Review articles

Origin and early evolution of the vertebrates: new insights from advances in molecular biology, anatomy, and palaeontology
Nicholas D. Holland1* and Junyuan Chen2
Summary Recent advances in molecular biology and microanatomy have supported homologies of body parts between vertebrates and extant invertebrate chordates, thus providing insights into the body plan of the proximate ancestor of the vertebrates. For example, this ancestor probably had a relatively complex brain and a precursor of definitive neural crest. Additional insights into early vertebrate evolution have come from recent discoveries of Lower Cambrian soft body fossils of Haikouichthys and Myllokunmingia (almost certainly vertebrates, possibly related to modern lampreys) and Yunnanozoon and Haikouella (evidently stem-group vertebrates). The earliest vertebrates had an unequivocally marine origin, probably evolved mineralised pharyngeal denticles before the dermal skeleton, and evidently utilised elastic recoil of the visceral arch skeleton for suction feeding. Moreover, the new data emphasise that the advent of definitive neural crest was supremely important for the evolutionary origin of the vertebrates. BioEssays 23:142151, 2001. 2001 John Wiley & Sons, Inc. Introduction The evolutionary origin of the vertebrates (used here in the broad sense to include hagfishes and lampreys) was vigorously debated during the latter part of the nineteenth century,(1) but excited little interest during much of the twentieth for several reasons. (1) Evolutionary reconstructions based on embryonic recapitulation had fallen into disrepute, (2) the New Synthesis was more concerned with small-scale evolution than big-picture phylogeny, and (3) no definitively vertebrate fossils had yet been found that antedated the Ordovician(2) or had even a hint of missing link about them. A strong revival of interest in the origin of the vertebrates, however, began about twenty years ago under the stimulus of new discoveries in molecular biology, anatomy, and palaeontology. Vertebrate origins can be sought at various levels within the tree of animal life; for example, one can debate whether vertebrates can be traced back to a creature resembling a larval deuterostome(3) or to a relatively complex urbilaterian.(4) Our focus is narrower here, however, namely on the origin of the vertebrates from their proximate invertebrate ancestor, which is widely assumed to have been an invertebrate chordate. Our purpose is to show how ideas about the origin of the vertebrates have recently been refined by molecular and morphological results from extant invertebrate chordates and by palaeontological discoveries, especially from the Lower Cambrian of south China. One of our major conclusions is that recent advances in biology and palaeontology support the idea that the advent of definitive neural crest was of overriding importance for the evolutionary origin of the vertebrate body plan.(5,6) Invertebrate chordates are not degenerated vertebrates Recent molecular data are inconsistent with the idea(7) that invertebrate chordates are degenerated vertebrates. In recent years, molecular studies have revealed that, in comparison to vertebrates, invertebrate chordates tend to have considerably fewer genes per gene family. This pattern is readily explicable if small gene families of an ancestral invertebrate chordate multiplied by genome or chromosome duplications in the early vertebrates.(8,9) The converse scenarionamely that a large number of vertebrate genes was reduced during the evolution of invertebrate chordateswould require a very improbable combination of gene removal events.(8) Thus there is little doubt that an invertebrate chordate ancestor gave rise to the vertebrates and not vice versa. The cephalochordate amphioxus as the best available model for the proximate ancestor of the vertebrates Living invertebrate chordates comprise the tunicates (the most familiar of which are the ascidians) and the cephalochordates

