e early animal evolution: emerging views from comparative

Early Animal Evolution: Emerging Viewsfrom Comparative Biology and Geology

Andrew H. Knoll1* and Sean B. Carroll2

The Cambrian appearance of fossils representing diverse phyla has longinspired hypotheses about possible genetic or environmental catalysts ofearly animal evolution. Only recently, however, have data begun toemerge that can resolve the sequence of genetic and morphologicalinnovations, environmental events, and ecological interactions that col-lectively shaped Cambrian evolution. Assembly of the modern genetic toolkit for development and the initial divergence of major animal cladesoccurred during the Proterozoic Eon. Crown group morphologies diversi-fied in the Cambrian through changes in the genetic regulatory networksthat organize animal ontogeny. Cambrian radiation may have been trig-gered by environmental perturbation near the Proterozoic-Cambrianboundary and subsequently amplified by ecological interactions withinreorganized ecosystems.

Near the mouth of the Kotuikan River, north-ern Siberia, steep cliffs punctuate the borealforest. At river level, carbonate rocks recordthe deposition of lime muds in a shallowseaway some 545 million years ago (Ma).Fossils are rare in these rocks, and those thatcan be found are simple. The only trace fos-sils are meanders of small worm-like crea-tures preserved on bedding surfaces. About3 m above this level, there is an abrupt shiftto shore-face sandstone; volcanic breccias tothe east date this horizon at 544 6 1 Ma (1).Above the sandstone ledge, variegated shalesform a steep shoulder above which rises awall of limestone and dolomite. The shalesrecord a flooding event, but as sedimentsaccumulated the sea grew shallower, so thatoverlying carbonates reflect marine environ-ments progressively closer to the shore. Nearthe top of the cliff, an erosional surfacerecords subaerial exposure—short-lived, asthe sea soon encroached again (2).

Beginning at the level of the sandstonebench, the rocks contain small skeletal fossils(3). In the lowermost beds, there are only a fewforms, cones of calcite little more than a milli-meter long. But as one ascends the cliff, theabundance and variety of these fossils increase.So, too, do the abundance and behavioral com-plexity of preserved tracks, trails, and burrows.Nearly 120 m above river level, rocks estimatedto be about 530 million years (m.y.) old containmore than 80 skeletal taxa. Some, like thosefound along the river, have a threefold symme-try that differentiates them from most animals

alive today. Others, however, include spiralshells that are recognizably molluscan and, a bithigher, arthropods and the bivalved shells ofbrachiopods.

This pattern is recorded in greater or lesserdetail in rocks of comparable age throughoutthe world. It marks the unfolding of animaldiversity popularly known as the Cambrian ex-plosion. The broad outline of Cambrian diver-sification has been known for more than acentury, and for almost that long scientists havedebated its biological interpretation and possi-ble causes. Beginning with Darwin (4), somehave argued that the appearance of explosiveevolution is illusory—that the apparently rapiddiversification of animals reflects massiverecord failure below the Proterozoic-Cambrianboundary (5). Others have accepted the realityof Cambrian radiation and ascribed it either tocauses intrinsic to animal biology or to extrinsic(environmental) triggers (6).

The debate about Cambrian evolutionmay be old, but only in the past decade havethe data necessary to weigh conflicting hy-potheses begun to emerge, issuing from abroad range of disciplines that includes notonly paleontology and stratigraphy, but alsogeochemistry, molecular systematics, and de-velopmental genetics. It is becoming increas-ingly clear that understanding the Cambrianexplosion requires that considerations of theCambrian fauna and the environments thatshaped it be complemented by new per-spectives on the late Proterozoic world. Tounderstand what actually transpired duringor what may have enabled the Cambrianexplosion, we must consider what animals,developmental and genetic mechanisms,and ecosystems were in place before it.Here, we review recent fossil, phyloge-netic, embryological, and paleoenviron-mental discoveries that are spawning newideas about early animal evolution.

Paleontology and the Pattern ofAnimal Diversification

The earliest Cambrian remains are not theoldest record of animals. Cambrian faunaswere preceded by the so-called Ediacaranbiota, preserved as impressions, casts, andmolds in uppermost Proterozoic rocks aroundthe world (7). Centimeter-scale, radiallysymmetrical impressions arguably formed bydiploblastic animals occur in rocks more than600 m.y. old (8). Radially symmetric fossilsalso predominate in younger Ediacaran fau-nas (575 to 544 Ma) (Fig. 1A), but they arejoined by a wider diversity of morphotypes(Fig. 1D) as well as trace fossils made at leastin part by bilaterian animals (Fig. 1F) (9).[Reports of older bilaterian traces (10) re-quire confirmation of age and interpretation.The unambiguous, abundant, and continuousrecord of bilaterian traces begins only nearthe end of the Proterozoic Eon.]

Although the stratigraphic distribution ofEdiacaran fossils is clear enough, their biolog-ical interpretation remains controversial, pro-viding what amounts to a paleontological Ror-schach test. Several distinct body plans arerepresented (11). Most radially symmetric fos-sils plausibly represent polypoid organisms orthe inflated holdfasts of colonial, diploblasticanimals—mostly unrepresented in the modernfauna. More complex fossils include a range offorms built of repeated, tube-like units (Fig.1D). In a stimulating, if controversial pro-posal, Seilacher (12) grouped such fossilsinto a clade that he christened the Ven-dobionta and viewed as an extinct experimentin multicellular organization. Others havequestioned this interpretation, assigning var-ious forms to colonial diploblasts or to stemmembers (13) of several bilaterian clades (11,14, 15). It is genuinely difficult to map thecharacters of Ediacaran fossils onto the bodyplans of living invertebrates. Long viewed asthe principal problem of interpreting Ediaca-ran assemblages, this difficulty increasinglyappears to be their central point. Much opin-ion supports the broad view that both extinctdiploblastic-grade animals and bilaterianstem groups [for example, the mollusk-likeKimberella (15)] are represented. Trace fos-sils record a modest diversity of (mostly)simple bilaterians (16). Crown group proto-stomes or deuterostomes may also lurk inEdiacaran-aged rocks but at present, evidenceof such animals remains equivocal.

