients re-main localized. If communication is by diffusionof transcription factors, for instance, it would bedifficult to constrain their effect and it would beimpossible to produce a sharply defined boundedpart, character, or pattern element. Some mecha-nism for establishing boundaries is therefore nec-essary and several quite different mechanismsappear to exist. Boundary sharpening of gradientscan be achieved by positive feedback and lateralinhibition processes of the general kind that arebelieved to be involved in the formation of zebrastripes and insect segments (Bard, ’77, ’81; Mein-hardt, ’82). In the case of symmetry systems, it isthe large physical distance between the two bandsof a system that provides an effective barrier. Oneband can be subject to developmental influencesthat simply decay below some threshold beforethey reach the region where the other band de-velops. This is in some sense a trivial mechanismand it would probably not be useful for producingclosely spaced or densely packed features, unlessthe decay can vary sharply in space, or thresholdsare very sharply defined (and cells can differenti-ate between minute differences in a gradient), orunless those structures are not determined simul-taneously. However, when different features aredetermined sequentially, so that one is fixed be-fore the process that determines the next one be-gins, it should be possible to develop tightlypacked features without sharp boundaries to thespread of information. Finally, it is possible tohave physical barriers that block communicationbetween different parts of a developing field. Insuch a case the field would be effectively compart-mentalized into developmentally independentunits and the boundaries of the compartmentwould at least in part define the boundaries ofstructures and parts that subsequently develop.Butterfly wings become compartmentalized thisway, as we will see later.

The evolution of serial homologyIn butterfly wing patterns the symmetry sys-

tem bands do not run continuously across thewing but are interrupted and dislocated at thewing veins so that the band is broken up into aseries of short pattern elements (Fig. 1B). Thewing veins appear to act as barriers to communi-cation between different parts of the wing epi-thelium and they effectively compartmentalize thewing with regard to pattern formation. Evidencefor this compartmentalization comes from several

sources: pattern elements are often abruptly trun-cated precisely at the wing veins; experimental per-turbation by damaging the wing epithelium canseverely distort the pattern that develops but thiseffect remains restricted to the wing compart-ments in which the damage is localized; geneticstudies have revealed that there are genes thatcan alter the pattern within a single compartmentwithout affecting the pattern elsewhere on thewing (Nijhout et al., ’90; Nijhout, ’91); finally, spe-cies-specific patterns are often characterized byspecific modifications of the pattern within asingle compartment (Nijhout, ’91). Thus observa-tional, experimental, and genetic evidence indi-cates that veins somehow constrain patternforming processes to areas within their bound-aries. We have preliminary evidence from elec-tron microscopy that gap junctions, which areabundant in the epithelial cells of the wing, areactually absent in epithelial cells near the wingveins. These junctions are required for cytoplas-mic communication between cells, and in theirabsence diffusible signals cannot get from one cellto another. Gap junction-less regions of epithe-lium at the wing veins would thus act as effec-tive barriers to the diffusion of morphogeneticsignals, and communication across such a bar-rier would be difficult.

Symmetry systems evolved long before they be-came compartmentalized by the wing veins. In thevast majority of moths, for instance, symmetry sys-tem bands run smoothly across the wing withoutinterruption or dislocation. The origin of compart-mentalization of the pattern can be understood bycomparative studies of patterns in the moths andtheir sister group, the caddisflies (Trichoptera).Although caddisflies do not have symmetry sys-tems, many have a color pattern made up of ir-regular random bands, called ripple patterns, andthese bands are always interrupted and truncatedat the wing veins. Many moths have similar ripplepatterns and wherever these are found they arealways truncated at the wing veins, even in spe-cies that have symmetry system bands that areuninterrupted by wing veins (Nijhout, ’94). Fromperturbation studies we know that during ontog-eny, ripple patterns are determined before the el-ements of the nymphalid groundplan. This findingimplies that in moths the wing is compartmen-talized for color pattern formation at the timeripple patterns are determined but not some timelater when the symmetry systems are determined.The simplest mechanism to account for this is thatgap junctions are absent in the epithelium around

ELEMENTS OF BUTTERFLY WING PATTERNS 223

the wing veins during the early stages of colorpattern determination, and that gap junctionsform in these areas during the later stages of pat-tern determination.

The ability to form compartments at the wingveins is evidently very old, predating the originof the Lepidoptera, and was thus present in thelineage of moths within which the butterfliesevolved.2 What happened in the course of butter-fly evolution is that the compartmentalization per-sisted during a longer period of ontogeny so thatit came to overlap the time at which the symme-try systems were determined. Compartmentaliza-tion of the wing for symmetry systems could thusbe due to a simple heterochronic shift in the tim-ing of gap junction inactivation. The consequenceof this compartmentalization, however, has provento be completely out of proportion to the simplicityof the mechanism, as it enabled the production ofan unprecedented and unrivalled diversificationof patterns.

Because there is no communication betweencompartments, it is difficult for processes in onecompartment to influence those in adjacent ones.Only factors that are shared by two regions of thewing before compartments are formed, or that aretransmitted through the extracellular mediumand can thus avoid compartment boundaries, canhave common causal effects on the events in twocompartments. In either event, uncoupling be-tween compartments assures that independentinstances of pattern formation take place in each.If these instances use exactly the same develop-mental information then one would expect identi-cal patterns to be produced in each compartment.Therefore, the net results of compartmentaliza-tion is the serial repetition of pattern elements ineach compartment, each series corresponding toone of the original symmetry system bands.

