JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 288:1–20 (2000)

© 2000 WILEY-LISS, INC.

Plasticity and Constraints in Developmentand Evolution

JASON HODIN*Science and Math, Seattle Central Community College, Seattle,Washington 98122

ABSTRACT Morphological similarities between organisms may be due to either homology orhomoplasy. Homologous structures arise by common descent from an ancestral form, whereashomoplasious structures are independently derived in the respective lineages. The finding that simi-lar ontogenetic mechanisms underlie the production of the similar structures in both lineages is notsufficient evidence of homology, as such similarities may also be due to parallel evolution. Parallel-isms are a class of homoplasy in which the two lineages have come up with the same solutionindependently using the same ontogenetic mechanism. The other main class of homoplasy, conver-gence, is superficial similarity in morphological structures in which the underlying ontogenetic mecha-nisms are distinct. I argue that instances of convergence and parallelism are more common than isgenerally realized. Convergence suggests flexibility in underlying ontogenetic mechanisms and maybe indicative of developmental processes subject to phenotypic plasticity. Parallelisms, on the otherhand, may characterize developmental processes subject to constraints. Distinguishing between ho-mology, parallelisms and convergence may clarify broader taxonomic patterns in morphological evo-lution. J. Exp. Zool. (Mol. Dev. Evol.) 288:1–20, 2000. © 2000 Wiley-Liss, Inc.

As the fields of developmental and evolution-ary biology continue to converge, an underlyingpattern is beginning to emerge: namely that theastonishing diversity of morphological variationin plants and animals is built on a scaffolding ofa seemingly quite limited set of developmentalprograms. This pattern was predicted at least asfar back as the mid-1970s by Emile Zuckerkandl(’76), at which time he wrote:

Functional innovation at the morphologi-cal level does not appear to require any func-tional innovation at the molecular level. Onemay wonder whether the formation, in thecourse of evolution, of a limb of a land verte-brate from the fin of a fish requires the ap-pearance of new proteins endowed with novelfunctions. I am inclined to believe that it doesnot. (p 404)

So when we catch our collective breath from“startling” findings such as the apparent conser-vation of Hox and Pax gene functions, let us stepback for a moment and consider the implications.There are optimists who feel that since the set ofdevelopmental mechanisms appears to be limited,we are likely to come to an understanding of thebasic nature of metazoan development and evolu-tion via a reductionist developmental and molecu-

lar approach. I argue, on the contrary, that sinceinnovations are manifest at the morphologicallevel, it is necessary to integrate a morphologicalwith a molecular approach if we hope to uncoverany such underlying principles. Specifically, I feelthat the study of incidents of parallel and conver-gent evolution may offer the best approach to ac-complish this integration.

Before continuing, I now provide definitions forseveral key terms. “Parallelism” is independentevolution using the same mechanism. This is con-trasted with “convergence”: independent evolutionusing an alternate mechanism. The third term inthis class is “reversal,” or a return to an ances-tral mechanism (a special case of parallelism).“Homoplasy” is a broader term covering parallel-ism, convergence and reversal: similarity not re-sulting from common ancestry. The definition of“homology” is certainly more contentious (re-viewed in Donoghue, ’92; Abouheif et al., ’97). Itentatively adopt the definition of Van Valen (’82):

Grant sponsor: National Institutes of Health; Grant number:HD07183.

*Correspondence to: J. Hodin, Science & Math, Seattle CentralCommunity College, 1701 Broadway, Seattle, WA 98122.E-mail: hodin@ alumni.washington.edu

Received 14 August 1999; Accepted 14 September 1999

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“resemblance caused by a continuity of informa-tion.” This definition highlights an important dis-tinction between homology and homoplasy, as thelatter involves a definite historical discontinuityof information. We should proceed with the real-ization that in practice the distinction betweenhomology and homoplasy is often far from clearand that other definitions of homology (such asthe “building block hypothesis” of Wagner, ’95)may often prove more useful as heuristic devices.

The term “mechanism” in the context of con-vergence and parallelism is in need of some clari-fication. In an entirely reductionist approach, “thesame mechanism” refers to identical genetic bases.For example, paedomorphosis (reproduction at animmature morphological stage) has evolved sev-eral times independently in tiger salamanders (re-viewed in Shaffer and Voss, ’96). Under the strictreductionist view, these independent instances ofpaedomorphosis are considered to have evolved inparallel only if they are characterized by muta-tions in the same gene or genes. Yet, discovery ofthe genetic bases for homoplastic changes in manyorganisms is not practically feasible. Thus, partlyfor practical reasons, I adopt a developmental defi-nition of mechanism. If the cell biological and mor-phogenetic changes are identical, then I considerit to be an example of a parallelism. Furthermore,since developmental mechanisms often appear tobe characterized by dense networks of cross-regu-latory and feedback interactions among genes,changes in several different members of a givengene network could produce identical morphoge-netic phenotypes. I consider such examples to beparallel evolution at the level of the developmen-tal mechanism. In the case of the tiger sala-manders, precocious sexual development versusdelayed adult development represent different de-velopmental mechanisms leading to a similar pae-domorphic phenotype and, therefore, an exampleof convergent evolution at the level of the devel-opmental mechanism. Alternatively, all tiger sala-manders might be characterized by delayed adultdevelopment via blocks in the metamorphic path-way. Even if these blocks were at different pointsin the metamorphic pathway (which would be anexample of convergence at the genetic level, sincemutations in different genes would underly theinstances of homoplasy), the developmental tra-jectories would be similarly altered and thuswould represent a case of parallel evolution at thelevel of the developmental mechanism. Althoughthe specific developmental mechanisms leading topaedomorphosis are poorly understood among

various tiger salamanders (Shaffer and Voss, ’96),when salamanders as a whole are considered, dif-ferent steps along the thyroid hormone responseaxis are blocked in different paedomorphic spe-cies (Frieden, ’81; Yaoita and Brown, ’90), leadingto a similar metamorphic failure. Thus althoughthe genetic bases of the paedomorphic phenotypeare clearly different (i.e., convergent at the geneticlevel) in different salamander species, similar de-velopmental mechanisms (at the level of the genenetwork) appear to be involved, so I consider thesedisparate cases of paedomorphosis to have evolvedin parallel. In order to learn how development mayinfluence evolution, we should focus on similari-ties and differences at the level of the develop-mental mechanism (rather than, say, the geneticlevel1) when making evolutionary comparisons.

In Part I of this paper, I concentrate on somehigh profile examples in the literature where ho-mology has been cited as the cause of similaritybetween arthropods and chordates. It seems thatthe possibility that these similarities are due toparallel or convergent evolution has been dis-missed too readily. I argue that the apparentunderestimation of the role of homoplasy in met-azoan evolution skews our perspective on how de-velopment evolves. In Part II, I explore thepossibility that the study of well-documented casesof homoplasy not only yields useful informationon evolutionary patterns, but also offers signifi-cant insights into the ways in which interactingnetworks of regulatory genes work to produce com-plex morphological structures. I distinguish be-tween cases of parallel evolution, which mayindicate the presence of developmental constraints(a bias in the production of phenotypic variationdue to ontogenetic factors; Maynard-Smith et al.,’85), from instances of convergent evolution, which,I hypothesize, correlate with phenotypic plasticity(environmentally induced alterations in morphol-ogy within a genotype) in underlying developmen-tal mechanisms.

PART I: HAS NOTHING INTERESTINGHAPPENED SINCE THE CAMBRIAN?

With the advent of DNA sequence analysis, theword “homology” is suddenly in every molecularbiologist’s vocabulary. References to “the verte-brate homolog” of, say, a Drosophila gene are notonly commonplace but, indeed, are becoming the

1Still, considering homplasy at the genetic level is useful in othercontexts, such as understanding the evolution of protein function (seebelow).

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cornerstone of discourse in modern developmen-tal genetics. In addition to the inherent problemsin using the same term to describe similarity attwo different levels (morphological and molecu-lar), there is a disturbing trend towards relaxingthe rigor applied to the term “homology,” evenwhen conversation is restricted to sequence simi-larity. Thus, whatever Genbank spits out becomesa homolog of the gene being studied, with somegenes being “more homologous” than others. Ihave no problem with the notion of degrees of ho-mology (see Roth, ’84), but as do Aboueif et al.(’97) and Doolittle (’86), I strenuously object to theequation of similarity with homology, for it leavesout the essential historical component of the term.The result appears to be that the concept of homo-plasy has been trampled underfoot on the yellowbrick road to a unifying principle of development.2

The fact that developmental networks (such asNotch/Delta, steroid hormones and their recep-tors; Pax6/eyes absent; patched/hedgehog) are uti-lized in a multitude of tissues within a singleorganism points to their essentially modular na-ture. The analogy here is to the concept of serialhomology. I believe that herein lies the basis ofmuch of the confusion in the literature of late re-garding “process homology” (see Gilbert et al., ’96).If a developmental module can be redeployedwithin an organism in the development of a vari-ety of different tissues, then we should use ex-treme caution in assigning the term “homology”to the discovery of the use of the same module intwo different organisms. The latter discovery sug-gests that the module was present in the com-mon ancestor, but it does not show, for example,that the morphogenetic process in which it is usedwas also present in that common ancestor andregulated by the module in question (see alsoAbouheif et al., ’97).

It could be argued that it all started with theHox genes. The discovery that both flies and micenot only have similar Hox gene clusters, but alsothat they are ordered in the genome and expressedin the embryo in a similar manner, sent shock-waves through the developmental biology commu-

nity. What followed was a flurry of conclusions(not hypotheses or suggested experimental tests)that the deuterostome-protostome ancestor hadthe full complement of Hox genes expressed in anested anterior-posterior progression. Data onthree more phyla (for a total of 5 out of about 30)led Slack and colleagues (’93) to suggest that thisconserved expression of Hox genes is the “zootype”:the primary synapomorphy (shared, derived fea-ture) uniting the metazoa! Recently, De Robertis(’97) stated that the similar expression patternsof the engrailed gene in insects and cephalo-chordates “tells us that segmentation was presentin the common ancestor from which the insect andchordate lineages diverged 500 million years ago”(my emphasis). There is no indication that theseauthors ever considered the possibility that thesesimilarities might be due to parallel evolution.

Hox genes and primary axis specificationWhat is the appropriate test of the hypothesis

that nested expression of Hox genes defines themetazoan body plan? The Hox gene cluster shouldbe present in every metazoan, and the genesshould be expressed during development in anested orientation along the primary axis. Since1993, Hox gene expression patterns have been ex-amined in at least three other phyla: Cnidaria,Echinodermata and Urochordata (Fig. 1). Theexpression pattern of the only Hox gene (an ap-parent group 1/labial ortholog) that has been ex-amined in ascidians (a urochordate) is similar tothat of other chordates (Katsuyama et al., ’95).Hox genes are apparently not expressed at all dur-ing primary axis formation in the cnidarians thathave been examined, much less in nested sets (P.Cartwright, personal communication). As for echi-noderms, only two of the ten identified Hox genesin the sea urchin Strongylocentrotus purpuratusare expressed during embryonic development (Are-nas-Mena et al., ’98), and the functions of neitherare consistent with a role in positional identityalong the primary axis (Ishii et al., ’99). Hox genesare expressed at metamorphosis in cnidarians andechinoderms, but it is not clear if the genes areinvolved in axial patterning at these later stages.In either case, these results appear to contradictthe concept of the zootype. Still, it is possible thatthe protostome-deuterostome ancestor had an em-bryonic, nested expression of Hox genes and thatthis pattern was lost in the highly derived echi-noderm lineage. Clearly further studies with ad-ditional phyla are warranted (Fig. 1). Recentevidence suggesting that acoel flatworms occupy

2A particularly cogent example comes from a paper by Haerry andGehring (’97, p 12) in which they speculate that “Hoxa-4 may repre-sent a closer relative to the Drosophila Dfd gene than Hoxb-4, sincethe Hoxa-4 intron is functional in Drosophila, whereas that of Hoxb-4 is not.” This is essentially the same fallacious argument used toascribe to sonic hedgehog the status of closest relative of Drosophilahedgehog on the basis of both being involved in appendage develop-ment. The argument becomes circular when it is then concluded thatinsect and vertebrate limbs are homologous. I discuss two furtherexamples (tinman/Nkx2-5 and sine oculis/Six3) below.