Early Cambrian fossil assemblages aredistinctly different. Although at least some

1Department of Organismic and Evolutionary Biology,Harvard University, 26 Oxford Street, Cambridge, MA02138, USA. 2Howard Hughes Medical Institute, Lab-oratory of Molecular Biology, University of Wiscon-sin–Madison, 1525 Linden Drive, Madison, WI 53706,USA.

*To whom correspondence should be addressed.

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Ediacarans survived into the Cambrian (17),the Cambrian fauna consists predominantlyof stem and crown group members of extantbilaterian phyla, along with diverse spongesand rarer cnidarians and ctenophores (18).Mineralized skeletons are widely distributed,if formed by a minority of Cambrian species.Trace fossils independently record a notableexpansion of behavioral complexity withinbilaterian animals (19).

New windows on Proterozoic biology. TheBurgess Shale has achieved almost iconic statusamong fossil assemblages, and for good reason:it preserves a remarkably detailed record ofCambrian diversification. Burgess fossils post-date the beginning of the Cambrian Period byas much as 40 million years, however, so therecent documentation of Burgess-like preserva-tion in somewhat older shales is welcome. Es-pecially in the Chengjiang (China) and SiriusPasset (Greenland) faunas (18, 20), superblypreserved fossils show that body plans of Bur-gess complexity already existed late in the Ear-ly Cambrian—still as much as 20 to 25 millionyears after the start of the period. Although notwidely appreciated, carbonaceous shales insome latest Proterozoic successions also pre-serve compressed macroscopic fossils inminute detail (Fig. 1B). Particularly in China,these deposits contain diverse algae and, per-haps, simple animals (21). But, despite excep-tional preservation, these assemblages are notknown to include arthropods or any of theother bilaterian complexities that character-ize taphonomically comparable Cambrianshales.

A similar picture is emerging from calci-fied fossils in terminal Proterozoic carbon-ates. It has been known for nearly 30 yearsthat the problematic animal Cloudina formeda weakly mineralized skeleton by the enzy-matic precipitation of calcite in an organicmatrix (22). Grotzinger and colleagues (23)have now shown that Cloudina was only oneof a moderately diverse group of animals thatformed preservable skeletons in the latestProterozoic ocean (Fig. 1C). Calcified fossilsare abundant and well preserved in carbonateplatform settings, where they are commonlyassociated with microbial reefs. Sponges, cni-darian-grade animals, and bilaterians may allbe represented in this assemblage, but likeEdiacaran casts in interbedded sandstonesand organic compressions in contemporane-ous shales, these fossils bear only a limitedsimilarity to the diverse protostomes and deu-terostomes preserved in younger rocks.

Postmortem replication by phosphate min-erals is responsible for some of the best Cam-brian fossil preservation, and exquisitelyphosphatized remains have recently beenfound in terminal Proterozoic rocks, as well(24). The fossils include diverse protists, an-atomically preserved algae, and, remarkably,spheroidal fossils interpreted as early cleav-

age stages of animal embryos (Fig. 1E). Pre-served embryos display egg case ornamenta-tion and an unusual cleavage geometry foundtoday among the arthropods, consistent withother paleontological inferences (11, 14, 15)that bilaterian cladogenesis began well beforethe Cambrian. At present, however, the adultbody plan associated with these fossils re-mains unknown, limiting phylogenetic infer-ence. Terminal Proterozoic phosphorites donot include the fossils of crown group bilat-erians that are so obvious in their Cambriancounterparts.

Thus, while paleontological discoveriesshed increasing light on biological diversity justbefore the Cambrian explosion, new fossilshave not extended bilaterian crown group mor-phologies deeply below the Proterozoic-Cam-brian boundary. They do, however, allow us toreject the episodically popular view that thelack of recognizable crown group bilaterians inProterozoic rocks can be attributed to a dearthof suitable rocks or preservational opportunities(5). Latest Proterozoic sedimentary rocks arewidely distributed and, commonly, well pre-served and exposed. They are full of body andtrace fossils that reflect the complete spectrumof fossilization mechanisms responsible for thesucceeding Cambrian record. Crown group bi-laterians may yet be demonstrated in terminalProterozoic rocks, but the diversification ofcrown groups was principally a Cambrianevent. Cambrian diversification thus reflects anevolutionary milestone regardless of the lengthor character of earlier animal history.

Stratigraphy, geochronology, and a new

sense of evolutionary pattern. Fossils can beassembled into meaningful evolutionary pat-terns only when ordered with respect to timeand environment. Among the most importantrecent advances, therefore, is the establishmentof a robust geochronology for Proterozoic andCambrian evolution. This advance has twocomponents. First, it has been recognizedthat the isotopic compositions of C and Srin carbonate rocks and organic matter varygreatly through this time interval. Thesevariations provide a means of correlating ter-minal Proterozoic and Cambrian rocks accu-rately among many localities (25, 26). Or-ganic-walled microfossils provide additional(and independent) biostratigraphic con-straints (27 ). Second, high-precision U-Pbdates on zircons in volcanic rocks have pro-vided accurate ages of sedimentary succes-sions that contain both fossils and a record ofSr and C isotopic shifts. Together, these pro-vide an unprecedented sense of the timing ofearly animal diversification (1, 23, 28).