Division of Marine Biology, Scripps Institution of Oceanography. Nanjing Institute of Geology and Palaeontology, Academia Sinica, China. Funding agencies: US NSF (Grant IBN96-309938), the National Foundation of Natural Sciences of China (Grant 49872004), and the Ministry of Science and Technology of China (Grant G2000077700). Correspondence to: Nicholas D. Holland, Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, CA 920930202.
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(amphioxus = lancelets). Within the chordates, tunicates are considered to be the sister group of the cephalochordate plus vertebrate clade.(10 12) On the one hand, with existing technology, ascidians, as compared to amphioxus, have embryos that are easier to work with experimentally and thus more favorable for functional and genetic studies.(13) On the other hand, ascidians have apparently tended toward simplification both morphologically (e.g. atrophy or loss of coeloms and posterior gut) and genetically (divergence of Hox genes and loss of some of them).(14) It is thus likely that cephalochordates, in comparison to ascidians, conserve a wider suite of primitive chordate characters. Cephalochordates and vertebrates have been evolving separately for at least half a billion years, which is ample time for independent gain or loss of characters and for the possible disjunction of genetic-level homologies from morphological homologies. Even so, living invertebrate chordates, and especially the cephalochordate amphioxus, afford the best available glimpse of the proximate invertebrate ancestor of the vertebrates and are likely to exemplify many of the starting conditions at the dawn of vertebrate evolution. Amphioxus is a marine filter feeder, a few centimeters long, that spends most of its time in shallow burrows, but sometimes swims by eel-like undulations.(15) A living adult amphioxus is translucently white and has the appearance of a small fish without any obvious lateral fins, jaws, or paired sense organs at the anterior end. The salient chordate features of amphioxus are a notochord, a hollow dorsal neural tube, pharyngeal gill slits, and segmented muscle blocks called myomeres (this last character can be considered general for chordates only if one assumes that tunicate tail muscles represent degenerated myomeres). Homologies between amphioxus and vertebrates, as exemplified by brain regions and neural crest-related tissue During the last decade, data suggesting new body part homologies and supporting old ones have come from studies of molecular genetics (Table 1) and from microanatomical studiesespecially computer-assisted, three-dimensional reconstructions based on serial transmission electron microscopy. Much of this information has been reviewed elsewhere,(16 18) and only highlights will be mentioned here, with special attention to the evolutionary origin of major brain regions and neural crest in the vertebrates. The amphioxus central nervous system consists of a dorsal hollow tube with a slight anterior dilation (the cerebral vesicle). This gross morphological simplicity has been the cause of heated debates over which, if any, of the major brain regions of vertebrates might be present in amphioxus. Fortunately, much of this controversy has been settled by the molecular and microanatomical studies summarised in Fig. 1. Data from both approaches indicate that amphioxus has a diencephalic

Table 1. Gene expression relevant for body part homologies between amphioxus and vertebrates
Body part 1. Brain Gene expression Otx BF1 Hox3 Hox1 Hox4 Islet Pax6 Pax6 Msx Hedgehog HNF3 Netrin distal-less snail Pax3/7 Msx Brachyury (T) HNF3 Pax1 Pax2/5/8 Pax2/5/8 Pax2/5/8 Nk2-1 Thyroid peroxidase Xlox, Ilp Neurogenin paired eyes. pineal photoreceptor cells. pronephros. thyroid gland. pancreatic endocrine cell. References 23 24 25 14 26 27 28 28 29 30 31 32 22 33 34 29 35 31 36 20 20 20 37 38 39 40

2. Frontal eyea 3. Lamellar organb 4. D/V pattern of nerve cord

5. Neural crest precursor

6. Notochord 7. Gill slits 8. Hatschek's nephridiumc 9. Endostyled 10. Gastric endocrine cellse

a b

Likely homolog Likely homolog c Likely homolog d Likely homolog e Likely homolog

of vertebrate of vertebrate of vertebrate of vertebrate of vertebrate

forebrain, a hindbrain, and (more arguably) a small midbrain. Within the amphioxus diencephalon, there is a frontal eye (presumably homologous to vertebrate paired eyes) and a lamellar organ (the likely homologue of vertebrate pineal photoreceptors).(19) Amphioxus appears to lack a telencephalic forebrain, and there is probably no specialised isthmocerebellar region with organiser properties.(20,21) It is thus likely that both of these features are vertebrate innovations. Developmental gene expression patterns in amphioxus also indicate the presence of an embryonic cell population with some similarities to vertebrate neural crest. During vertebrate development, the neural crest cells originate at the boundary between the neural plate and epidermis, where they express a characteristic suite of genes. During amphioxus embryology, a similarly located population of cells expresses homologues of several of these neural-crest-specific genes (Table 1). During subsequent vertebrate development, the neural crest cells migrate and differentiate, thereby contributing to many structures (e.g. cranium, branchial arch skeleton, and sensory