4 J. HODIN

a phylogenetic position near the protostome-deu-terostome divergence (Ruiz-Trillo et al., ’99) makethem an especially useful taxa for examining suchissues.

Still, from the available data some interestingpatterns are apparent. For example, the nestedexpression of at least the posterior class of Hoxgenes may have been independently derived in thearthropod lineage. Averof and Akam (’95) foundthat the anterior borders of the Antp, Ubx, andabd-A expression domains all coincided in the first

thoracic segment of Artemia. In more derivedarthropods (including insects and other crusta-ceans), the expression of these posterior Hox genesis nested, with the canonical, nonoverlapping an-terior borders (Averof and Akam, ’95; Averof andPatel, ’97). While the ancestral state remainsequivocal until more basal arthropod species areexamined, the intriguing possibility is raised thatthere may be something about the organizationof Hox genes in a cluster that predisposes themto be expressed in a nested anterior-posterior pro-gression with respect to their chromosomal posi-tion (known as “colinearity”).3 Put simply, nestedHox gene expression may be a character subjectto parallel evolution, not only within the Arthro-poda, but indeed among the “higher” metazoa asa whole. This hypothesis is eminently testable:we simply need to examine more species. Recently,Abzhanov and Kauffman (’99) have examined thepatterns of expression of the anterior class Hoxgenes in the developing head of the isopod crus-tacean Porcellio scaber, and compared these pat-terns to the previously reported insect Hox geneexpression patterns. Surprisingly, while colinear-ity of the three head Hox genes examined wasstill observed (as it is in insects), different Hoxgenes were expressed in homologous segments.For example, while in insects, the maxillary andlabial mouth parts express the Hox gene pro-boscipedia, the homologous appendages in the iso-pod were found to express Sex combs reducedinstead. The authors concluded that arthropodhead segmentation must have evolved prior to thesegmental expression of the anterior class Hoxgenes. In this scenario, colinearity of anterior classHox gene expression in crustaceans and insectsevolved in parallel.

Furthermore, while colinearity of the anteriorborders of Hox gene expression is a fairly consis-tent pattern, the functions of these Hox genes donot always correspond to this pattern. For ex-ample, while the anterior borders of Hoxa7,Hoxb6, Hoxb7, and Hoxb9 expression in the mouseare at different axial positions, mutations in eachof these genes result in defects in the same verte-brae (Chen et al., ’98). As these authors note, simi-

Fig. 1. Metazoan phylogeny (after Ruppert and Barnes,’94; Philippe et al., ’94; Halanych et al., ’95; Aguinaldo etal., ’97; Ruiz-Trillo et al., ’99). Here I have represented ahodge-podge of these various phylogenetic hypotheses. Dif-ferent topologies yield similar conclusions. Underlined phylahave Hox genes expressed colinearly during primary axisformation, while the italicized phyla do not (see the text).In the other taxa (neither underlined nor italicized), Hoxgene expression patterns have not been reported. Examina-tion of Hox gene expression in phyla such as Chaetognatha,Rotifera, Priapulida, and Acoela would more fully test the“zootype” hypothesis (Slack et al., ’93). Solid bars denotephyla showing a metameric pattern of engrailed expression.Open bars denote phyla that do not show such a metamericengrailed pattern. The echinoderms are represented by anopen bar since the presumed ancestral condition is non-metameric expression of engrailed (see Fig. 2). Although chi-tons (Mollusca) show a metameric engrailed expressionpattern (Jacobs et al., ’94; Jacobs, personal communication),I consider the ancestral state to be equivocal for molluscs,in which metamerism is generally believed to be a derivedcondition (see the text). “P” and “D” denote the protostomeand deuterostome clades, respectively.

3There are two known counter-examples to colinearity. The C.elegans ceh-13 Hox gene is out of order with respect to chromosomalposition (reviewed in Bürglin and Ruvkun, ’93), but is still expressedand functions in the anterior of the animal (Brunschwig et al., ’99),as is expected from its sequence similarity to other anterior-classHox genes. The Hox-B1 gene in mice is a bona fide class 1 Hox geneand is ordered on the chromosome in the expected position, yet itsanterior border of expression lies posterior relative to that of Hox-A2and -B2 (reviewed in Krumlauf, ’93).

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lar results have been obtained from other Hoxgene knockouts. Again, the possibility arises thatHox genes are predisposed to be expressed coli-nearly, despite the fact that they do not alwaysfunction colinearly.

These studies on Hox gene expression patternsin metazoan embryos exemplify some shortcom-ings in the field of comparative developmentalbiology. For example, we currently have no satis-factory biochemical model to explain colinearity.Until we understand the mechanistic basis forcolinearity, it seems premature to conclude thatcolinearity in arthropod and chordate embryos isdue to shared history. The scenarios outlinedabove for the possible parallel evolution of poste-rior and anterior class Hox gene colinearity inarthropods represents another possibility. A secondcommonly encountered problem in comparativestudies is the assignment of molecular orthologiesto related genes in different phyla (I discuss sev-eral additional examples below). With the Hoxgenes in particular, it is often difficult to assignrelatedness among members of the vertebrate Hoxcluster and their counterparts in other phyla. Re-ports of gene cloning in development and cellbiology journals rarely include alignment compari-sons beyond the most similar gene and often uti-lize only the default alignment settings in the onealignment program of choice. Furthermore, differ-ent alignment programs and different alignmentparameters can yield trees with very different to-pologies. Thus it is extremely difficult to judge thebasis on which authors claim that such-and-sucha fly gene is the “closest homolog” of a mouse gene,claims that subsequently propagate through theliterature (see footnote 2). Relatedness among dif-ferent Hox genes is generally assigned on the ba-sis of similarity within the highly conserved“homeodomain,” the region responsible for DNAbinding. Consensus sequences for the assignmentof Hox genes to so-called “paralog groups” (relatedgenes that can be traced back to gene duplicationevents) are based on just a few nucleotides in thehomeodomain and adjacent regions (Sharkey etal., ’97). Needless to say, the greater the time sincetwo Hox genes diverged, the more difficult it is toassess relatedness. Finally, Avise points out (’94,p 12–13) that clusters of related genes are sub-ject to concerted evolution (unequal crossing overleading to greater sequence similarity betweenneighboring genes than one would predict basedon the time since gene duplication). Thus, ortholo-gous genes (which, by definition, diverged morerecently, at around the time the species being com-

pared diverged from one another) can end up be-ing less similar than genes within a cluster (whichformed long before the divergence of the speciesin question). In such instances, of which the Hoxgene cluster may represent such an example, as-signing “homology” to pairs of genes in differentphyla is at best a questionable exercise.

Engrailed and segmentation: homologyor homoplasy?

Of the three phyla from which we have signifi-cant functional data on developmental genes(nematodes, arthropods, and chordates), two ofthem are characterized by overt segmentation.Segmentation in the strictest sense (“a precise anddefinite repetition of all [mesodermal] structuresin each [body region]”; Willmer, ’90, p 40) prob-ably only applies to annelids, arthropods and chor-dates, while metamerism (serial repetition of somebody structures) is more widespread in the Meta-zoa. It is found, for example, in turbellarian flat-worm guts, nemertine gonads, rotifers, strobilizingcnidarian medusae, much of the Vendian fauna,brittle star and chiton body plates, and tripartitelophophorates, chaetognaths and pterobranchs.According to the suggestion of De Robertis (seeabove and ’97), these disparate cases of metamer-ism might all (with the exception of cnidarians andthe Vendian fauna) be variations on a segmentedbody plan already present in the protostome-deu-terostome ancestor. Therefore, segmentation inchordates and arthropods are considered homolo-gous. This implies that segmentation (indeed anytrace of metamerism) has been lost numeroustimes in the evolution of “higher” metazoans buthas been retained in the chordate and arthropodlineages. Again, the evidence presented for this“ancient segmentation” view is that the engrailedgene in annelids, arthropods and lower chordatesis expressed in segmentally-iterated stripes (Hol-land et al., ’97; Fig. 1).

These data suggest a hypothesis that segmen-tation is the ancestral condition (ancient segmen-tation) and a null hypothesis of parallel evolution.So how do we go about testing this hypothesis?Without any paleontological data, this hypothesisis very difficult to test directly. I propose that welook to the more widespread occurrence of meta-merism among metazoans as an evolutionarymodel. Are independently evolved metamericstructures built using similar (parallel) or differ-ent (convergent) developmental pathways? To an-swer this question one should: (1) establish viaan independent phylogeny that metamerism in the

6 J. HODIN

group of interest is likely to be a derived condi-tion; (2) examine the developmental expression ofengrailed in the group of interest, as well as in anon-metameric outgroup; and (3) compare theseresults to engrailed expression patterns in ceph-alochordate and insect embryos. Absence of astriped engrailed expression pattern in the groupof interest tells you very little, but a striped pat-tern suggests that engrailed is a character sub-ject to parallel recruitment in a similar process(metameric organization), thus casting doubt onthe ancient segmentation hypothesis.

Let us examine the occurrence of metamerismin the arm plates of brittle stars, which is almostcertainly a derived condition within the echino-derms (from phylogenetic as well as from paleon-tological data; Ruppert and Barnes, ’94). Althoughthere is no striped pattern of engrailed during de-velopment in sand dollars and starfish (C. Lowe,personal communication), brittle stars show an ob-vious striped pattern of engrailed expression dur-ing arm growth (Lowe and Wray, ’97; Fig. 2). Theseresults suggest that a striped pattern of engrailedmay have evolved independently within echino-derms in the brittle stars, suggesting that expres-sion of engrailed in stripes is not always evidenceof shared history (though this conclusion would bebolstered if engrailed in crinoids is shown not tobe expressed in stripes; see Fig. 2). Similar cepha-lochordate and arthropod engrailed patterns mightalso be due to parallel evolution. Furthermore, Chi-

ton embryos show a striped engrailed expressionpattern (Jacobs et al., ’94, personal communication),and metamerism in chitons is generally believedto be a derived condition (Kozloff, ’90; Willmer, ’90;Ruppert and Barnes, ’94).

Interestingly engrailed expression is also de-tected within the nervous system in brittle stars(Lowe and Wray, ’97), as well as in arthropods,annelids, and chordates. This suggests a possibleexplanation for why one might expect parallel evo-lution of striped engrailed patterns. Generally,metameric body structures contain iterations ofsubsets of neurons. This is true in annelid andarthropod segments (Snodgrass, ’38) as well asbrittle star arms (Lowe and Wray, ’97). So, ifengrailed was expressed in these nervous systemsancestrally, one would expect to then find ametameric organization of engrailed in derived,metameric nervous systems. This would putengrailed in the right place (in a segmentally it-erated pattern) at the right time (during the evo-lutionary elaboration of segmentally repeatedmesodermal structures) to be co-opted for use inthe specification of other, associated metamericstructures (such as mesodermal tissues). In thisview, the ancestral condition is engrailed expres-sion in nervous systems, and later, parallel co-option of engrailed for specification of metamericstructures. Again, this hypothesis is testable byexamining the expression of engrailed in addi-tional metameric and non-metameric organisms.

In summary, I concur with Abouheif et al. (’97)that without placing evolutionary-developmentalcomparisons in an explicitly phylogenetic context,it is impossible to distinguish between homologyand homoplasy. Furthermore, the general assump-tion that such similarities are due to homology iscreating a bias in research programs in this rela-tively young field.

PART II: PARALLEL (ANDCONVERGENT) NETWORKS

Pax6 in protostomes and deuterostomes:parallelism or homology?

One of the classic examples of convergence in themetazoa is the evolution of the complex eye. Be-cause the ultrastructure and developmental trajec-tories of invertebrate and vertebrate eyes are sodifferent, the independent evolution of these struc-tures had been widely accepted. Therefore, it wassurprising to find that the Pax6 gene is a key regu-lator of eye development in both mice and fruit flies(Quiring et al., ’94). These results led Quiring et al.

Fig. 2. Echinoderm phylogeny (Smith, ’97) showing therelationships among the five extant classes. Ophiuroids areunique among the echinoderms in having strongly metamericarm skeletons (often called “vertebrae”; Ruppert and Barnes,’94). Chris Lowe (personal communication) examined en-grailed expression in echinoderms from three of these classesand found that a metameric pattern of engrailed expressionwas present in the arms of ophiuroids (solid bar), but not inechinoids or asteroids (open bars).