In consequence, an expanded view of termi-nal Proterozoic-Cambrian evolutionary historyis emerging. Independent of any molecularclock considerations, fossils of red algae andother protists indicate that the major radiation ofeukaryotic life—a divergence that includes theanimals (29)—began no later than 1200 to 1000Ma (30). Multicellularity and clade diver-gence came early to several algal groups andmay have done so in the animal clade as well,but at present the fossil record is silent onthese issues. The widespread preservation ofmillimeter-scale lamination in strata older

Fig. 1. The nature of the terminal Proterozoic fossil record. (A) Ediacaria, a radially symmetrical castpreserved on the underside of a sandstone bed, Rawnsley Quartzite, South Australia. (B) Macro-scopic alga preserved as a carbonaceous compression in shales of Doushantuo Formation, China.(C) Calcified fossils in limestones of the Nama Group, Namibia. (D) Pteridinium, a frondoseEdiacaran fossil consisting of three vanes built of repeating units (two visible in specimen) that arejoined along a central axis. (E) Phospatized animal egg and early cleavage-stage embryo, Doush-antuo Formation. (F) Simple trace fossils of bilaterian animals, Rawnsley Quartzite. Bar 5 2.5 cmfor (A), 3 mm for (B), 1.5 cm for (C) and (D), 250 mm for (E), and 2 cm for (F).

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than 600 m.y. suggests that any pre-Ediaca-ran metazoans must have been rare, small, orgossamer forms unlikely to be preserved.Should evidence of such organisms be found,it will likely occur as phosphatized remains inNeoproterozoic carbonate rocks (24).

Paleontologists have come to terms with therelatively short time frame of Ediacaran biolo-gy. Diverse Ediacaran assemblages from Aus-tralia, northern Russia, and Namibia were alldeposited within the last 15 to 20 million yearsof the Proterozoic Eon. Less completely assim-ilated are the implications of new radiometricdates for Cambrian evolution. Although mostEdiacaran fossils have no post-Proterozoicrecord, they were not immediately succeeded inlowermost Cambrian rocks by diverse crowngroup bilaterians. Earliest Cambrian assem-blages contain few taxa, and the diversity oftrace and body fossils grew only over a pro-tracted interval (3, 26, 31, 32). Hyoliths andhalkierids (extinct forms thought to be relatedto mollusks), true conchiferan mollusks and,perhaps, chaetognaths enter the record duringthe first 10 to 12 million years of the Cambrian,but crown-group fossils of most other bilaterianphyla appear later: the earliest body fossils ofbrachiopods, arthropods, chordates, and echino-derms all post-date the beginning of the periodby 10 to 25 million years (Fig. 2) (32). Tracefossils suggest earlier appearances for somegroups, notably arthropods (31), but the obser-vation remains that the Early Cambrian con-tains considerable time for the assembly anddiversification of crown group morphologies.

From this overview, we can begin to ap-preciate the Cambrian explosion as a biolog-ical event. It does not represent the origin oflife or of the animal clade. Nor, given the thepresence of mollusks near the beginning ofthe Cambrian and probable stem bilateriansin Ediacaran rocks, does it appear to representthe initial divergence of major animal clades.Rather, the Cambrian explosion records theradiation of bilaterian animals (and sponges)with modern body plans (33)—the diversifi-cation of crown groups within clades thatdiverged earlier (34). Equally important,Cambrian fossils record the initial assump-tion by animals of the prominent ecologicaland biogeochemical roles they play in mod-ern ecosystems (31).

The Genealogy and Genetics ofAnimal AncestorsBiological explanations for the patterns ob-served in the early fossil record must addressboth historical and mechanistic questions. Whatare the phylogenetic relationships among ani-mal ancestors and their modern descendants?And, because morphological diversity is theproduct of genetic differences in development,what developmental genetic mechanisms gov-erned the origin and diversification of variousforms? Progress in both systematics and devel-opmental genetics has revolutionized our per-spective on animal relationships and providednew hypotheses about early animal evolution(35).

Metazoan phylogeny: Redrawing the tree.

Constructing an accurate picture of metazoanrelationships has been challenging, and thereare many alternative pictures of animal phy-logeny. With most of the 35 or so modernmetazoan phyla represented or inferred by theend of the Cambrian, the age, rate of diver-gence, and number of branches in this treeimpose both technical and theoretical limita-tions on its resolution (36–38). Nonetheless,new molecular phylogenies based on 18S ri-bosomal RNA sequences suggest that the Bi-lateria should be reorganized into three greatclades: the deuterostomes, lophotrochozoans,and ecdysozoans (Fig. 2) (39). This phylog-eny, which has received some independentsupport (40), overturns traditional trees, nest-ing groups once viewed as primitive, such asthe flatworms and nematodes, well within thetwo protostome branches (Fig. 2) (34, 41).More recently, it has been suggested thatacoel flatworms could represent the earliestextant bilaterians (42), a clade that branchedoff the Bilateria before the radiation of thethree major clades. These new views of themetazoan tree have important implicationsfor the ancestry and evolution of Bilateriananimals (43).

One implication of the new bilaterian phy-logeny is that early-diverging groups withinderived clades may display ancestral and de-rived characters in combination. This appearsto be the best explanation for the long-stand-ing difficulties concerning the phylogeny ofthe lophophorates. These animals have beenasserted to be basal deuterostomes on mor-

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Fig. 2. Animal diversity acrossthe Proterozoic-Cambrian transi-tion. Phylogeny based on (39,40); time scale and diversity his-tory of animal classes and ordersfrom (1, 23, 27). First appearanc-es of crown and possible stemrepresentatives of phyla fromreferences cited in text, as wellas (119) for the proposed Ediaca-ran stem echinoderm and (120),for biomarker molecules ascribedto poriferans. Carbon isotopicrecord for terminal Proterozoicand Cambrian carbonates fromreferences cited in text, plus(121).


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phological grounds (44 ). Yet, molecularphylogenies (45, 46 ) and, more recently,Hox gene characters (40), place them with-in the protostomes. Debate has focused onwhich set of observations is correct, but itis now possible to rationalize both morpho-logical and molecular data (Fig. 3). Simi-larly, because priapulids display radialcleavage, yet also molt and possess Hoxgenes characteristic of ecdysozoans (40),this group may have diverged first amongthe Ecdysozoa (Fig. 3).