Individuation of serial homologuesThe compartments do not, however, contain ex-

actly the same developmental information. Theydiffer in size and shape and those differences canhave profound effects on diffusion-dependent de-velopmental events. The size and shape of a de-velopmental field affects the shape of diffusiongradients that develop within it and this altersthe position and shape of thresholds. Thus, simpleand unavoidable differences in the initial condi-

tions of compartment formation should affect pat-tern development in each compartment differently.These differences can cause misalignment of thereplicates that develop in each compartment, sothat a single smoothly continuous band is now bro-ken up into unconnected and dislocated elements.Differences in initial and boundary conditions canalso cause the replicate elements in each compart-ment to differ in shape and size, even though theyall share the same genetic determinants.

In each compartment there will therefore besystematic differences in the pattern that devel-ops. Subsequent evolutionary events can thengradually reduce or magnify these differences. Inorder for genetic changes to alter the pattern inone compartment and not in another there has to

2Exactly which family of moths forms the sister group to the but-terflies is still unresolved and a matter of much speculation andcontroversy.

Fig. 8. In the butterflies, the wing pattern is compart-mentalized by the wing veins so that the position, pigmenta-tion, and shape of the bands in a compartment can be modifiedindependently of those in an adjoining compartment (afterNijhout, ’94).

224 H.F. NIJHOUT

be a non-linear association between genetic varia-tion and pattern variation so that a small geneticchange can have a big effect in one location on thewing and a small effect elsewhere. Such non-linearities are actually part of the very nature ofa diffusion-threshold process (Nijhout and Paul-sen, ’97; Klingenberg and Nijhout, ’99) and arecharacteristic of many other types of develop-mental and pattern-forming processes as well(Murray, ’89). Indeed, genetic studies of Heliconiushave shown that genes have evolved that affectthe pattern in one compartment only (Nijhout etal., ’90; Nijhout, ’91). It is, therefore, possible forthe pattern elements in each compartment to ac-quire characteristics that are not shared by theirhomologues in other compartments, and to ac-quire a modicum of genetic and developmentalindependence from its serial homologues. The de-gree to which mutation and selection can alterone pattern element without affecting other pat-tern elements within its compartment, or otherpattern elements in its homologous series, de-pends on the number of shared developmental de-terminants. These shared determinants areexpressed as genetic correlations among patternelements (Paulsen and Nijhout, ’93; Paulsen, ’94;and see section “Pattern elements as characters”earlier in this article).

Each pattern element shares a few relativelyweak genetic correlations for size, shape, and po-sition with its serial homologues and with itsneighbors in a compartment, but is otherwise re-markably free of such correlations (Paulsen, ’94).This finding reveals that the normal variation insize, shape, and position of different pattern ele-ments is not dominated by the effects of the many

Fig. 9. Derivation of the dead-leaf mimicking wing pat-tern of Kallima inachus from the elements of the nymphalidgroundplan (Fig. 1B). The sequence of patterns shown is notintended to represent an evolutionary transformation series

but is given to illustrate how each element of the pattern ismodified and displaced to produce the overall image of aveined leaf.

developmental factors the two have in common,but by factors that are unique to each element ofthe pattern. By compartmentalization and subse-quent evolution each pattern element has acquiredunique morphological properties and unique pat-terns of variation, and through this uniqueness ithas acquired an identity that sets it apart fromall the other elements of the color pattern.

E PLURIBUS UNUM & EX UNO PLURIAThe hypothesis presented here for the origin and

individuation of pattern elements emerges fromstudies of comparative morphology, genetics, anddevelopment, and proposes the following scenariofor the evolution of this evolvable system. The ori-gin of individuated pattern elements probably be-gan with a simple system of circular patterns, allformally identical to one another like the stripesof a zebra or the spots of a leopard. The self-sym-metrical nature of these original patterns led totheir fusion into parallel systems of paired bands(Fig. 6). By interactions with the asymmetrical de-velopmental environment of the wing these simplebands each acquired different characteristics, sothat distinctive systems of patterns evolved alongthe proximo-distal axis of the wing. Subsequentcompartmentalization of the wing surface allowedthe development of each segment of a band to beuncoupled from that of its neighbors. In each com-partment the developmental environment thendiverged sufficiently so that each pattern elementwas able to acquire a unique morphology (Fig. 8)and independent genetic variation. The conse-quence of the developmental uncoupling of pat-tern elements was a system of morphologicalparts virtually unencumbered by mutual con-

ELEMENTS OF BUTTERFLY WING PATTERNS 225

straints on their variation and evolution. Eachelement could be independently modified in form,color, and position, and the ensemble is able tobuild up a virtually unlimited diversity of colorpatterns (e.g. Fig. 9).

ACKNOWLEDGMENTSI would like to thank Louise Roth, Dan McShea,

and Chris Klingenberg for critical comments onthe manuscript, and Monique Nijhout for draw-ing Figure 3.

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