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(’94) and others to propose that the vertebrate andinvertebrate eyes are, indeed, homologous. Exami-nation of the expression patterns of Pax6 in a widevariety of invertebrate embryos showed that Pax6expression always correlated with eye development(reviewed in Callaerts et al., ’97).

This example really brings into focus the prob-lems encountered with the use of the word “homol-ogy” to describe both molecules and morphology.Yes, the “homologous gene” (properly, the “ortho-logous gene”) is used to build both the fly and themouse eye. But are they used in the same way?The appropriate way to address this question isnot to see if the mouse gene works in fly eye de-velopment. The mouse gene was found to regu-late eye development in Drosophila (Halder et al.,’95), yet there is a functional Pax6 in C. elegans,an organism that lacks eyes altogether (see be-low). Based upon its sequence similarity, I wagerthat C. elegans Pax6 would also work in fly eyes.A positive result tells you only that the biochemi-cal properties of the protein have been conserved,not necessarily that its function within a certainmorphological structure has also been conserved.The commonplace use of the same gene within anorganism performing distinct functions in a mul-titude of tissues reveals why this experiment isgenerally uninformative with respect to evolution-ary history (see also Abouheif et al., ’97). Instead,one way to address the potential similarity infunction of fly and mouse Pax6 is to examine theblack box in between transcription factor and mor-phological structure.

There are two ways to account for the similar-ity in Pax6 expression in invertebrate and verte-brate eyes. In the first scenario (which I call the“ancient eye” scenario), the protostome-deuteros-tome ancestor had an eye of sorts under the con-trol of Pax6 (as, for example, a regulator of alight-sensitive, proto-rhodopsin gene), and thisassociation continued and was reinforced as therespective eyes increased in complexity. In the sec-ond scenario (which I call the “parallel recruit-ment” scenario), the Pax6 regulatory network wasindependently recruited to function in invertebrateand vertebrate eye development. If the only de-scribed function of Pax6 was in eye development,then the ancient eye scenario seems fairly likely.This is not the case: Pax6 is also involved in brain,nose, and pancreas development in mice (reviewedin Dahl et al., ’97) and head and sensory neurondevelopment in C. elegans (Chisholm and Horvitz,’95; Zhang and Emmons, ’95). Different Pax6 al-leles in flies show embryonic, larval or pupal

lethality, head defects, and supernumerary anten-nae in addition to eye defects (Shatoury, ’63; re-viewed in Harris, ’97). In fact, if metazoan Pax6expression patterns are considered as a whole, in-volvement in the development of anterior struc-tures is probably as accurate a characterizationof similarity as involvement in eye development.

In 1994, Quiring and colleagues proposed thatPax6 may be the master regulator of eye develop-ment in mice and flies, and perhaps in all meta-zoans. The justification for bestowing this titleupon Pax6 was the finding that ectopic expres-sion of the mouse or fly Pax6 gene in, for example,the fly leg primordium results in an ectopic eyeon the adult leg. It seems to me that the conceptthat there is a master regulator of eye develop-ment at all predisposes us to think of metazoaneye development as representing one conserveddevelopmental program. First, homozygous nullmutations in mouse Pax6 do not block formationof the optic cup, suggesting that there is some-thing that acts earlier than Pax6 in mouse eyedevelopment. Furthermore, ectopic expression ofPax6 is not the only way to produce ectopic eyesin flies. Ectopic expression of eyes absent (Boniniet al., ’97) and dachshund (Shen and Mardon, ’97)also have this effect (and like good members of agene network, dachshund and eyes absent havealso been shown to regulate Pax6 expression, andvice-versa: Halder et al., ’95; Bonini et al., ’97;Shen and Mardon, ’97). Also, a mouse sine oculisfamily member, Six3, can induce ectopic eyes infish (Oliver et al., ’96). So, if Pax6 is not the mas-ter regulator of eye development, what does it do?It does regulate vertebrate lens crystallin gene ex-pression (reviewed in Cvekl and Piatigorsky, ’96).Also, Sheng et al. (’97) showed that Pax6 directlyregulates rhodopsin 1 expression in Drosophilaphotoreceptor cells and that the promoter ele-ments that bind Pax6 are also found in some ver-tebrate opsin regulatory regions. This suggests aplausible scenario for the function of the Pax6regulatory network in eye development. The Pax6/dachshund/eyes-absent/sine oculis regulatory net-work may have been involved originally in speci-fication of various anterior structures in primitivemetazoans, and since it was in the right place, itwas recruited to regulate the expression of struc-tural genes in the eye (such as lens crystallins,and perhaps opsins) in invertebrates and verte-brates independently (“right place at the righttime” hypothesis; see below). Uncovering the na-ture of this regulatory network, as well as itsdownstream targets, should allow us to distin-

8 J. HODIN

guish between the parallel recruitment and an-cient eye scenarios.

Recent data on the functions of sine oculis fam-ily members may support the parallel recruitmentscenario. Oliver and colleagues (’95a,b) foundthree mouse genes with close sequence similar-ity to sine oculis, which they named Six1, 2, and3. Six3 is expressed in the developing eye andother anterior structures, while Six1 and 2 areexpressed in other anterior structures but not inthe developing eye. Of these three genes, Six3 hasthe lowest percent sequence similarity with sineoculis, yet Oliver et al. (’95b) conclude that Six3is the “functional homolog” of sine oculis sinceboth are expressed during eye development. As itturns out, there is another Drosophila gene inthis family called optix which is also expressedduring fly eye and head development. Sequencecomparisons with the Six genes revealed thatoptix, not sine oculis, is the Six3 ortholog in flies(Toy et al., ’98). However, this still leaves us withthe unsavory situation that sine oculis is involvedin eye development while its mouse orthologs(Six1 and 2) are not. The question is: what wasthe role of sine oculis in the protostome-deuteros-tome ancestor? The most obvious answer is that,like all the members of this family, it was prob-ably involved in anterior development and laterwas recruited into the development of the fly, butnot the mouse, eye.

If the ancient eye scenario is correct, it tells usthat when you have a system that works, you con-serve it. The parallel recruitment scenario, by con-trast, offers us a model for understanding howcomplex structures are built in multicellular or-ganisms. If squid, flies, and mice have all comeup with the same solution to the same problemindependently, this suggests that metazoan eyedevelopment is “constrained” in some fashion. Thiscould be for two (not necessarily mutually exclu-sive) reasons. First, if we hypothesize an eyelessmetazoan in which these genes were already ex-pressed in the head, they would be in the rightplace (the head) at the right time (namely, thetime at which eyes evolved in the respective lin-eages) to be recruited for an additional role in eyedevelopment (as described above). Alternatively,there may be something about the biochemicalnature of the Pax6 network that makes it par-ticularly good at regulating the inductive inter-actions characteristic of both vertebrate andinvertebrate eye development. Once again, our un-derstanding of the biochemistry of this system isfar too rudimentary to be able to evaluate this

possibility. Developmental constraint in this con-text refers to the predisposition of the develop-mental system to utilize the Pax6 network in eyeevolution. Put another way, constraint in devel-opmental programs (such as the Pax6 network)may be especially likely to lead to parallelisms inmorphological evolution.

Developmental constraints andparallel evolution

I believe that the clearest definition of develop-mental constraints was proposed by MaynardSmith et al. (’85) as a bias in the production ofphenotypic variation due to ontogenetic factors.Wagner and Misof (’93) noted that “constraint” canmake itself evident at the level of the phenotypiccharacter (morphostatic constraints) or the under-lying developmental processes (generative con-straints). Morphostatic constraints generallyimply variability in generative processes and vice-versa. Von Dassow and Munro (’99) provide a use-ful example. Different insects utilize distinctgenerative processes to specify segmental identityduring embryogenesis. In the short-germ insects,typified by Drosophila melanogaster, all the seg-ments are specified nearly simultaneously in anoncellular, syncitial environment (reviewed inLawrence, ’92). By contrast, in the long-germ in-sects, typified by the grasshopper Schistocerca, thesegments are specified in a gradual anterior-to-posterior progression in a cellular environment(reviewed in Patel, ’94; Tautz et al., ’94). Still, bothprocesses eventually generate very similar-look-ing segmented embryos, with the same numbersof head, thoracic and abdominal segments. InWagner and Misof ’s terminology, substantialvariation in developmental (generative) processesunderlie the production of a constrained segmen-tal (morphostatic) phenotype.

On the other hand, variation in the axial posi-tion and morphology of insect appendages (mor-phostatic variability, such as the number of wingsand wing-like structures in different insect groups)is associated with tight conservation of Hox geneexpression patterns (a possible generative con-straint). I believe that the explanation for thesefindings is that constraint at each level underliesthe essential modularity of development. In thecase of morphostatic constraints, a phenotypiccharacter is maintained despite variations in thetrajectories of development, such as heterochro-nies or canalization in the face of environmentalheterogeneity and the production of segmentedinsect embryos. In the case of generative con-

PLASTICITY AND CONSTRAINTS 9

straints, conserved developmental pathways areutilized for the production of different phenotypiccharacters, such as Pax6 function in the verte-brate pancreas and the fly eye and appendagevariability in insects.

Although developmental constraints are ofteninvoked to explain biases in the patterns of mor-phological variation, in practice it is far fromstraightforward to determine if the lack of vari-ability in a morphological feature is due to con-straints or, for example, stabilizing selection onthat morphology (see Maynard-Smith et al., ’85).The presumed explanation for this difficulty is thegeneral lack of knowledge about the mechanisticbases for supposed constraints. Recently, however,some work on trade-offs (van Noordwijk and deJong, ’86; Houle, ’91; de Jong and van Noordwijk,’92) in insect development has shed some light ona potential mechanism for constraints. Nijhoutand Emlen (’98) and Klingenberg and Nijhout (’98)noticed trade-offs in the growth of morphologicalfeatures in horned scarab beetles and buckeye but-terflies. The beetles have two distinct malemorphotypes: larger beetles have long horns andsmall eyes while smaller beetles have short hornsand large eyes. Selection for beetle lines with longor short horns always yielded beetles with, respec-tively, decreased and increased eye size. Further-more, the application of juvenile hormone (JH)during larval development (which is known to con-trol the difference between the two morphotypes)results in short-horned beetles developing a largerbody size. These beetles also develop large eyes,while females (which never produce horns) showno significant change in eye size with JH treat-ment. In these experiments, the sizes of other bodyparts were not affected. It seems that eye size andhorn size are truly (and negatively) coupled. Ex-periments with the buckeye butterfly showed thatremoval of the hindwing primordium in a cater-pillar resulted in an adult with significantly in-creased weights of the forewing, thorax, andforeleg. Again, other body parts were unaffected.Interestingly, the increased forewing and forelegweights were only apparent on the same side ofthe animal as the hindwing removal: the forew-ing and foreleg on the opposite side of the animalwere essentially unaffected (controls demonstratedthat this was not simply due to surgery). Thesefindings led the authors to conclude that there iscompetition for a local resource in these insect lar-vae, a form of internal trade-offs. Alterations inthe size of one body part appear to be constrainedby correlated alterations in the size of nearby body

parts. Such constraints apparently influenceevolvability (evolutionary potential; see below)since horn size is quite variable among relatedhorned beetle species, but is always negatively cor-related with eye size (D. Emlen, personal com-munication). Wagner et al. (’97) also discuss anexample of mechanisms of constraints with re-spect to digit formation in salamanders (see alsoWilkins, ’98; von Dassow and Munro, ’99).