Clues about Urbilateria: Rocks, Hox,and molecular clocks. The three-branchedbilaterian tree (34, 39) provides no extantcandidates for the sort of animal that couldrepresent the hypothetical last common an-cestor of protostomes and deuterostomes,dubbed Urbilateria (47). Even if the recentbasal placement of acoel flatworms is con-firmed, they may represent some stage ofearly bilaterian evolution, but not the lastcommon ancestor of the three great clades.Our approach to Proterozoic animal ances-tors, therefore, relies on phylogenetic infer-ences drawn from extant animals aboutgene content, development, and potentialmorphological features. Given a picture ofUrbilateria, we can then probe more deeplyto identify some of the innovations that

must have occurred early in the evolutionof bilaterians by comparing these Urbilat-erian features with those of diploblasticanimals (for example, Cnidaria). This, inturn, makes it possible to examine innova-tions that accompanied the radiation of bi-laterians into major clades and their ulti-mate diversification within the Cambrian.

The shared genetic regulatory repertoireof protostomes and deuterostomes revealsthat Urbilateria possessed a sizable array ofintercellular signaling systems (transforminggrowth factor–B, Wnt, hedgehog, Notch, epi-dermal growth factor receptor) and a large,diverged complement of transcription factorgene families (Hox, Pax, bHLH, zinc finger,and various homeobox-type genes such as theDistal-less, engrailed, and LIM-type genes).How might the existence of these shareddevelopmental regulatory genes relate to po-tential morphological features of Urbilateria?As progress has been made in understandingthe developmental regulatory mechanismsunderlying embryonic axis formation, thepatterning of the rostral-caudal axis, and or-ganogenesis in both protostomes (primarilythe arthropod Drosophila melanogaster) anddeuterostomes (primarily vertebrates), anumber of remarkable similarities haveemerged. These include: the deployment of

an extensive cluster of Hox genes along themajor rostral–caudal axis (48, 49); the de-ployment of ParaHox cluster in different an-teroposterior regions of the gut (50); the or-ganization of the dorsal/ventral axis by thehomologous transforming growth factor–bprotein family members Dpp/BMP and theirinteracting ligands Sog/Chordin (47); the uti-lization of similar genetic machinery for theformation of appendages (Dll ) (51, 52), eyes[Pax-6/eyeless; (53, 54)], and mesodermalderivatives including muscles (55–57) andheart [tinman/NKX. 2.5; (58)]; and the peri-odic expression of the engrailed-related (59)and hairy/her1 (60, 61) genes in selectedtaxa. These developmental genetic similari-ties have led to the hypothesis that Urbilaterianot only possessed all of the genes sharedbetween arthropods and chordates, which iscertain, but also some of the morphologicalcharacters or structures that these genes reg-ulate, such as photoreception organs, append-ages, a heart, and some type of metamerism(47, 62, 63).

While the existence of such features inearly bilaterians cannot be confirmed withoutdirect fossil evidence, it seems unlikely thateach of these genetic functions in regulatingsimilar processes evolved convergently inprotostomes and deuterostomes, especially inlight of emerging evidence that similaritiesextend beyond individual genes to interactingnetworks of developmental regulators (52,64–66) and to key structural genes they reg-ulate [for example, opsins (64)]. Yet, at thesame time, it is difficult to imagine highlycomplex eyes, appendages, or hearts in an-cestral bilaterians. One possibility, that dis-misses convergence but takes a more moder-ate view of the anatomical potential, is thatstem bilaterians possessed some differentiat-ed but primitive forerunners of these struc-tures: perhaps a simple outgrowth of the bodywall or a circumoral tentacular feeding struc-ture rather than a modern locomotory ap-pendage, a contractile muscle regulating he-macoel fluids rather than a modern heart, anda simple photoreceptor complex rather thanan optically sophisticated eye. The develop-ment of these structures could constrain ge-netic evolution enough to conserve the func-tions of regulatory genes across the Bilateriawithout requiring modern morphologies inancestral animals.

Conjectures about how Urbilateria gener-ated their primary embryonic axes, germ lay-ers, body cavity, and bilateral symmetry de-pend upon inferences about which mecha-nisms observable today are primitive andwhich are derived. The three-clade bilateriantree opens the door to viewing the basicdevelopmental characteristics of deuteros-tomes as ancestral features of bilaterians rath-er than deuterostome synapomorphies (43).Urbilateria may, thus, have been small, bilate-

Fig. 3. Anatomical, developmental, and genetic innovations in the evolution of Bilateria. Inferencesabout the evolution of bilaterian features have been drawn from comparisons of developmentalmechanisms and genetics among sponges, cnidarians, ctenophores, and selected protostomes anddeuterostomes and mapped onto one presumed phylogeny [figure modified from (69)]. We notethat the relationships among lower metazoans and bilaterians and the evolution of particularcharacters (highlighted in blue) are both uncertain and of central importance. Characteristics sharedbetween protostomes and deuterstomes are deduced to have existed in some form in their lastcommon ancestor, Urbilateria. The evolution of gene functions in controlling specific developmen-tal features are shown in red. Figure based on references (34, 39, 40, 50, 52, 63, 69), and text.


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rally symmetrical creatures with radial cleav-age, coelomic cavities formed by the out-pouching of the archenteron (enterocoely),and anterior tentacular appendages formed bycoelomic outpouching.

Thus, at the genetic, morphological, anddevelopmental levels, Urbilateria were fairlysophisticated, not flat amorphous worms. Pa-leontology indicates that these ancestors livedbefore the Cambrian—at least 560 Ma andpossibly more than 600 Ma. Molecular clockestimates are controversial and yield dispar-ate results (5, 67, 68), but even those esti-mates most nearly concordant with paleonto-logical data suggest a prostome-deuterostomesplit at least 100 m.y. before bilaterians ap-pear in the fossil record (67). Collectively,then, available data suggest that two substan-tial periods of bilaterian evolution precededthe Cambrian: the evolution of the bilaterianstem lineage leading to Urbilateria and thesubsequent diversification of the three majorbilaterian clades. We must understand whathappened during these two intervals to delim-it what eventually transpired in the Cambrian.