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Figure 1. Dorsal views of anterior end of developing central nervous system of A: amphioxus and B: lower vertebrate. In A, the cerebral vesicle is stippled, and the numbered rectangles represent the muscular somites. In B, abbreviations are: TFB, telencephalic forebrain; DFB, diencephalic forebrain, MB, midbrain; HB, hindbrain (r1r6, rhombomeres 16); SC, spinal cord. The morphological features at center are the following proposed homologies:(37) EYE, amphioxus frontal eye versus vertebrate paired eye; PPR, pineal photoreceptors in amphioxus lamellar body versus vertebrate epiphysis; SRF, source of Reissner's fiber from amphioxus infundibular organ versus vertebrate subcommissural organ; OT, optic tectum (``? '' indicates this homology has been questioned, Ref. 86); PMC, anterior limit of primary motor center. Rostrocaudal expression domains of amphioxus and vertebrate developmental genes are indicated, respectively, to the left of A and to the right of B. Orthologous genes (references in Table 1) have matching colors; the diagram does not indicate that the Islet gene is expressed in a segmented pattern in register with the first eight somites of amphioxus and in each of the eight rhombomeres of vertebrates.

ganglia). In the amphioxus embryo, some of the cells at the edges of the neural plate are incorporated into the dorsal region of the neural tube, while the adjacent epidermal cells migrate over the neural plate by means of lamellipodia.(22) In contrast to vertebrate neural crest cells, these amphioxus cells neither enter the blastocoel nor differentiate into a wide variety of cell types. However, despite the limited repertory of the amphioxus cells, it is tempting to speculate that the proximate invertebrate ancestor of the vertebrates had comparable cells in the border zone separating the neural plate and epidermis and that this cell population constituted the evolutionary precursor of definitive vertebrate neural crest. Unsettled issues of homology between amphioxus and vertebrates Despite the progress summarised in Table 1, some important homologies between amphioxus and vertebrates remain

controversial (Table 2). Often these questions involve deciding whether a particular character is primitive in amphioxus (and, by extension, was present in the proximate ancestor of the vertebrates) or appeared later in the cephalochordate line of descent. For example, it remains controversial whether features like the peripharyngeal space (atrium), the extension of the notochord to the anterior end of the animal, and the myomeres in the head region are primitive or novelties, possibly related to a predominantly burrowing life style. Further, although amphioxus is often described as heartless,(18) the fine structure of the subendostylar vessel is similar to that of the ascidian heart and might qualify as a homologue of the vertebrate myocardium.(41) Finally, there is much confusion about the composition of amphioxus visceral arch skeleton. Although these fairly rigid elements have been termed chitinous(42) or cartilaginous,(43) they are more likely composed of mucoscleroproteins(18) that have not yet been well characterised chemically.

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along the dorsal edge of the animal (an inefficient arrangement for opposing muscular contraction), except at the anterior end.(52) The possible occurrence of sediment deposited internally near the anterior end might indicate the presence of a pharynx.(51) Although the fossils show no trace of any visceral clefts or arches, the anteroventral surface bears a fringe of short projections, which might conceivably have been associated with pharyngeal openings(51) or gills.(52) The anterior end of the body bears a small pair of anterior protrusions of uncertain function; their depiction(53) as eyestalks, while intended to be humerous, runs the risk of being taken at face value by many readers. In summary, the cephalochordate affinity of Pikaia is at best only weakly indicated by the characters visible in fossils discovered so far. Therefore, some have speculated that Pikaia represents an even more basal chordate(45) (possibly derived from vendozoans, which are enigmatic fossils from the late Precambrian)(54) or possibly a protostome-like creature.(55) It has even been suggested that the anteroposterior axis has been reversed in the conventional interpretation and that Pikaia fossils might represent the remains of hagfish-like animals.(56)

Table 2. Outstanding questionsa about amphioxus anatomy

Question 1. Is the presence of myomeres at the anterior end primitive or derived? 2. Is the extension of the notochord to the anterior end primitive or derived? 3. Is the presence of the peripharyngeal space (atrium) primitive or derived? 4. Is the cyclopic frontal eye primitive or derived from originally paired eyes? 5. Does amphioxus have a homolog to at least part of the vertebrate heart? 6. What is the composition of the visceral arch skeleton? 7. Does the circulatory system have an endothelial lining? References