The “right place at the right time”hypothesis, heart evolution, and

developmental constraintsAlthough morphologically quite distinct, devel-

opment of both the vertebrate and fly heart isregulated by members of the NK-2 family of genes:tinman in flies and Nkx2–5 (among others) inmice. This finding has led to the now popular no-tion that the protostome-deuterostome ancestorhad a NK-2–regulated heart, which contrasts withthe traditional view of convergence between thesetwo organs (e.g., Beklemishev, ’69). Nkx2–5 andtinman are first expressed broadly in the visceralmesoderm and only later restricted to cardiac me-soderm. Ranganayakulu and colleagues (’98) setout to test whether the roles of NK-2 genes inflies and mice are due to homology or to parallelevolution by substituting the mouse gene for thefly gene in transgenic flies. Surprisingly, while themouse gene rescued the visceral mesoderm defectsfound in tinman mutants, it failed to rescue theheart defects! Furthermore, they mapped the do-main of the tinman protein responsible for theheart function to a small region at the N-termi-nus, a region of the protein that shows no detect-able amino acid similarity with Nkx2–5. Thesefindings suggest that while one of the ancestralroles of NK-2 genes in the protostome-deuteros-tome ancestor may have been in visceral meso-derm development, Nkx2–5 and tinman appear tohave been independently co-opted for heart de-velopment in the two lineages, an example of par-allel evolution.4 Further evidence to support thisidea comes from sequence comparisons of Nkx2–5and tinman. Here, again, as was the case for theSix genes discussed above, we find a situation inwhich the true orthologs (the genes showing thehighest sequence similarity to one another) do notappear to share the same role. All the NK-2 genes

4Of course, the formal possibility exists that the control sequencesfor heart within the two proteins have diverged enough (from a com-mon ancestor’s control sequence) to make it unrecognizable not onlyby DNA alignment methodology, but also by the functional analysisdescribed above.

10 J. HODIN

involved in vertebrate heart development are ac-tually closer relatives of the fly ventral nervoussystem defective and bagpipe genes (Rangan-ayakulu et al., ’98), which function in the nervoussystem and visceral mesoderm, respectively.

In this parallel recruitment scenario for heartevolution, the “developmental constraint” is anexample of the NK-2 genes being expressed in the“right place at the right time” (i.e., in the vis-ceral mesoderm during the time that heartsevolved) in both the protostome (e.g., insect) anddeuterostome (e.g., vertebrate) lineages. Metazoanhearts arise from visceral mesoderm, and NK-2family genes were probably involved ancestrallyin the determination of these tissues. This shouldbe considered a bona fide constraint on heart evo-lution, since it certainly represents a “bias [in]the production of variant phenotypes caused bythe…dynamics of the developmental system”(Maynard-Smith et al., ’85, p 266). Furthermore,the reiterated use of a gene first in, say, a field ofcells, and later in only a subset of the progeny ofthose cells is commonplace in animal development[e.g., Distal-less in proximo-distal axis specifica-tion in insect appendages early, and later in thedifferentiation of distal pattern elements in thebutterfly wing (Carroll et al., ’94; see below); therole of the C. elegans Hox gene mab-5 in severaldistinct steps (with distinct functions) in male tailmorphogenesis (Salser and Kenyon, ’96)].

A biochemical analog of the “right place at theright time” scenario appears to hold for nuclearhormones receptors and their ligands. It appearsthat in at least two instances, distantly relatedmembers of the nuclear hormone receptor super-family have acquired the ability to bind chemi-cally similar ligands. The insect ecdysone receptorand its relatives bind steroids (Kliewer et al., ’99),but are in a different sub-clade than the verte-brate steroid receptors; the vertebrate retinoid-Xreceptor and retinoic acid receptor are distantrelatives, but both bind 9-cis-retinoic acid (Escrivaet al., ’97; Laudet, ’97). These might be examplesof “right ligand binding region configuration at theright time” and are probably examples of parallelevolution at the level of three-dimensional pro-tein structure (see also, e.g., Govindarajan andGoldstein, ’96; Graumann and Marahiel, ’96). Thisphenomenon, whereby unrelated or distantly re-lated genes have evolved similar biochemical ac-tivity independently, has recently been referredto as “non-orthologous gene displacement,” andwas shown to be quite common in a comparisonof the proteins encoded by the fully sequenced ge-

nomes of two species of bacteria, Haemophiliusinfluenzae and Mycoplasma genitalium (Kooninet al., ’96).5 For example, RNase H activity is con-ferred by a typical RNase H ortholog in H.influenzae, while an unrelated protein with simi-larity to the 5´-3´ exonuclease domain of DNApolymerase I performs this role in M. genitalium.

I believe the tinman story is only the tip of theiceberg. Examples of parallel evolution are com-monplace, at least among the limited instancesin which people have explicitly looked for them.Although there is substantial precedence for theconcept that the same genes and proteins are uti-lized repeatedly in morphological evolution (Jacob,’77), it is possible that current evolutionary theory,with its emphasis on parsimony, may not be wellequipped to deal with the numerous findings ofparallellisms (and convergence) in morphologicalevolution. Here I cite some of the examples that Ihave come across in my search of the literature,as well as an example from my own work on ova-rian development in insects. I discuss these ex-amples because in each case, the finding ofparallel evolution may indicate constraints onmorphological evolution. Identifying the nature ofthese constraints may offer insights into the evo-lutionary potential (“evolvability”) of these lin-eages (see below).

Morphogenetic hormones anddevelopmental constraints

Mangroves are a polyphyletic assemblage, withthe mangrove phenotype having evolved indepen-dently in 16 families (Tomlinson, ’86; Juncosa andTomlinson, ’88; Chase et al., ’93), six of which showvivipary (Tomlinson, ’86). Farnsworth and Farrant(’98) performed an outgroup comparison of man-groves and non-mangroves in four of the six vi-viparous families, monitoring the relationshipbetween abscisic acid (ABA) levels and the evolu-tion of vivipary and the corresponding loss of seeddormancy. ABA has been shown to be involved inthe desiccation tolerance of dormant seeds as wellas in plant responses to flooding and high salin-ity (reviewed in Kermode, ’95). While one non-vi-viparous mangrove (Sonneratia alba) showedlevels of embryonic ABA more akin to its non-man-grove sister group, each of the independentlyevolved viviparous mangrove species showed re-

5Eleven such cases were found, as compared to the 233 proteinpairs shown to be orthologous between the two species. Extrapolat-ing, the authors predict that such cases in animal genomes shouldnumber in the hundreds. Cases of both parallelisms and convergenceare grouped in this analysis.

PLASTICITY AND CONSTRAINTS 11

duced levels of embryonic ABA. Therefore, thesefour independent cases of evolution of viviparousmangroves appear to include a common mecha-nistic basis, as parallel hormonal modifications inthe embryo have evolved in concert with the evo-lution of vivipary.

In insects, a unique type of vivipary is found insome species of gall midges (dipterans from thefamily Cecidomyiidae), which have evolved facul-tative larval reproduction (called “paedogenesis”)at least two times independently (Jaschof, ’98;Hodin and Riddiford, submitted). In both taxa,paedogenesis involves the precocious differentia-tion of the ovary in early-stage larvae (reviewedin Went, ’79). The resulting oocytes undergo par-thenogenetic activation, and the embryos developin the hemocoel (open body cavity) of the motherlarva. Ultimately the larvae hatch, consume themother from the inside and burst out of the emptycuticle. The newly emerged larvae repeat the pae-dogenetic life cycle if resources are abundant, butdelay ovarian differentiation and metamorphoseinto a winged adult under poor food conditions.In two of the species with independently evolvedpaedogenesis (Heteropeza pygmaea and Mycophilaspeyeri), precocious ovarian differentiation is as-sociated with up-regulation of the Ecdysone Re-ceptor (EcR) and Ultraspiracle (USP) proteinswithin 12 hours after larval birth (Hodin andRiddiford, submitted). EcR and USP heterodi-merize to form the functional receptor for 20-hydroxyecdysone (Yao et al., ’93), which is criticalfor molting and metamorphosis in insects. InDrosophila melanogaster, the ovary lacks both EcRand USP during most of larval life, then early inthe final instar both appear and ovarian differen-tiation begins (Hodin and Riddiford, ’98). Simi-larly, in both paedogenetic gall midge species,larvae that are destined to metamorphose (poorfood conditions) express only low levels of EcR andUSP in the ovarian primordium until metamor-phosis. Thus the ontogenetic mechanisms under-lying paedogenesis (precocious activation of EcRand USP) seem to have evolved in parallel withinthe gall midges. Since ecdysteroids appear at everymolt in insects, heterochronies in tissue differen-tiation in insects may commonly be accomplishedby changes in the timing of EcR/USP expression.In other words, the use of the ecdysteroid-recep-tor system to evolve heterochronic changes mayrepresent a local evolutionary optimum (SewallWright, ’32): in this case, a simple developmentalswitch may cause a macroevolutionary change inmorphology and life history. It is the modular na-

ture of adult organ formation in metamorphosinginsects that would make this scenario possible.

The previous two examples, viviparous man-groves and paedogenetic insects, have major evo-lutionary alterations in life history patterns incommon that involve modifications in hormonalsystems. The evolution of neoteny in salamanders(Frieden, ’81; Yaoita and Brown, ’90) and directdevelopment in frogs (Hanken et al., ’97; Jenningsand Hanken, ’98) and possibly sea urchins (Saitoet al., ’98) similarly involve hormonal modifica-tions. Perhaps this is not surprising. Since lifehistory transformations in a wide variety of mul-ticellular organisms tend to be regulated by hor-mones, modifications in hormone release and/orthe cellular response to hormones are a likely fo-cus for evolutionary change in these systems (seealso Matsuda, ’82; Nijhout, ’99). Furthermore, Ibelieve that these would appropriately be consid-ered “constraints” on life history evolution, as evo-lutionary patterns are certainly biased in theseinstances.

Close ecological associations andparallel evolution

Close interactions between organisms can oftenlead to reiterated evolutionary patterns and mayhence indicate constraints. Becerra (’97) andBecerra and Venable (’99) investigated the chemi-cal bases of the shifts in host plant use by fleabeetles (genus Blepharida) on their host plants,New World frankincense and myrrh (genus Bur-sera). Burseras use a variety of terpenes as chemi-cal defenses, and distantly related burseras seemto have evolved the use of certain types of terpe-nes independently. Consequently, some beetle spe-cies that ancestrally appear to have exploited onlyone sub-clade of Bursera can colonize new plantsthat fortuitously (for the beetles) produce a simi-lar class of terpenes. In this case, parallel evolu-tion of terpene production within the burseras canexplain macroevolutionary trends within the ble-pharid flea beetles. The distribution of flea beetlesis to some degree constrained by the evolutionarypatterns in terpene production in burseras.

Endoparasitism has evolved in hymenopteranwasps at least eight times independently from ec-toparasitic ancestors (reviewed in Strand andGrbic, ’97) and in each case is associated with aremarkably similar suite of developmental modi-fications. Early embryogenesis in hymenopterans(as exemplified by honeybees; DuPraw, ’67; Fleig,’90; Fleig et al., ’92; Binner and Sander, ’97) gen-erally follows the Drosophila pattern (reviewed in

12 J. HODIN

Lawrence, ’92): a large yolky egg, syncytial earlynuclear divisions, cytokinesis at the “cellular blas-toderm” stage, and segmental patterning via a cas-cade of regulatory interactions from pair rule tosegment polarity to homeotic genes (maternal andgap genes have been difficult to find outside higherDiptera, but see Wolff et al., ’98). Ectoparasitichymenopterans also appear to share the canoni-cal Drosophila pattern (Grbic and Strand, ’98).Many endoparasites, however, have undergoneradical shifts in the patterns of early development.Most endoparasites develop from tiny eggs withlittle or no yolk and cellularize early (Strand andGrbic, ’97; M. Strand, personal communication).These parallel modifications are likely related tothe fact that endoparasitic embryos take up nu-trients from the fluids of the host and can there-fore develop into sizable larvae from small eggs.Early cellularization might also be a function ofsmall egg size. Most strikingly, in the two inde-pendently evolved endoparasites that have beenexamined for expression of patterning genes, bothappear to have lost pair rule gene functions, andthe earliest evidence of the segmentation cascadeis the expression of segment polarity genes (Grbicand Strand, ’98; Grbic et al., ’98). A similar situa-tion appears to hold in so-called “short-germ” in-sects (such as in grasshoppers; reviewed in Patel,’94; Tautz et al., ’94), in which segments are addedprogressively throughout embryogenesis. Perhapsthe more complex segmentation hierarchy is onlyrequired when patterning “long-germ” embryos(typified by Drosophila), in which all the segmentsare specified during a very short developmentalperiod. Unlike some of the previous examples, theparallel alterations in early embryogenesis in en-doparasites seem to indicate a relaxation of con-straints on early embryogenesis in each of theselineages, leading to parallel losses of developmen-tal processes in these insects.