The Diploblast-Bilaterian gap: Pre-Edia-caran genetic and developmental innovation.Comparisons of bilaterians to other metazoans,particularly at the anatomical and genetic lev-els, suggest that there was a tremendous amountof developmental evolution within the inter-node between the last common ancestor ofdiploblastic animals such as the Cnidaria andUrbilateria (50, 69). The discontinuity betweencnidarian and bilaterian body plans as well asthe probable complexity of Urbilateria suggeststhat the evolution of bilaterian characters musthave required many innovations, including athird germ-layer, bilateral symmetry, a central-ized nerve cord, a through gut, various primi-tive organs, and the genetic systems to organizeand pattern these features (Fig. 3). It is not clearto what degree any extant taxon represents anintermediate in the anatomical or genetic evo-lution of bilaterian organization. The phylumCtenophora (comb jellies) has been proposed asa sister group to the Bilateria (70, 71), and theacoel flatworms as an early branch of the Bila-teria (42). Both groups possess characters thatcan place them within the Cnidaria-Urbilateriainternode (Fig. 3). However, the phylogeneticplacement of the ctenophores remains contro-versial, and other data variously suggest closerelationships to cnidarians, placozoans (72), oreven deuterostomes (44). Similarly, the place-ment of the acoels awaits further examination.

Whatever the phylogenetic relationshipamong bilaterians and the diploblastic phyla,there was clearly a significant expansion ingenomic information within the internodethat connects the bilaterian clade to otherbranches. For example, surveys of Hox genediversity in cnidarians have suggested thatthe last common ancestor of cnidarians andbilaterians had as few as one [probably two,

(73–76)] ortholog of bilaterian Hox genes,only one ancestral Pax homeobox gene (77),a smaller array of intercelluar signaling mol-ecules, and generally fewer transcription fac-tor types than we know existed in Urbilateria.A limited survey of ctenophores also did notreveal an expanded Hox family (78). Thedifferences in genome content and regulatorygene family size and diversity between extantdiploblastic animals and bilaterians appearmuch greater than those known among anybilaterian taxa (except chordates). At the Hoxgene level alone, it is certain that at leastseven genes had evolved by duplication andwere functionally diverged in Urbilateria, farmore genes than existed in the common an-cestor of diploblasts and bilaterians (40). Itseems inescapable then that the phylogeneticinternode leading to Urbilateria represents asubstantial period of pre-Ediacaran history.Moreover, we can speculate that a spectrumof intermediate and divergent body planscould have existed that represented differentstages of the developmental and genetic evo-lution of Bilateria. These predicted bodyplans provide both a challenge and searchimages for paleontology.

The role of the expanding systems of sig-naling proteins and transcription factors inthe evolution of stem bilaterians requires con-sideration of the primitive mode of bilateriandevelopment, a question which has recentlyreceived considerable attention (43, 69, 79–84). It has been argued that most bilaterians,except (ironically) insects and vertebrates,share mechanisms of embryonic specificationtermed type I embryogenesis that are distinctfrom the much less ordered styles of embry-onic development in diploblastic organisms(80). In type I development, a series of 10 orso divisions produces cell lineages whichgive rise to particular cell types that are gen-erally invariant within species. The positionand fate of these lineages are specified byshort-range interactions among adjacent ornearby cells. The shared bilaterian signalingsystems and cell-type specific transcriptionfactors appear sufficient to generate a smallfeeding animal composed of the basic gut,muscle, neural, and ectodermal cell typesfound in all bilaterians. Based on the wide-spread occurrence of type I embryogenesis inextant phyla, Davidson et al. (80) argued thatthis is the ancestral mode of embryogenesisthat operated in early microscopic bilaterians.

The short-range regulatory interactionsthat operate in type I embryogenesis are notsufficient, however, to generate the macro-scopic body plans characteristic of most mod-ern bilaterians. Therefore, the evolution oflarger body plans must have involved furtherdevelopmental innovations. Davidson et al.(80) proposed that the key innovation in theorigin of macroscopic body plans was theevolution of so-called “set-aside” cells. These

cells are released from the constraints onproliferation and specification imposed bytype I processes and become organized intothe adult bilaterian by regional pattern-form-ing mechanisms. Furthermore, it has beenargued that maximal indirect development, inwhich the adult develops from an imaginalrudiment of a larva, is a general, widespread,and ancestral mode of bilaterian development(82). This view has been challenged, basedon concerns that the widespread occurrenceof planktotrophic larvae may be convergent(33, 81, 83–85).

Evidence to support the view that type Iembryogenesis is primitive and was sufficientfor micrometazoan development has recentlyemerged from analysis of Hox gene utilizationin embryos that give rise to maximal indirectlydeveloping larvae. In modern bilaterians, highlyconserved Hox gene products sculpt the mor-phology of adult bodies regulating diverse setsof target genes in a region-specific manneralong the rostral-caudal axis. However, in thedevelopment of the echinoderm pluteus-stagelarva, only two of the conserved set of tendeuterostome Hox genes are deployed (86).This demonstrates that the generation of a free-living feeding bilaterian may not require a clus-ter of Hox genes. Similarly, Hox genes arelargely not required for embryogenesis in thenematode Caenorhabditis elegans (87), nor arethey deployed during polychaete annelid larvaldevelopment (88). If type I embryogenesis isindeed primitive, these observations could indi-cate that the evolution of Hox gene diversityoccurred in concert with the set-aside progeni-tor cells to generate adult body patterns in stembilaterians. If, on the other hand, the earliest bila-terians developed directly from embryos toadults (as, for example, extant acoels do), thenthe restrictions imposed by type I embryogen-esis must have been circumvented before theevolution of set-aside cells. In either scenario,all the extant developmental regulatory mecha-nisms for generating macroscopic animalswould have been put in place, setting the stagefor the bilaterian radiation. Such a view doesnot require that adult body plans evolved inde-pendently in all bilaterian phyla; in fact, theportrait of Urbilateria painted in previous para-graphs makes that unlikely.