8, 44 32 45 19, 32 41 18

15, 18

a Two old controversies about amphioxus anatomy have recently been settled. First, the excretory tubules are mesodermal(46) and not ectodermal as often claimed, and, second, the so-called ``fin rays'' of amphioxus are not homologous to either the radials or the dermal fin rays of vertebrates. Instead, the striations in amphioxus median fins are either ciliary rootlets within epidermal cells(47) or the apposed walls of contiguous fin box coeloms or, under some physiological conditions, retromesothelial accumulations of haemal fluid projecting into the fin box coeloms.(48)

Definite and proposed chordates from the early Palaeozoic

During the last twenty years, one of the outstanding advances in vertebrate palaeontology has been the discovery of soft body fossils of euconodonts, the oldest one dating from the Ordovician. There is little doubt that these fossils represent vertebrate chordates, probably the most basal gnathostomes. Euconodont fossils have been comprehensively reviewed recently(49) and will not be covered here. Instead, we will concentrate on putative chordate fossils from the Cambrian.

Euconodont animals

During the last decade, soft body fossils of presumed chordates have been discovered from the Lower Cambrian (520 million years ago) of south central China. These are the remains of animals that averaged a few centimeters long and inhabited a subtidal, eutrophic marine environment where the bottom was soft and where the water was relatively calm and well oxygenated.(57) They evidently lived close to the sea floor, where occasional episodes of anoxia and rapid burial led to their fossilisation in mudstones.(57)

Lower Cambrian fossils from China

The Middle Cambrian Pikaia from the Burgess Shale

In the 1970s, Conway Morris informally reinterpreted Pikaia, a Mid-Cambrian Burgess Shale fossil originally classified as an annelid worm, as a cephalochordate. Subsequently, this reinterpretation became almost universally accepted after its unqualified and forceful endorsement by Gould.(50) Pikaia fossils, of which over a hundred are now known,(51) are amphioxus-like in their shape, size, and inconspicuous median fins. Pikaia was probably a suspension and/or deposit feeder and is widely represented as swimming by eel-like undulations, although short- or long-term burrowing cannot be categorically ruled out. Faintly V-shaped myomeres occur all along the anteroposterior axis, and a putative notochord runs

The first Lower Cambrian chordate to be described was Yunnanozoon. The initial description was based on only three specimens, which were not designated to any particular phylum.(58) Subsequent studies of numerous additional specimens indicated that Yunnanozoon was an invertebrate chordate(59,60) showing evidence of myomeres, an endostyle, a notochord, a metameric series of gonads, and a pharynx supported by a non-mineralised visceral arch skeleton. Following its description as a chordate, Yunnanozoon was variously interpreted as a cephalochordate,(61) as a very basal deuterostome possibly derived from a vendozoan,(45) as a hemichordate,(62) as an urbilaterian,(63) and as a worm-like animal of unknown affinity.(64) These diverse views will not be detailed here, because they have been largely superceded by insights gained from the 1999 discovery of more than three hundred exquisitely preserved fossils of Haikouella,(65,66) which has a body plan quite similar to that of Yunnanozoon.

Yunnanozoon and Haikouella

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Figure 2. Soft body fossil Haikouella in approximate side view with anterior toward left (the cephalopharyngeal region of this specimen is slightly twisted and thus appears in ventrolateral view; posterior end slightly folded over). GO, gonad; MG, midgut; MM, myomere; NO, notochord; VS, visceral arch skeleton. 5 mm scale bar. The inset is an enlarged lateral view of the anterior cephalopharyngeal region of a different specimen of Haikouella showing the brain (arrow); 1 mm inset scale bar.