Evolvability and developmental constraintsWagner and Altenberg (’96, p 970) define evolv-

ability (evolutionary potential) as “the genome’sability to produce adaptive variants when actedon by the genetic system.” Developmental con-straints can influence evolvability either by hin-dering the ability of a genome to produce a givenadaptive variant or by predisposing genomes toproduce a given variant by a defined mechanisticroute (which, I argue, may likely lead to parallelevolution of this adaptive variant in related lin-eages). As a possible example of the latter, let usconsider the evolutionary potential of bacterial β-

galactosidase. Although bacteria do not undergodevelopment per sé, a recent study by Hall andMalik (’98) offers some insights into evolvability,and thus developmental constraints. An E. coligene known as “evolved β-galactosidase” (Ebg) isessentially incapable of effectively utilizing β-ga-lactoside sugars. Extensive mutagenesis hasshown that only mutations causing amino acid re-placements at two of the 15 active site residuesof Ebg can restore effective function. Furthermore,a phylogenetic comparison of 13 related bacterialβ-galactosidase genes revealed that the β-galac-tosidase consensus differs from Ebg only in thesesame two active site residues. It seems that theevolutionary potential of Ebg to evolve into a func-tional β-galactosidase is limited to these twoamino acid replacements. This may well be an ex-ample of a local optimum; perhaps other activeconformations are possible but may involve toomany intermediate evolutionary steps to make theevolutionary jump likely. Alternatively, there maysimply be no possible alternate configurations. Ineither case, this seems to be a nice biochemicaldemonstration of an evolutionary constraint. Ananalogous situation may explain the remarkablesimilarity between the independently evolved anti-freeze glycoproteins in Antarctic notothenoid fishand Arctic cod. Although both proteins are char-acterized by Thr-Ala-Ala amino acid repeats, thenotothenoid gene evolved from an ancestral trypsi-nogen gene (Chen et al., ’97a), while the cod geneis completely unrelated to trypsinogens (Chen etal., ’97b). Therefore, the similar functional require-ments in the two fish have led to the entirely in-dependent evolution of Thr-Ala-Ala–containingproteins. Like the Ebg gene example, either thebiochemical options for this particular functionalrole (antifreeze) are quite limited, or it is rela-tively easy (perhaps due to codon bias) to evolveThr-Ala-Ala repeats.

Occasionally parallel evolution may simply bethe result of chance. I believe that the use of al-dehyde dehydrogenase as a lens crystallin in squidand elephant shrews represents such a case(Tomarev et al., ’91; Wistow and Kim, ’91; Gra-ham et al., ’96). There are many different meta-bolic enzymes utilized as lens crystallins indifferent taxa, but the set of possible proteins isprobably somewhat limited. Presumably squid andelephant shrews just happened to come up withthe same solution. Yet some cases of multiple simi-lar transformations within a single lineage can-not be explained as chance occurrences. Skeletalchanges in plethodontid salamanders (Wake, ’91),

PLASTICITY AND CONSTRAINTS 13

cleavage modifications in direct developing echi-noids (Wray and Bely, ’94), and extreme dimor-phism in male fig wasps (Cook et al., ’97) are afew striking examples. Possibly, each of the simi-larly transformed lineages was responding to simi-lar selection pressures and either evolved differentsolutions to the same problem (convergence) orevolved the same solutions (parallelism). If thesolutions are convergent, then developmental con-straints are probably not acting on the structurein question. By contrast, if the solutions repre-sent parallelisms, developmental constraints maybe involved.

Still, developmental constraint is not the onlyexplanation for multiple parallel transformationswithin a lineage. A purely adaptationist explana-tion is also possible: namely, the parallel trans-formations in question may represent the bestpossible solutions in the presence of similar se-lection pressures.

Developmental constraints in practiceSo how can developmental constraints be posi-

tively identified? If different types of developmen-tal perturbations (teratogens, environmentalshifts, transgenics) tend to produce a similar setof phenotypes, developmental constraints may beinvolved. The development of the vertebrate skel-etal axis is a nice example. To date, about 50% ofthe mouse Hox genes have been knocked out, andsome patterns are beginning to emerge. Skeletaldefects appear to be concentrated at certain “hotspots” (Chen and Capecchi, ’97), notably theboundaries between different vertebral types (e.g.,shifts in the lumbo-sacral border). In addition, seg-mental fusions (e.g., rib fusions) appear repeat-edly. Interestingly, Russell (’56) noted very similarresults for mouse embryos exposed to X-rays whileundergoing organogenesis (6.5–12.5 days after fer-tilization). Irradiations at earlier and later stageshad little or no effect on skeletogenesis, suggest-ing that the explanation for the similarity betweenX-irradiation and Hox mutants is not as simpleas representing X-ray–induced lesions in Hoxgenes. An analogous situation occurs with Droso-phila Hox mutations at the bithorax locus, inwhich the haltere-bearing segment (T3) is trans-formed to a wing-bearing segment (T2). Eithertemperature shock or ether application to embryosduring a critical period around the time of blasto-derm formation yields bithorax phenocopies (Cap-devila and Garcia-Bellido, ’74; and referencestherein; see also below).

My interpretation of these data is that axial pat-

terning in both flies and mice is a process subjectto developmental constraints and that these con-straints are manifest by a limited subset of axialdefects following diverse perturbations. The im-plicit prediction is that the vertebrate and arthro-pod lineages are each characterized by parallelvariations in axial morphology. Recent data fromAverof and Akam (’95) and Averof and Patel (’97)on Hox gene expression domains in various crus-taceans suggest that the extreme variation in crus-tacean appendages overlays a stereotyped patternof variations in Hox gene expression. How thesedifferences fall out phylogenetically is not clear,so the jury is still out on arthropod axial parallel-isms. Interestingly, a very similar scenario has un-folded during vertebrate axial evolution. Forexample, while the transition between the cervi-cal and thoracic vertebrae occurs at widely diver-gent axial positions across vertebrates, the anteriorborder of the Hoxc-6 expression domain is at thesomite level corresponding to this morphologicaltransition in all cases examined to date (frog, fish,mouse and birds; Gaunt, ’94; Burke et al., ’95).

Since the seemingly parallel alterations in axialevolution in both arthropods and vertebrates aremirrored in the transgenic and developmental per-turbation experiments cited above, I conclude thataxial evolution in these lineages is to some de-gree constrained by the expression patterns andfunctions of Hox genes.

Phenotypic plasticity andconvergent evolution

Convergence is a widespread phenomenon inboth plant (reviewed in Niklas, ’97) and animal(reviewed in Moore and Wilmer, ’97; Conway Mor-ris, ’98) evolution. Yet very few studies have ex-plicitly examined the developmental mechanismsunderlying the production of supposedly conver-gent structures. In a landmark study of themechanisms of homoplasy in salamanders, Wake(’91) identified several cases in which superficiallysimilar morphologies were produced by very dif-ferent developmental trajectories, including theindependent acquisition of vertebral joints bythree distinct mechanisms and the evolution ofvertebral elongation in tropical salamanders byeither the addition of vertebrae (as in Oedipina)or by elongation of individual vertebrae (as inLineatriton). Such instances of convergence withinrelatively closely related taxa indicate that theunderlying developmental system is somewhatflexible, in contrast to the developmental con-straints indicative of parallelisms. If we take this

14 J. HODIN

one step further, developmental mechanismsshown to be convergent may indicate flexibilityin developmental trajectories within a genotype,a phenomenon known as phenotypic plasticity (en-vironmentally based phenotypic differences withina genotype; reviewed in Schlichting and Pigliucci,’98). Turning this argument around, I hypothesizenot only that organisms exhibiting phenotypicplasticity for a trait tend to express interspecificvariability (genetically fixed differences betweenspecies) for that trait, but also that the mecha-nisms underlying such variability are convergent.A corollary of this hypothesis is that lineages inwhich phenotypic plasticity is the ancestral con-dition are characterized by multiple instances ofconvergent evolution (Moore and Willmer, ’97). Totest these hypotheses I have searched the litera-ture for cases in which both the mechanisms un-derlying phenotypic plasticity for some feature andthe mechanisms underlying interspecific variabil-ity for that same feature are known. Additionally,in my own work I have investigated variability inovariole number (an indicator of ovary size) infruit flies and honeybees with the same issue inmind. It should be noted that in many instances,so-called convergent structures might be similarat only a very superficial level. For the term tohave any relevance with respect to evolutionarypatterns, use of the term should be restricted tosimilar structures that might have some aspectof their function in common (such that similar evo-lutionary forces might be expected to act on thetwo independently evolved structures).

Some experiments conducted almost 50 yearsago by Waddington (’56) still have not been ad-equately accounted for by a developmental geneticmodel. Application of ether to fruit fly embryosresults in some adults developing a bithorax-likephenotype (see above). After selection for animalsthat responded in this way, Waddington (’56) no-ticed that after only eight generations some fliesdeveloped the bithorax-like phenotype without anyether application at all! Waddington coined the term“genetic assimilation” to describe the phenomenon,and analysis of the spontaneous bithorax-like stockrevealed that it was caused by a single dominantallele. Therefore, this mutation must have arisenfortuitously in the course of selection (otherwiseit would have been detected in the parental popu-lation). In a second round of selection, a similar-looking, constitutive, bithorax-like mutation arosein generation 29. This second mutation was alsoa single dominant allele but at a completely dif-ferent locus. Recently, Gibson and Hogness (’96)

showed that the plastic bithorax-like response toether6 seems to result from localized loss of Ultrabi-thorax expression in the haltere primordium (whichis normally required to repress wing formationthere; see above). Since the developmental bases ofthe bithorax-like phenotype was not investigatedfor the constitutive lines, we cannot distinguish be-tween parallelism and convergence in this case.Later, I discuss the possibility that genetic assimi-lation might represent a generic mechanism of evo-lutionary change. While reading the followingexamples, the reader should keep in mind the pos-sibility that genetic fixation of phenotypically plas-tic traits might have occurred by a process akin toWaddington’s genetic assimilation (West-Eberhard,’89; Schlichting and Pigliucci, ’98).

Buckeye butterflies exhibit seasonal phenotypicplasticity (“seasonal polyphenism”) for the colorof the ventral hindwing: beige in the summer (thelinea morph), dark reddish-brown in the fall (therosa morph). The shift between the two forms islargely photoperiod dependent. When Rountreeand Nijhout (’95a) removed the brains of linea-destined pupae, they tended to produce the rosamorph, but injection of 20-hydroxyecdysone (whichis not produced in brainless animals) rescued thelinea morph. When levels of ecdysteroids weremeasured in rosa- and linea-destined pupae, rosa-destined pupae had much lower levels of ecdy-steroids during the “critical period” (the time wheninjection of 20-hydroxyecdysone into brainless pu-pae rescued the linea morph). Therefore, the de-velopmental basis of the plastic response appearsto be the suppression of ecdysteroid secretion dur-ing the critical period in rosa-destined pupae.There is also intra-specific variation for this plas-tic response, and there exists a constitutive rosamorph in natural populations. Surprisingly, con-stitutive rosa pupae do not show the suppressionof ecdysteroids characterizing the plastic response.Instead, their ecdysteroid profiles resemble thoseof linea-destined pupae (Rountree and Nijhout,’95b). Transplantation of constitutive rosa wingprimordia into linea-destined pupae resulted inthe rosa phenotype, indicating that the constitu-tive rosa phenotype is due to some factor or factorswithin the wing disc itself, and is thus distinctfrom the mechanism that produces the plastic re-sponse (ecdysteroid levels; though the constitu-

6I use the term ”phenotypic plasticity“ broadly, to refer to any en-vironmentally induced alteration in morphology within a genotype.Thus, while fruit flies in their natural habitats would never encoun-ter ether, I consider the phenotypic effects of ether application tofruit fly embryos to be an example of phenotypic plasticity.

PLASTICITY AND CONSTRAINTS 15

tive rosa phenotype may be due to alterations inthe ecdysteroid response pathway specifically inthe wing). Since the rosa morph is due to the ex-pression of two additional ommochrome pigmentsin the wing (Nijhout, ’97), it will be interesting tosee how the expression of enzymes involved in thesynthesis of these pigments is differentially regu-lated in the plastic and constitutive rosa morphs.In any case, the plastic and constitutive rosa mor-phs are produced by convergent developmentalmechanisms.