Urbilateria branches out: Proterozoiccladogenesis. The extent of bilaterian radi-ation at the dawn of the Cambrian is uncer-tain. However, fossils as well as molecularclock estimates suggest that the proto-stome-deuterostome divergence, the sepa-ration of the two protostome clades, andsome radiation within these clades, predat-ed the Cambrian. Thus, the developmentalcharacters that distinguish major cladeswould have also evolved by this time. Spi-ral cleavage, molting, and other featuresassociated with specific protostome cladesmust already have been in place (Fig. 3).


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While there is now a better prospect forinferring sister group relationships among ex-tant taxa, the origins of the various bilaterianbody plans remain obscure. We can group pri-apulids with arthropods or annelids with mol-lusks, but the morphological transitions thatgave rise to the phyla cannot be extrapolatedfrom their living descendants. It will require afar richer Proterozoic and earliest Cambrianfossil record to solve the problem paleontologi-cally. And the keys to the origin of body plansare not likely to be the sort of genetic innova-tions that occurred in the bilaterian stem lin-eage; protostomes and basal deuterostomeshave fairly comparable batteries of develop-mental regulatory genes (40, 49). Rather, thedifferences in body plans are likely to havearisen at the level of the regulatory networksthat organize pattern formation. This idea isillustrated most clearly by studies on the diver-sification of one of the most successful bilat-erian body plans: the arthropods.

Cambrian diversification: So many arth-ropods, so little time. Arthropods are the mostabundant Cambrian fossils, and by the time ofthe Burgess Shale, they had evolved a numberof different body plan designs and many vari-ations upon these themes (18, 89–91). Thesister group to the arthropods, the lobopo-dans, is also well known, which has inspiredefforts to synthesize a picture of arthropodstem-lineage evolution from lobopodan-likeancestors (92). The modern descendants ofthese forms, including the chelicerates, crus-tacea, myriapods, insects, and onychophoraillustrate all the elements of the body plandiversity apparent in Cambrian assemblages,

including the major differences in tagmosis,segment and appendage number, and limbmorphology (Fig. 4, right). Investigations intothe developmental mechanisms that specifydifferences among extant arthropods (andonychophorans) can thus provide a windowon the developmental processes underlyingthe Cambrian radiation.

The most obvious trend in the evolution ofthe onychophoran-arthropod clade has been theevolution of increased segment diversity from agenerally homonomous condition in basalforms to various heteronomous forms in Cam-brian and recent taxa. Lobopodans had only afew appendage types and a homonomous trunk,whereas arthropods are more distinctly tagma-tized and possess a wider array of appendagetypes. Since segment and appendage diversityin the highly derived insects is regulated by Hoxgenes (93), most work has focused on the rolesthese genes play in the development and evo-lution of arthropod diversity. Three generalpoints have emerged from comparative studiesof Hox gene organization and expression inchelicerates (94, 95), crustacea (96–98), myri-apods (99), insects (100), and onychophora(99). First, the entire onychophoran-arthropodclade possesses essentially the same set of Hoxgenes that pattern the main body axis (Fig. 4;left) (99). Thus, Cambrian and recent diversityevolved around an ancient and conserved set ofHox genes. Second, the increase in segmentdiversity is correlated with changes in the rela-tive domains of Hox gene expression along themain body axis (Fig. 4, right). This result isillustrated by the evolution of appendage diver-sity among the crustacea in which the modifi-

cation of thoracic limbs into feeding appendag-es correlates precisely with the modification ofHox gene expression (96). And third, changesin the morphology of homologous appendagesare correlated with changes in the array ofgenes that are regulated by the same Hox gene(101).

The mechanisms underlying the evolutionof developmental changes in the arthropods,while generally limited thus far to inferencesabout Hox genes, may, nonetheless, havesome general explanatory power. First, theconservation of the Hox gene family bothwithin and among phyla suggests that mostbody plan evolution arose in the context ofvery similar sets of Hox genes, and thus wasnot driven by Hox gene duplication. Second,the trend toward the evolution of heterono-mous body plans is apparent in other groups,such as annelids. Third, in other derivedgroups that have been examined such as thevertebrates, the correlation between diversi-fication of Hox gene expression patterns andthe evolution of anterior-posterior pattern-ing along the body axis applies (102). Theseobservations suggest that bilaterian bodyplan diversification has occurred primarilythrough changes in developmental regulatorynetworks rather than the genes themselves,which evolved much earlier.

The Environmental Context of AnimalDiversificationIf assembly of the developmental toolkit wasnot by itself the trigger for the Cambrian explo-sion, can we identify environmental events thatcould have released the morphogenetic poten-

Fig. 4. Evolution of the ony-chophoran-arthropod cladeand Hox gene regulation.(Left) The last common an-cestor of protostomes anddeuterostomes possessed atleast seven diverged Hoxgenes. The common ances-tor of the protostomes pos-sessed an additional centralclass gene that gave rise tothe Ubx and abdA genes inthe onychophoran-arthro-pod ancestor. The diversityof this clade has evolved around a conserved set of nine Hoxgenes. (Right) Modern onychophorans and arthropods differin segment number, identity, and morphology. Within aspecies, different Hox genes sculpt the morphology of bodyparts. The relative boundaries of Hox genes (depicted incolors corresponding to the gene identities shown on the left;many expression patterns are not yet known) have divergedamong arthropods but often correlate with transitions inappendage morphology. The heteronomy and diversificationof the arthropod body plan through diversification of Hoxgene regulation illustrates in general that regulatory changesin conserved developmental genes underlie body plan evo-lution. Hox gene evolution scenario relies on (40). Geneexpression data is summarized from (94–97, 99).