(One dissenting view(67) admits that Haikouella and Yunnanozoon may be chordate-like anteriorly, but implies that they resemble segmented protostomes posteriorly and might represent a very basal group of deuterostomes.) A fossil of Haikouella is illustrated in Figure 2, and more detail is shown diagrammatically in Figure 3 (differences between Haikouella and Yunnanozoon are noted in the figure caption). Haikouella has a cephalopharyngeal region (approximately the anterior third of the body), a more inflated myomeric region (roughly the posterior two-thirds), and a very

short caudal projection. There is a short zone of overlap between the posterior extremity of the pharynx and the most anterior myomeres. Medially, Haikouella bears inconspicuous dorsal, caudal and ventral fins without striations, and there are no lateral appendages. The two dozen bilaterally arranged myomeres of Haikouella are located between the fifth visceral arch and the base of the caudal projection. Because successive myomeres are separated by relatively straight myosepta, instead of the more familiar chordate V- or W-shaped myosepta, some have

Figure 3. Diagrammatic side view of Haikouella (somewhat exaggerated in the dorsoventral dimension) with anterior toward the left. AB, anterior branchial vessel; AN, anus; AP, atriopore (limits of atrium not shown); BC, buccal cavity; BR, brain; BT, buccal tentacle; CF, caudal fin; CP, caudal process; DA, dorsal aorta; DF, dorsal fin; EG, endostyle gland; ES, endostyle; GO, gonad; HT, heart; IN, intestine; LE?, lateral eye?; MG, midgut; MM, myomere; NO, notochord; PD, pharyngeal denticle; PH, pharynx; PV, possible protovertebrae; SC, spinal cord; VA, ventral aorta; VF, ventral fin; VS, visceral arch skeleton. In comparison to Haikouella, Yunnanozoon differs in the following ways: the nervous and circulatory systems are not visible; there is no caudal process; the presumed buccal cavity is extended as a short flared proboscis (anterior organ) not fringed with buccal tentacles; there is an inconspicuous velum-like structure posteriorly in the buccal cavity; no anterior glands have been found associated with the endostyle; there are seven pairs of pharyngeal arch skeletons with cross banding but lacking the posterior fringe of stubs; the pharyngeal denticles are located near the posterior end of the pharynx; and there are thirteen pairs of metamerically arranged gonads.

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questioned the muscular nature of Haikouella myomeres.(67) Because young larvae of amphioxus swim effectively by undulations powered by a series of myomeres separated by straight myosepta,(68) there is no biomechanical reason, however, for disqualifying Haikouella myomeres on the basis of their uncomplicated myosepta. At the level of the fifth and sixth myomeres, there appear to be four bilateral pairs of gonads on the ventral side of the animal. A notochord extends along about 85% of the body, starting in the caudal projection and stopping short of the slightly bulbous anterior end of the body. The notochord runs along the ventral edge of the myomere series and is thus (like the notochord of Pikaia) not an optimal compression strut for opposing muscular contractions.(67) In the myomeric region of the body, the notochord is lightly banded segmentally, possibly due to the presence of cartilaginous protovertebrae(66) similar to those of lampreys. The anteroventral mouth of Haikouella is fringed with approximately twelve short buccal tentacles and is possibly supported by a cartilaginous band.(66) The buccal cavity leads into a spacious pharynx supported by six bilateral pairs of visceral arches. The skeleton of each visceral arch is a linear series of about twenty-five dark discs alternating with lucent spaces. This cross-banding resembles the mucocartilage supporting the gill arches of lamprey larvae. In Haikouella, each visceral arch skeleton is fringed posteriorly with short stubs (two per dark disc) that might have been the basal supports for gill filaments not visible in the fossils. It is not known whether the visceral clefts were coextensive with the spaces between successive visceral arch skeletons or were smaller openings in the living animal. A few minute denticles are located in the pharynx at the level of the third visceral arch. These are cone-shaped and may have been mineralised. Although nothing definite is known about the diet of Haikouella, the pharyngeal denticles may have been used for puncturing the tough outer layers of ingested algae and protozoa to facilitate the entry of digestive enzymes. A pair of ridges runs ventrally along the pharynx, probably indicating the course of an endostylar groove (homologous to the vertebrate thyroid). Each of the proposed endostylar ridges expands anteriorly into possible glands that have no clear counterparts in the endostyles of other chordates. Posterior to the pharynx, there is a short esophagus, a longer midgut, and an even longer intestine that terminates in an anus just ventral to the caudal projection. The midgut sometimes appears spiral, although it is not certain whether this represents a spiraled gut wall or helically wound gut contents, as occurs in amphioxus.(18) The central nervous system of Haikouella comprises a neural tube just dorsal to the notochord. In a few of the fossils, the anterior end of the neural tube appears to be dilated into a two- or three-lobed brain (Fig. 2, inset, arrow) located anterior to the tip of the notochord. In a single fossil, a structure that might be one of two paired eyes is located on the side of the head, although the contralateral eye is not visible. In the pharyngeal region, the circulatory system consists of a ventral aorta with a large posterior dilation interpreted as the heart. The ventral aorta also has small swellings along its length in register with each visceral arch, possibly comparable to the branchial hearts of amphioxus.(18) Although branchial arch vessels are presumed to have been present in Haikouella, they are not visible in the fossils. At the anterior end of the pharynx, however, a conspicuous anterior branchial vessel on either side of the body connects the ventral and dorsal aortae. The pharynx is presumably enclosed in an atrium as there is an atriopore-like structure situated about midway along the ventral side. An atrium, if present, might have helped protect the gills from the substratum during burrowing; however, the unstreamlined body plans of Yunnanozoon and Haikouella make it more likely that the animals swam slowly or hovered close to the ocean bottom and perhaps rested on it temporarily. To date, the placement of Haikouella within the chordates has been left as an unresolved trichotomy with the cephalochordates and vertebrates (sensu lato).(69) Consequently, we have evaluated the more exact phylogenetic position of Haikouella by a cladistic analysis based on the same 103 characters and the same method (PAUP 3.1.1) recently used to place the euconodonts within the chordates.(49) Our analysis (Fig. 4) identifies Haikouella as the sister group of the vertebrates (sensu lato). By criteria for recognising stemand crown-groups,(70) Haikouella can also be considered a stem-group vertebrate.