Colder developmental temperatures result inincreases in body size and wing size in fruit flies.The increases in wing size due to this plastic re-sponse in Drosophila melanogaster have beenshown by several researchers to result from in-creases in cell size rather than cell number (Par-tridge et al., ’94 and references therein). Partridgeand colleagues (’94) found that selection at inter-mediate temperatures for increased wing size alsocauses increases in cell size. De Moed and col-leagues (’97) showed that genetically fixed differ-ences in wing size among three D. melanogasterpopulations reared in a common garden were ex-plained by differences in cell number. Therefore,genetically fixed variability in wing size can becaused by either increases in cell size (the selec-tion experiments of Partridge et al., ’94) or in-creases in cell number [as de Moed et al. (’97)found in natural populations], and is thus anotherexample where marked plasticity is correlatedwith convergent evolution.

Terminal height and leaf number are phenotypi-cally plastic characters in two closely related spe-cies of Lobelia (Lobeliaceae), L. cardinalis and L.siphilitica (Pigliucci and Schlichting, ’95; Pigliucciet al., ’97). There is also substantial genotypic vari-ability for these characters. When the growth tra-jectories of several genotypes of each species wereexamined, these authors noted that different geno-types can converge on similar terminal phenotypesby different developmental routes. For example,similar final height could be reached either by aspurt of rapid growth early in ontogeny or by sus-tained slower growth.

The insect ovary is a modular structure madeup of ovarioles, each of which can independentlymature eggs in an assembly-line fashion. There-fore, ovariole number correlates with reproductiveoutput, and hence, presumably, fitness. Still, theremay be trade-offs associated with having largerovaries, such as decreased flight maneuverability(Berrigan, ’91) or developmental production costs(Nijhout and Emlen, ’98). Ovariole number var-

ies widely within drosophilids (reviewed in Maho-wald and Kambysellis, ’80) and is even quite vari-able within the melanogaster species group. Forexample, while the mean ovariole number variesamong populations of D. melanogaster from 16 to23 per ovary, the most derived member of the spe-cies group (Caccone et al., ’96), the island speciesD. sechellia, has only 8–9 ovarioles per ovary. Fur-thermore, ovariole number is determined during lar-val development, and D. melanogaster expressesmarked phenotypic plasticity for ovariole numberwhen reared at various temperatures or nutrientconditions (Savilev ’28; Delpuech et al., ’95;Moreteau et al., ’97; Morin et al., ’97). Hodin andRiddiford (submitted) examined the mechanisticbasis for plasticity and interspecific variation forovariole number in flies of the melanogaster spe-cies group by comparing the trajectories of ova-rian growth and differentiation in the variousphenotypic and genotypic contexts. We found thatthe mechanisms underlying the plastic decreasein ovariole number under different rearing condi-tions are mostly distinct from the mechanisms ex-plaining interspecific variation in ovariole number.Thus, once again, plasticity was correlated withmechanistic convergence in this system.

Still, there are exceptions to this trend. For in-stance, honeybee larvae of a given genotypedevelop either into a queen with hundreds of ova-rioles or into a worker with generally fewer than10 ovarioles, depending on larval nutrition. Hart-felder and Steinbrück (’97) and Schmidt Capellaand Hartfelder (’98) have shown that this differ-ence is due to differential cell death in the ova-rian primordia of workers in the honeybee Apismellifera carnica. In addition, the workers of dif-ferent geographical races of A. mellifera differ inmean ovariole number, with the most strikinginstance being workers of the Cape honeybeeA.m.capensis, which have about twice as manyovarioles as do most other races, including theirneighbor the African honeybee, A.m.scutellata (re-viewed in Ruttner, ’88). Our preliminary resultssuggest that the differences in ovariole numbersbetween the workers of these two African racesis also due to differential cell death (Hodin,Crewe, Riddiford, and Allsopp, unpublished).Therefore, the mechanism of plasticity appearsto be the same as that for intra-specific variationin this instance.

Another counterexample comes from work onbutterfly eyespots. Many butterflies have large cir-cular patterns on their wings known as eyespots,which are thought to play a role in predator avoid-

16 J. HODIN

ance by startling potential predators. In thesatyrine butterfly, Bicyclus anynana, there are twoseasonal morphs determined by temperature: awarmer wet season morph with larger eyespots,and a cooler dry season morph with smaller eyes-pots. Expression of the Distal-less (Dll) gene hadbeen previously shown to prefigure the positionof the eyespots in the wing primordia of the buck-eye butterfly (Carroll et al., ’94), and a similar pat-tern of expression is seen in the wet season morphin B. anynana (Brakefield et al., ’96). Pupal wingprimordia of dry season morphs show a smallerpatch of Dll expression prefiguring the position oftheir smaller eyespots. Brakefield and colleagues(’96) also examined butterfly lines selected at anintermediate temperature for constitutive expres-sion of either large or small eyespots. The consti-tutively large-spotted morph showed large patchesof Dll in the pupa, while the constitutively small-spotted morph showed small patches. Furthermore,recent work has demontrated that the size of theeyespots on the ventral wings is regulated byecdysteroids, and that differences in the timing ofecdysteroid release in the large and small eye spotselection lines mirror alterations in ecdysteroid re-lease of unslected lines reared at warmer and coolertemperatures, respectively (Koch et al., ’96; Brake-field et al., ’98). Thus, the mechanism of plasticity(early ecdysteroid release yielding large ventral eye-spots, apparently via large Dll patches; laterecdysteroid release yielding small ventral eyespots,apparently via small Dll patches) seems to be thesame as the mechanism of fixed, genetic differ-ences in the selection lines.

It should be noted that in the Dll example, aswell as in the example of the honeybees, there stillmay be instances of convergence within these taxo-nomic groups. Recall that on its own, Partridge etal. (’94) suggested that the mechanisms of plasticand genetically fixed differences in wing size werethe same. It was only after de Moed et al. (’97)examined three additional D. melanogaster popu-lations that convergence was apparent. Perhaps ifmore independent cases of increased or decreasedovariole number in bees, as well as interspecificvariation in eyespot size were examined, some in-stances of convergence would be found.

Still, in six cases in which (to my knowledge)plasticity and genetically fixed mechanisms ofvariation have been compared, convergent mecha-nisms were apparent in four of them. The signifi-cance of this finding is in the notion of evolvability,and I return to the concept of modularity to ex-emplify this point. Phenotypic plasticity is a spe-

cial case of modularity, where, for example, pho-toperiod shifts can produce radical changes inwing coloration while the development of manyother morphological features remains unaffected.Evolutionary changes in such developmental sys-tems are also characterized by flexibility, as evi-denced by the preponderance of convergence insuch cases. Parallelisms, on the other hand, arecharacteristic of developmental mechanisms thatare more constrained, such as the relationship be-tween Hox gene expression and appendage typein arthropods (Averof and Patel, ’97). In such situ-ations constraints may limit the scope of possiblevariation. Yet the arthropod segmental plan isclearly a successful one, so the constraint does notappear to be much of a hindrance. The “success”of constrained developmental programs may beattributed to one of two phenomena. First, the de-velopmental system may be primed to respond toselection in a given way, thus making the evolu-tionary options more limited but the process ofadaptation more efficient (a quicker route to anadaptive peak, in Wright’s terminology). Alterna-tively, constraints may necessitate the evolutionof novel solutions to the constraints, thus yield-ing a phenotype with higher fitness. The latterscenario may explain, for example, the evolutionof complex retinotectal projections built on the“simplistic” morphology of the salamander visualsystem, imposed by the constraint of large cell size(reviewed in Roth et al., ’97).

As for convergence and evolvability, recall theWaddington genetic assimilation experimentswhere a plastic ancestral population evolved rapidfixation of the bithorax-like phenotype under strongselection. As Schlichting and Pigliucci (’98) havenoted, the plasticity of the ancestral population ap-pears to have allowed for the rapid response toselection and that genetic assimilation likely rep-resents a relatively common mechanism for phe-notypic evolution. It is important to note here thatthe ancestral population apparently did not har-bor genetic variation for the constitutive bithorax-like phenotype: it arose de novo in the course ofselection. Whether convergent mechanisms arelikely to arise out of such plastic systems by ge-netic assimilation has not been addressed. In ad-dition, there are no available data on the frequencywith which assimilation experiments are success-ful (since, presumably, negative results are not re-ported). Thus it is difficult to determine howcommon a mechanism for phenotypic evolution ge-netic assimilation may actually represent.

PLASTICITY AND CONSTRAINTS 17

CONCLUSIONThere appears to be a common misunderstand-

ing in the literature about the definition of devel-opmental constraints. My understanding of theterm is that it does not represent prohibitions onthe evolution of certain features, just that the de-velopmental system is predisposed to respond toselection in a certain way. What exactly it is aboutdevelopmental systems that lead to constraintsis not known. I suggest that it is the modular na-ture of development that leads to such constraints,since these modules can be thought of (teleologi-cally) as “easy” ways to solve new problems. In-stances of convergence, by contrast, point to theabsence of such constraints and may be charac-teristic of processes subject to phenotypic plastic-ity. Detailed investigation into the nature ofparallelisms and convergences may represent thefirst step towards developing a predictive scienceof the mechanisms of evolution.

ACKNOWLEDGMENTSThis manuscript originated as a working paper

for the Modularity of Animal Form workshop, heldat Friday Harbor Laboratories, Friday Harbor, WA,in the fall of 1997. Discussions at the workshophelped focus my ideas. I am grateful to the orga-nizers, George von Dassow and Ed Munro, for in-viting me to participate in that stimulating event.In addition, I thank George and Mickey vonDassow, Jim Eubanks, Wesley Grueber, Dr. JoelKingsolver, Dr. Lynn Riddiford, Dr. James Truman,and Dr. Graeme Wistow for comments on themanuscript. I also thank Dr. Paulyn Cartwright,Dr. Doug Emlen, Dr. David Jacobs, Dr. Christo-pher Lowe and Dr. Michael Strand for allowingme to cite their unpublished findings. Finally, Iam grateful to the University of Washington zo-ology department, where this work was done.

LITERATURE CITEDAbouheif E, Akam M, Dickinson WJ, Holland PWH, Meyer

A, Patel NH, Raff RA, Roth VL, Wray GA. 1997. Homologyand developmental genes. Trends Genet 13:432–433.

Abzhanov A, and Kaufman TC. 1999. Homeotic genes andthe mandibulate head: divergent expression patterns of la-bial, proboscipedia and Deformed homologues in crustaceansand insects. Dev Biol 210:187.

Aguinaldo AMA, Turbeville JM, Linford LS, Rivera MC, GareyJR, Raff RA, Lake JA. 1997. Evidence for a clade of nema-todes, arthropods and other moulting animals. Nature387:489–493.

Arenas-Mena C, Martinez P, Cameron RA, Davidson EH.1998. Expression of the Hox gene complex in the indirectdevelopment of a sea urchin. Proc Nat Acad Sci USA95:1362–1367.

Averof M, Akam M. 1995. Hox genes and the diversification ofthe insect and crustacean body plans. Nature 376:420–423.

Averof M, Patel N. 1997. Crustacean appendage evolution as-sociated with changes in Hox gene expression. Nature388:682–686.

Avise JC. 1994. Molecular markers, natural history & evolu-tion. New York: Chapman & Hall.

Becerra JX. 1997. Insects on plants: macroevolutionary chemi-cal trends in host use. Science 276:253–256.

Becerra JX, Venable DL. 1999. Macroevolution of insect-plantassociations: the relevance of host biogeography to host af-filiation. Proc Nat Acad Sci USA 96:12626–12631.

Beklemishev WN. 1969. Principles of comparative anatomy ofinvertebrates, Vol 2. Edinburgh, Scotland: Oliver and Boyd.

Berrigan D. 1991. Lift production in the flesh fly, Neobellieria(=Sarcophaga) bullata Parker. Funct Ecol 5:448–456.

Binner P, Sander K. 1997. Pair-rule patterning in the honey-bee Apis mellifera: expression of even-skipped combinestraits known from beetles and fruitfly. Dev Genes Evol206:447–454.

Bonini NM, Bui QT, Gray-Board GL, Warrick JM. 1997. TheDrosophila eyes absent gene directs ectopic eye formationin a pathway conserved between flies and vertebrates. De-velopment 124:4816–4826.