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tial of these genes? The most likely candidatefor such an environmental gate keeper is mo-lecular oxygen (103). Simple biophysical con-siderations relate maximum body size to oxy-gen availability in animals that obtain oxygenby diffusion, a likely prospect in early metazo-ans (104). (The relationship applies principallyto bilaterian animals with potentially thick mus-cles and mesodermally derived internal organs;diploblasts can become macroscopic by limit-ing metabolically active cells to a thin superfi-cial layer that may be complexly folded toincrease surface area). A late Proterozoic in-crease in atmospheric oxygen levels could,thus, reconcile the known fossil record withmolecular inferences that call for an extendedinterval of metazoan prehistory, played out bysmall animals unlikely to be preserved (91,105).

Biogeochemical investigations of Neopro-terozoic rocks reveal patterns of stratigraphicvariation that stand out against the backdropof the past 2 billion years. In particular, un-usually strong positive excursions in the car-bon isotopic composition of carbonate min-erals and organic matter indicate that rates oforganic carbon burial in the late Proterozoicocean basins were episodically high (25,106). Insofar as the burial of photosyntheti-cally derived organic matter provides a mech-anism for oxygen build-up in the atmosphereand oceans (107), carbon isotopic data pro-vide direct empirical support for the oxygenfacilitation hypothesis. The biogeochemicalrecord of sulfur independently suggests thatoxygen increased in the late Proterozoic(108).

Oxygen would not in and of itself havecaused animals to evolve. Rather, it wouldhave removed an environmental barrier to the

evolution of large, metabolically active ani-mals. Canfield (109) has recently modeledearlier Proterozoic oceans as moderately oxicat the surface but anoxic below the mixedlayer, making predictions that can be testedagainst observations of Proterozoic sedimen-tary rocks. If confirmed, this model suggeststhat regardless of genetic potential, large bi-laterian animals could not have diversifiedbefore the late Proterozoic Eon.

Late Proterozoic carbon isotopic profilesdisplay strong negative as well as positive ex-cursions. Negative excursions are specificallyassociated with the major ice ages that markimmediately pre-Ediacaran time. Much re-search is currently focused on this unusual cou-pling of climate and biogeochemistry (110),and both paleoceanographic models and clus-tered phytoplankton extinctions (111) suggestthat these ice ages had a severe impact on thebiota—potentially applying brakes to early an-imal evolution (24, 110). All diverse Ediacaranfossil assemblages post-date the last major Pro-terozoic ice age.

Although environmental change can thushelp explain why large animals could flourishlate in the Proterozoic Eon, it does not appear toilluminate Cambrian diversification per se. Pos-sibly, once rising oxygen levels removed phys-ical barriers to the evolution of large size, noother extrinsic drivers were required; morpho-logical innovations such as complex nerve netsor sensory, locomotory, and feeding appendag-es made the Cambrian explosion inevitable.This may be, but one other recent biogeochemi-cal discovery strongly suggests that evolutionreceived a further nudge from the environment.

In the past few years, evidence has accu-mulated for a remarkable perturbation in thecarbon cycle close to the Proterozoic-Cam-brian boundary. Globally distributed sedi-mentary successions document a strong (7 to9 per mil) but short-lived negative excursionin the carbon-isotopic composition of surfaceseawater at the stratigraphic breakpoint be-tween Ediacaran-rich fossil assemblages andthose that document the beginning of trueCambrian diversification (112, 113). Thecauses of this event remain uncertain, but theonly comparable events in the more recentEarth history coincide with widespread ex-tinction—for example, the Permo-Triassiccrisis, when some 90% of marine speciesdisappeared, is marked by an excursion sim-ilar to but smaller than the Proterozoic-Cam-brian boundary event (114). An earliest Cam-brian increase in bioturbation shuttered thetaphonomic window on Ediacaran biology.Thus, while Chengjiang and Sirius Passetfossils indicate that Ediacarian-grade organ-isms were not ecologically important by thelate Early Cambrian, biostratigraphy admitsthe possibility that Ediacarans were eaten oroutcompeted by Cambrian animals. It is bio-geochemistry that lends substance to the hy-

pothesis that Ediacaran and Cambrian faunasare separated by mass extinction.

It is possible, then, that the evolution ofearly animals parallels the better resolved his-tory of land vertebrates (Fig. 5). Mammalsdiverged early in the Mesozoic Era, anda moderate diversity of mammalian stemgroups lived for millions of years in dinosaur-dominated ecosystems. Only with the demiseof the dinosaurs did mammals radiate to pro-duce the crown group diversity seen in Ter-tiary and modern faunas. Perhaps Ediacarananimals are the “dinosaurs” of the terminalProterozoic oceans, simple but successful or-ganisms that placed ecological constraints onbilaterian evolution. In this view, end-Prot-erozoic environmental perturbation createdecological opportunity, facilitating the simul-taneous diversification of multiple clades ofbilaterian survivors (113).

In reorganized Cambrian ecosystems,interactions among organisms further cata-lyzed biological diversification. Predationlikely facilitated the evolution of skeleton-ized invertebrates (115), and, despite theirearlier appearance, algae record a Cam-brian explosion of phytoplankton diversitythat parallels that of animals. Such obser-vations bring a new ecological dimensionto the story (27, 116 ), requiring that Cam-brian diversification be seen as an ecosys-tem-wide phenomenon that affected pro-tists, sponges and bilaterians alike. As not-ed above, morphological diversification inthe Cambrian oceans reflects changes ingene regulation. Therefore, the role of end-Proterozoic environmental disruption—likeoxygen before it—would have been to in-troduce a new selective landscape in whichthese novel variants could persist. As newbody plans evolved, the biological compo-nents of environment, themselves, becameprincipal features of this new landscape.

ConclusionsWe cannot yet claim to have solved the majorquestions of early animal evolution. But wehave reached a point where pathways to un-derstanding are becoming clearer. Testinghypotheses about the identity, age, complex-ity, and diversity of early animals and theenvironments in which they lived has becomean interdisciplinary exercise, and we see fourareas that are potentially the most productivepursuits. First, we need to mine the late Pro-terozoic record for bilaterian fossils and pa-leoenvironmental insights. Second, to under-stand the assembly of the bilaterian tool kit,investigations into the developmental genet-ics (and phylogeny) of sponges, cnidarians,and ctenophores are crucial. Third, the originof key developmental characters (for exam-ples, mesoderm, spiral cleavage, set asidecells) may be elucidated by broadening thebase of comparative developmental biology

Fig. 5. Comparison of evolutionary dynamicsacross the Proterozoic-Cambrian and Creta-ceous-Tertiary boundaries. The history of ter-restrial vertebrates, in which mammals radiatedonly after dinosaurs suffered mass extinction,may provide a framework for understandingthe successive radiations of Ediacarian-gradeand crown-group bilaterian animals.