Brakefield PM, Gates J, Keys D, Kesbeke F, Wijngaarden PJ,Monteiro A, French V, Carroll SB. 1996. Development, plas-ticity and evolution of butterfly eyespot patterns. Nature384:236–242.

Brakefield PM, Kesbeke F, Koch PB. 1998. The regulation ofphenotypic plasticity of eyespots in the butterfly Bicyclusanynana. Am Nat 152:853–860.

Brunschwig K, Wittmann C, Schnabel R, Bürglin TR, ToblerH, Müller F. 1999. Anterior organization of the Caenor-habditis elegans embryo by the labial-like Hox gene ceh-13. Development 126:1537–1546.

Bürglin TR, Ruvkun G. 1993. The Caenorhabditis eleganshomeobox gene cluster. Curr Opinion Genet Dev 3:615–620.

Burke AC, Nelson CE, Morgan BA, Tabin C. 1995. Hox genesand the evolution of vertebrate axial morphology. Develop-ment 121:333–346.

Caccone A, Moriyama EN, Gleason JM, Nigo L and PowellJR. 1996. A molecular phylogeny for the Drosophilamelanogaster subgroup and the problem of polymorphismdata. Mol Biol Evol 13:1224–1232.

Callaerts P, Halder G, Gehring WJ. 1997. PAX6 in develop-ment and evolution. Ann Rev Neurosci 20:483–532.

Capdevila MP, Garcia-Bellido A. 1974. Development and ge-netic analysis of bithorax phenocopies in Drosophila. Na-ture 250:500–502.

Carroll SB, Gates J, Keys DN, Paddock SW, Panganiban GE,Selegue JE, Williams JA. 1994. Pattern formation and eye-spot determination in butterfly wings. Science 265:109–114.

Chase MW, Soltis DE, Olmstead RG, Morgan D, Les DH,Mishler BD, Duvall MR, Price RA, Hills HG, Qiu YL. 1993.Phylogenetics of seed plants: an analysis of nucleotide se-quences from the plastid gene rbcL. Ann Missouri Bot Gard80:528–580.

Chen F, Capecchi MR. 1997. Targeted mutations in hoxa-9 andhoxb-9 reveal synergistic interactions. Dev Biol 181:186–196.

Chen F, Greer J, Capecchi MR. 1998. Analysis of Hoxa7/Hoxb7mutants suggests periodicity in the generation of differentsets of vertebrae. Mech Dev 77:49–57.

Chen L, DeVries AL, Cheng C-HC. 1997a. Evolution of anti-freeze glycoprotein gene from a trypsinogen gene in Antarc-tic notothenoid fish. Proc Nat Acad Sci USA 94:3811–3816.

18 J. HODIN

Chen L, DeVries AL, Cheng C-HC. 1997b. Convergent evolu-tion of antifreeze glycoproteins in Antarctic notothenoid fishand Arctic cod. Proc Nat Acad Sci USA 94:3817–3822.

Chisholm AD, Horvitz HR. 1995. Patterning of the Caenor-habditis elegans head region by the Pax-6 family membervab-3. Nature 377:52–55.

Conway Morris S. 1998. The crucible of creation: the Bur-gess Shale and the rise of animals. New York: Oxford Uni-versity Press.

Cook JM, Compton SG, Herre EA, West SA. 1997. Alterna-tive male tactics and extreme male dimorphism in fig wasps.Proc R Soc Lond B 264:747–754.

Cvekl A, Piatigorsky J. 1996. Lens development and crystallingene expression: many roles for Pax6. BioEssays 18:621–630.

Dahl E, Koseki H, Balling R. 1997. Pax genes and organo-genesis. BioEssays 19:755–765.

De Jong G, van Noordwijk AJ. 1992. Acquisition and alloca-tion of resources: genetic (co)variances, selection, and lifehistories. Am Nat 139:749–770.

Delpuech J-M, Moreteau B, Chiche J, Pla E, Vouidibio J,David JR. 1995. Phenotypic plasticity and reaction normsin temperate and tropical populations of Drosophila melano-gaster: ovarian size and developmental temperature. Evo-lution 49:670–675.

De Moed GH, de Jong G, Scharloo W. 1997. The phenotypicplasticity of wing size in Drosophila melanogaster: the cel-lular basis of its genetic variation. Heredity 79:260–267.

De Robertis EM. 1997. Evolutionary biology: the ancestry ofsegmentation. Nature 387:25–26.

Donoghue MJ. 1992. Homology. In: Keller EF, Lloyd EA, edi-tors. Keywords in evolutionary biology. Cambridge, MA:Harvard University Press. p 170–179.

Doolittle RF. 1986. Of URFs and ORFs: a primer on how toanalyze derived amino acid sequences. Mill Valley, CA: Uni-versity Science Books.

DuPraw EJ. 1967. The honeybee embryo. In: Wilt FH,Wessells NK, editors. Methods in developmental biology.New York: TY Crowell Co. p 183–217.

Escriva H, Safi R, Hänni C, Langlois M-C, Saumitou-LapradeP, Stehelin D, Capron A, Pierce R, Laudet V. 1997. Ligandbinding was acquired during evolution of nuclear receptors.Proc Nat Acad Sci USA 94:6803–6808.

Farnsworth EJ, Farrant JM. 1998. Reductions in abscisic acidare linked with viviparous reproduction in mangroves. AmJ Bot 85:760–769.

Fleig R. 1990. Engrailed expression and body segmentationin the honeybee Apis mellifera. Roux Arch Dev Biol 198:567–473.

Fleig R, Walldorf U, Gehring WJ, Sander K. 1992. Develop-ment of the Deformed protein pattern in the embryo of thehoneybee Apis mellifera L. (Hymenoptera). Roux Arch DevBiol 201:235–242.

Frieden E. 1981. The dual role of thyroid hormones in verte-brate development and calorigenesis. In: Gilbert LI, FriedenE, editors. Metamorphosis: a problem in developmental bi-ology. New York: Plenum Press. p 545–563.

Gaunt SJ. 1994. Conservation in the Hox code during mor-phological evolution. Int J Dev Biol 38:549–552.

Gibson G, Hogness DS. 1996. Effect of polymorphism in theDrosophila regulatory gene Ultrabithorax on homeotic sta-bility. Science 271:300–203.

Gilbert SF, Opitz JM, Raff RA. 1996. Resynthesizing evolu-tionary and developmental biology. Dev Biol 173:357–372.

Govindarajan S, Goldstein RA. 1996. Why are some proteinstructures so common? Proc Nat Acad Sci USA 93:3341–3345.

Graham C, Hodin J, Wistow G. 1996. A retinaldehyde dehy-drogenase as a structural protein in a mammalian eye lens:gene recruitment of eta-crystallin. J Biol Chem 271:15623–15628.

Graumann P, Marahiel MA. 1996. A case of convergent evolu-tion of nucleic acid binding modules. BioEssays 18:309–315.

Grbic M, Nagy LM, Strand MR. 1998. Development of poly-embryonic insects: a major departure from typical insectembryogenesis. Dev Genes Evol 208:69–81.

Grbic M, Strand MR. 1998. Shifts in the life history of para-sitic wasps correlate with pronounced alterations in earlydevelopment. Proc Nat Acad Sci USA 95:1097–1101.

Haerry TE, Gehring WJ. 1997. A conserved cluster ofhomeodomain binding sites in the mouse Hoxa-4 intron func-tions in Drosophila embryos as an enhancer that is directlyregulated by Ultrabithorax. Dev Biol 186:1–15.

Halanych KM, Bacheller JD, Aguinaldo AMA, Liva SM, HillisDM, Lake JA. 1995. Evidence from 18S Ribosomal DNAthat the lophophorates are protostome animals. Science267:1641–1643.

Halder G, Callaerts P, Flister S, Walldorf U, Kloter U, GehringWJ. 1995. Eyeless initiates the expression of both sine oculisand eyes absent during Drosophila compound eye develop-ment. Development 125:2181–2191.

Hall BG, Malik HS. 1998. Determining the evolutionary po-tential of a gene. Mol Biol Evol 15:1055–1061.

Hanken J, Jennings DH, Olsson L. 1997. Mechanistic basisof life-history evolution in anuran amphibians: direct de-velopment. Am Zool 37:160–171.

Harris WA. 1997. Pax-6: Where to be conserved is not con-servative. Proc Nat Acad Sci USA 94:2098–2100.

Hartfelder K, Steinbrück G. 1997. Germ cell cluster forma-tion and cell death are alternatives in caste-specific differ-entiation of the larval honey bee ovary. Invert Reprod Dev31:237–250.

Hodin J, Riddiford LM. 1998. The ecdysone receptor andultraspiracle regulate the timing and progression of ova-rian morphogenesis during Drosophila metamorphosis. DevGenes Evol 208:304–317.

Holland LZ, Kene M, Williams NA, Holland ND. 1997. Se-quence and embryonic expression of the amphioxusengrailed gene (AmphiEn): the metameric pattern of tran-scription resembles that of its segment-polarity homologin Drosophila. Development 124:1723–1732.

Houle D. 1991. Genetic covariance of fitness correlates: whatgenetic correlations are made of why it matters. Evolution45:630–648.

Ishii M, Mitsunaga-Nakatsubo K, Kitajima T, Kusunoki S,Shimada H, Akasaka K. 1999. Hbox1 and Hbox7 are in-volved in pattern formation in sea urchin embryos. DevGrowth Differ 41:241–252.

Jacob F. 1977. Evolution and tinkering. Science 196:1161–1166.

Jacobs DK, DeSalle R, Weeden C. 1994. Engrailed: homologyof metameric units, molluscan phylogeny and relationshipto other homeodomains. Dev Biol 163:536.

Jaschof M. 1998. Revision der “Lestremiinae” (Diptera,Cecidomyiidae) der holarktis. Stud Dipter Suppl 4:1–552.

Jennings DH, Hanken J. 1998. Mechanistic basis of life his-tory evolution in anuran amphibians: thyroid gland devel-opment in the direct-developing frog, Eleutherodactyluscoqui. Gen Comp Endocrinol 111:225–232.

Juncosa AM, Tomlinson PB. 1988. Systematic comparison andsome biological characteristics of Rhizophoraceae andAnisophylleaceae. Ann Missouri Bot Gard 75:1296–1318.

PLASTICITY AND CONSTRAINTS 19

Katsuyama Y, Wada S, Yasugi S, Saiga H 1995. Expressionof the labial group Hox gene HrHox-1 and its alterationinduced by retinoic acid in development of the ascidianHalocynthia roretzi. Development 121:3197–3205.

Kermode AR. 1995. Regulatory mechanisms in the transitionfrom seed development to germination: interactions betweenthe embryo and the seed environment. In: Kigel J Galili G,editors. Seed development and germination. New York:Marcel Dekker. p 273–332.

Kliewer SA, Lehmann JM, Willson TM. 1999. Orphan nuclearreceptors: shifting endocrinology into reverse. Science284:757–760.

Klingenberg CP, Nijhout HF. 1998. Competition among grow-ing organs and developmental control of morphologicalasymmetry. Proc Roy Soc Lond B 265:1135–1139.

Koch PB, Brakefield PM, Kesbeke F. 1996. Ecdysteroids con-trol eyespot size and wing color pattern in the polyphenicbutterfly Bicyclus anynana. J Insect Physiol 42:223–230.

Koonin EV, Mushegian AR, Bork P. 1996. Non-orthologousgene displacement. Trends Genet 12:334–336.

Kozloff EN. 1990. Invertebrates. San Francisco: Saunders.Krumlauf R. 1993. Hox genes and pattern formation in the

branchial region of the vertebrate head. Trends Genet9:106–112.

Laudet V. 1997. Evolution of the nuclear receptor superfam-ily: early diversification from an ancestral orphan receptor.J Mol Endocrinol 19:207–226.

Lawrence PA. 1992. The making of a fly: the genetics of ani-mal design. Boston: Blackwell Scientific Publications.

Lowe CJ, Wray GA. 1997. Radical alterations in the roles ofhomeobox genes during echinoderm evolution. Nature389:718–721.

Mahowald AP, Kambysellis MP. 1980. Oogenesis. In: Ash-burner M, Wright TRF, editors. The genetics and biology ofDrosophila, Vol 2D. New York: Academic Press. p 141–224.

Matsuda R. 1992. The evolutionary process in talitrid am-phipods and salamanders in changing environments, witha dicussion of “genetic assimilation” and some other evolu-tionary concepts. Can J Zool 60:733–749.

Maynard-Smith J, Burian R, Kauffman S, Alberch P, CampbellJ, Goodwin B, Lande R, Raup D, Wolpert L. 1985. Develop-mental constraints and evolution. Q Rev Biol 60:265–287.

Moore J, Willmer P. 1997. Convergent evolution in inverte-brates. Biol Rev 72:1–60.

Moreteau B, Morin J-P, Gibert P, Pétavy G, Pla E, DavidJR. 1997. Evolutionary changes in nonlinear reactionnorms according to thermal adaptation: a comparison ofthe Drosophila species. C R Acad Sci Paris, Sciences de laVie 320:833–841.

Morin J-P, Moreteau B, Pétavy G, Parkash R, David JR. 1997.Reaction norms of morphological traits in Drosophila: adap-tive shape changes in a stenotherm circumtropical species?Evolution 51:1140–1148.

Nijhout HF. 1997. Ommochrome pigmentation of the lineaand rosa seasonal forms of Precis coenia (Lepidoptera:Nymphalidae). Arch Insect Bioch Physiol 36:215–222.

Nijhout HF. 1999. Control mechanisms of polyphenic devel-opment in insects. BioScience 49:181–192.

Nijhout HF, Emlen DJ. 1998. Competition among body partsin the development and evolution of insect morphology. ProcNat Acad Sci USA 95:3685–3689.

Niklas KJ. 1997. The evolutionary biology of plants. Chicago:University of Chicago Press.

Oliver G, Wehr R, Jenkins NA, Copeland NG, Cheyette NG,

Hartenstein V, Zipursky SL, Gruss P. 1995a. Homeoboxgenes and connective tissue patterning. Development121:693–705.

Oliver G, Mailhos A, Wehr R, Copeland NG, Jenkins NA,Gruss P. 1995b. Six3, a murine homologue of the sine oculisgene, demarcates the most anterior border of the develop-ing neural plate and is expressed during eye development.Development 121:4045–4055.

Oliver G, Loosli F, Koster R, Wittbrodt J and Gruss P. 1996.Ectopic lens induction in fish in response to the murinehomeobox gene Six3. Mech Dev 60:233–239.

Partridge L, Barrie B, Fowler K, French V. 1994. Evolutionand development of body size and cell size in Drosophilamelanogaster in response to temperature. Evolution 48:1269–1276.

Patel NH. 1994. The evolution of arthropod segmentation:insights from comparisons of gene expression patterns. DevSuppl 201–207.

Philippe H, Chenuil A, Adoutte A. 1994. Can the Cambrianexplosion be inferred through molecular phylogeny? DevSuppl 15–25.

Pigliucci M, diIorio P, Schlichting CD. 1997. Phenotypic plas-ticity of growth trajectories in two species of Lobelia in re-sponse to nutrient availability. J Ecol 85:265–276.

Pigliucci M, Sclichting CD. 1995. Ontogenetic reaction normsin Lobelia siphilitica (Lobeliaceae): response to shading.Ecology 76:2134–2144.

Quiring R, Walldorf U, Kloter U, Gehring WJ. 1994. Homol-ogy of the eyeless gene of Drosophila to the Small eye genein mice and Aniridia in humans. Science 265:785–789.

Ranganayakulu G, Elliott DA, Harvey RP, Olson EN. 1998.Divergent roles for NK-2 class homeobox genes in cardio-genesis in flies and mice. Development 125:3037–3048.

Roth G, Nishikawa KC and Wake DB. 1997. Genome size,secondary simplification and the evolution of the brain insalamanders. Brain Behav Evol 50:50–59.

Roth VL. 1984. On homology. Biol J Linn Soc 22:13–29.Rountree DB, Nijhout HF. 1995a. Hormonal control of a sea-

sonal polyphenism in Precis coenia (Lepidoptera: Nymph-alidae). J Insect Physiol 41:987–992.

Rountree DB, Nijhout HF. 1995b. Genetic control of a sea-sonal morph in Precis coenia (Lepidoptera: Nymphalidae).J Insect Physiol 41:1141–1145.

Ruiz-Trillo I, Riutort M, Littlewood DTJ, Herniou EA, BaguñaJ. 1999. Acoel flatworms: earliest extant bilaterian metazoans,not members of Platyhelminthes. Science 283:1919–1923.

Ruppert EE, Barnes RD. 1994. Invertebrate zoology, 6th edi-tion. New York: Saunders College Publishing.

Russell LB. 1956. X-ray induced developmental abnormali-ties in the mouse and their use in the analysis of embryo-logical patterns. J Exp Zool 131:329–394.

Ruttner F. 1988. Biogeography and taxonomy of honeybees.New York: Springer Verlag.

Saito M, Seki M, Amemiya S, Yamasu K, Suyemitsu T,Ishihara K. 1998. Induction of metamorphosis in the sanddollar Peronella japonica by thyroid hormones. Dev GrowthDiffer 40:307–312.

Salser SJ, Kenyon C. 1996. A C. elegans Hox gene switcheson, off, on and off again to regulate proliferation, differen-tiation and morphogenesis. Development 122:1651–1661.

Saviliev VI. 1928. Manifold effects of the gene vestigial in Droso-phila melanogaster. Trud Leningr Obst Estestv 58:65–88.

Schlichting CD, Pigliucci M. 1998. Phenotypic evolution: areaction norm perspective. Sunderland, MA: Sinauer.

Schmidt Capella IC, Hartfelder K. 1998. Juvenile hormone

20 J. HODIN

effect on DNA synthesis and apoptosis in caste-specific dif-ferences of the larval honey bee (Apis mellifera L.) ovary. JInsect Physiol 44:385–391.

Shaffer HB, Voss R. 1996. Phylogenetic and mechanisticanalysis of a developmentally integrated character complex:alternative life-history modes in ambystomatid salamanders.Am Zool 36:24–35.

Sharkey M, Graba Y, Scott MP. 1997. Hox genes in evolu-tion: protein surfaces and paralog groups. Trends Genet13:145–151.

Shatoury HH. 1963. The development of the “eyeless” condi-tion in Drosophila. Caryologia 16:431–437.

Shen W, Mardon G. 1997. Ectopic eye development in Droso-phila induced by directed dachshund expression. Develop-ment 124:45–52.

Sheng G, Thouvenot E, Schmucker D, Wilson DS, DesplanC. 1997. Direct regulation of rhodopsin 1 by Pax6/eyeless inDrosophila: evidence for a conserved function in photore-ceptors. Genes Dev 11:1122–1131.

Slack JM, Holland PW, Graham CF. 1993. The zootype andthe phylotypic stage. Nature 361:490–492.

Smith AB 1997. Echinoderm larvae and phylogeny. Ann RevEcol Syst 28:219–241.

Snodgrass RE. 1938. Evolution of the Annelida, Onychophora,and Arthropoda. Smithsonian Miscellaneous Collections, no.3483 97(6):1–159.

Strand MR, Grbic M. 1997. The development and evolutionof polyembryonic insects. Curr Topics Dev Biol 35:121–159.

Tautz D, Friedrich M and Schröder R. 1994. Insect embyro-genesis: which is ancestral and which is derived? Dev Suppl193–197.

Tomarev SI, Zinovieva RD, Piatigorsky J. 1991. Crystallinsof the octopus lens: recruitment from detoxification enzymes.J Biol Chem 266:24226–24231.

Tomlinson PB 1986. The biology of mangroves. Cambridge:Cambridge University Press.

Toy J, Yang JM, Leppert GS, Sundin OH. 1998. The Optx2homeobox gene is expressed in early precursors of the eyeand activates retina-specific genes. Proc Nat Acad Sci USA95:10643–10648.

van Noordwijk AJ, de Jong G. 1986. Acquisition and allo-cation of resources: their influence on variation in lifehistory tactics. Am Nat 128:137–142.

Van Valen L 1982. Homology and causes. J Morph 173:305–312.

Von Dassow G, Munro E. 1999. Modularity in animal devel-opment and evolution: elements of a conceptual frameworkfor EvoDevo. J Exp Zool (Mol Dev Evol) 285:307–325.

Waddington CH. 1956. Genetic assimilation of the bithoraxphenotype. Evolution 10:1–13.

Wagner GP. 1995. The biological role of homologues: a build-ing block hypothesis. N Jb Geol Paläont Abh 19:279–288.

Wagner GP, Misof BY. 1993. How can a character be develop-mentally constrained despite variation in developmentalpathways? J Evol Biol 6:449–455.

Wagner GP, Altenberg L. 1996. Complex adaptations and theevolution of evolvability. Evolution 50:967–976.

Wagner GP, Kahn P, Naylor G, Blanco M, Misof BY. 1997.Evolution of Hoxa-11 expression in amphibians: is the sala-mander autopod an innovation? Am Zool 37:173A.

Wake DB. 1991. Homoplasy: the result of natural selection,or evidence of design limitations? Am Nat 138:543–567.

Went DF. 1979. Paedogenesis in the dipteran insect Heteropezapygmaea: an interpretation. Int J Invert Reprod 1:21–30.

West-Eberhard MJ. 1989. Phenotypic plasticity and the ori-gins of diversity. Ann Rev Ecol Syst 20:249–278.

Wilkins AS. 1998. Homology. BioEssays 20:1052–1053.Willmer P. 1990. Invertebrate relationships: patterns in ani-

mal evolution. New York: Cambridge University Press.Wistow G, Kim H. 1991. Lens protein expression in mam-

mals: taxon-specificity and the recruitment of crystallins. JMol Evol 32:262–269.

Wolff C, Schroder R, Schulz C, Tautz D, Klingler M. 1998.Regulation of the Tribolium homologues of caudal andhunchback in Drosophila: evidence for maternal gradientsystems in a short germ embryo. Development 125:3645–3654.

Wray GA, Bely AE. 1994. The evolution of echinoderm develop-ment is driven by several distinct factors. Dev Suppl 97–106.

Wright S. 1932. The roles of mutation, inbreeding, cross-breed-ing, and selection in evolution. Proc VI Internat Cong Genet1:356–366.

Yao TP, Forman BM, Jiang Z, Cherbas L, Chen JD, McKeownM, Cherbas P, Evans RM. 1993. Functional ecdysone re-ceptor is the product of EcR and ultraspiracle genes. Na-ture 366:476–479.

Yaoita Y, Brown DD. 1990. A correlation of thyroid hormonereceptor gene expression with amphibian metamorphosis.Genes Dev 4:1917–1924.

Zhang Y, Emmons SW. 1995. Specification of sense-organ iden-tity by a Caenorhabditis elegans Pax-6 homologue. Nature377:55–59.

Zuckerkandl E. 1976. Programs of gene action and progres-sive evolution. In: Goodman M, Tashian RE, editors. Molecu-lar anthropology: genes and proteins in the evolutionaryascent of the primates. New York: Plenum Press. p 387–447.

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Erratum

Hodin J. 2000. Plasticity and constraints in development and evolution.J Exp Zool (Mol Dev Evol) 288:1–20.

The sentences on page 8, in paragraph 2 of column 2 reads:

In the short-germ insects, typified by Drosophila melanogaster, all thesegments are specified nearly simultaneously in a noncellular, syncitialenvironment (reviewed in Lawrence, ’92). By contrast, in the long-germinsects, typified by the grasshopper Schistocerca, the segments are speci-fied in a gradual anterior-to-posterior progression in a cellular environ-ment (reviewed in Patel, ’94; Tautz et al., ’94).

The sentences should read:

In the long-germ insects, typified by Drosophila melanogaster, all thesegments are specified nearly simultaneously in a noncellular, syncitialenvironment (reviewed in Lawrence, ’92). By contrast, in the short-germinsects, typified by the grasshopper Schistocerca, the segments are speci-fied in a gradual anterior-to-posterior progression in a cellular environ-ment (reviewed in Patel, ’94; Tautz et al., ’94).

The terms “long-germ” and “short-germ” were reversed. The germ-typeclassification is key to the point being made in this paragraph: namely,showing that a similar-looking segmental plan can be formed by two ratherdifferent ontogenetic routes).

The author regrets the error.