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to less well-studied taxa such as mollusks,polychaete annelids, and invertebrate deuter-ostomes (117). And finally, understandingthe molecular developmental basis of evolu-tionary change is crucial. The architectureand evolution of the genetic regulatory net-works that pattern animals must be elucidatedin much greater depth if we are to reconstructthe genetic history of the major transitions inanimal evolution.

We can see clearly now that intrinsic andextrinsic hypotheses are not really alternativeways of explaining animal diversification.There were certainly intrinsic catalysts of earlyanimal evolution. The assembly and regulatorydiversification of the genetic toolkit for animaldevelopment undoubtedly underpin Proterozoicand Cambrian evolution. And the evolution ofcomplex appendages, organs, and sophisticatednervous and musculo-skeletal structures musthave facilitated diversification (118). Yet, ex-trinsic events also helped to shape early animalevolution altering environments in ways thatdoomed some clades and created opportunityfor others.

Discipline-bound intrinsic or extrinsic ex-planations of early animal history fail not somuch because they are wrong as because theyare incomplete. The Cambrian explosion—the stratigraphic pattern seen in those cliffsalong the Kotuikan River—is the historicalproduct of the interplay between genetic pos-sibility and environmental opportunity, am-plified by ecological interactions to extendacross all of biology.

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R E V I E W

The Evolution of DinosaursPaul C. Sereno

The ascendancy of dinosaurs on land near the close of the Triassic nowappears to have been as accidental and opportunistic as their demise andreplacement by therian mammals at the end of the Cretaceous. Thedinosaurian radiation, launched by 1-meter-long bipeds, was slower intempo and more restricted in adaptive scope than that of therian mam-mals. A notable exception was the evolution of birds from small-bodiedpredatory dinosaurs, which involved a dramatic decrease in body size.Recurring phylogenetic trends among dinosaurs include, to the contrary,increase in body size. There is no evidence for co-evolution betweenpredators and prey or between herbivores and flowering plants. As themajor land masses drifted apart, dinosaurian biogeography was moldedmore by regional extinction and intercontinental dispersal than by thebreakup sequence of Pangaea.

During the past 30 years, intensified paleon-tological exploration has doubled recordeddinosaurian diversity (1) and extended theirgeographic range into polar regions (2). Ex-ceptional fossil preservation has revealedeggshell microstructure (3), nesting patternsand brooding posture among predators (4),and epidermal structures such as downy fila-ments and feathers (5, 6). Analysis of bonemicrostructure and isotopic composition hasshed light on embryonic and posthatchinggrowth patterns and thermophysiology (7).Footprint and track sites have yielded newclues regarding posture (8), locomotion (9),and herding among large-bodied herbivores(10). And the main lines of dinosaurian de-scent have been charted, placing the afore-mentioned discoveries in phylogenetic con-text (11).

The most important impact of this en-riched perspective on dinosaurs may be itscontribution to the study of large-scale evo-lutionary patterns. What triggers or drivesmajor replacements in the history of life?How do novel and demanding functionalcapabilities, such as powered flight, firstevolve? And how does the breakup of asupercontinent affect land-based life? Thecritical evidence resides in the fossilrecord—in the structure, timing, and geog-raphy of evolutionary radiations such asthat of dinosaurs.

Early Dinosaurs: Victors by AccidentDid dinosaurs outcompete their rivals or sim-ply take advantage of vacant ecologicalspace? The ascendancy of dinosaurs on landtranspired rather rapidly some 215 millionyears ago, before the close of the Triassic.Herbivorous prosauropods and carnivorouscoelophysoid ceratosaurs spread across Pan-gaea, ushering in the “dinosaur era”: a 150-

million-year interval when virtually all ani-mals 1 m or more in length in dry landhabitats were dinosaurs.

Dinosaurs, the descendants of a single com-mon ancestor, first appeared at least 15 millionyears earlier but were limited in diversity andabundance (Fig. 1). Well-preserved skeletonsdiscovered recently in 230-million-year-oldrocks (mid-Carnian in age) provide a glimpseof a land radiation already underway (12). Themost fundamental adaptations for herbivoryand carnivory among dinosaurs had alreadyevolved. A novel means for slicing plant matter,utilizing inclined tooth-to-tooth wear facets, isfully developed in the meter-long herbivorePisanosaurus, the oldest known ornithischian(Fig. 1, left; Fig. 2, node 1; Fig. 3A, feature 4).Jointed lower jaws and a grasping hyperextend-able manus for subduing and eviscerating preyare present in the contemporary predatorsEoraptor and Herrerasaurus, which are theoldest well-preserved theropods (Fig. 1, right;Fig. 2, node 41; Fig. 3B, features 11 and 12).

Traditional scenarios for the ascendancy ofdinosaurs that invoke competitive advantage(13) have difficulty accommodating the sub-stantial temporal gap (15 million years or more)between the initial radiation of dinosaurs andtheir subsequent global dominance during thelatest Triassic and Early Jurassic (14). Oppor-tunistic replacement of a diverse array of ter-restrial tetrapods (nonmammalian synapsids,basal archosaurs, and rhynchosaurs) by dino-saurs is now the most plausible hypothesis (11,14, 15). This pattern is broadly similar to thereplacement of nonavian dinosaurs by therianmammals at the end of the Cretaceous. Recent

Department of Organismal Biology and Anatomy,University of Chicago, 1027 East 57th Street, Chicago,IL 60637, USA.


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e early animal evolution: emerging views from comparative

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