Cathaymyrus

This species, which is only known from a single fossil, is almost certainly a Lower Cambrian chordate. Cathaymyrus has been interpreted in two very different ways, however: (1) as a cephalochordate with a notochord stopping short of the anterior extremity,(71) and (2) as an incomplete specimen of Yunnanozoon preserved in a peculiar orientation.(60) The fossil is indistinct at its anterior end, and discovery of additional specimens will be needed to distinguish between these two possibilities.

These Lower Cambrian species are each known from a single fossil in which only a few anatomical features are clearly identifiable.(72,73) Myllokunmingia and Haikouichthys (Fig. 5) are fusiform animals with a cephalopharyngeal region (approximately the anterior third of the body) followed by a region of conspicuous Z-shaped myomeres. There is a definite median dorsal fin and a much more questionable pair of ventrolateral fins.(73) The dorsal fin of Haikouichthys (but not of Myllokunmingia) shows fine striations that might be fin rays or

Myllokunmingia and Haikouichthys

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radials, although they are disconcertingly inclined anteriorly instead of posteriorly. The presence of caudal and ventral fins cannot be judged because the fossils are incomplete at the posterior end. The pharynx is perforated with a maximum of six visceral clefts in Myllokunmingia (indicated by a series of rather equivocal branchial pouches) and nine in Haikouichthys (indicated by a more convincing visceral arch skeleton). The visceral arch skeleton in Haikouichthys was probably cartilaginous, but lacks cross banding. Fossils of both Haikouichthys and Myllokunmingia include structures interpreted as the remains of cartilaginous skulls,(72,73) although additional fossil material is needed to verify that these structures are not preparation artifacts. Neither species shows any evidence of pharyngeal denticles. Aside from reasonable evidence for an intestine in Myllokunmingia, other features of the internal morphology are very indistinct in both species and require confirmation from additional fossils. One artist's conception of Myllokunmingia includes paired eyes,(74) a not unreasonable assumption, but an assumption nonetheless. Because the fossils are incomplete anteriorly, there is no evidence for their feeding mode, although there has been speculation that they were active predators.(75) There can be little doubt that Myllokunmingia and Haikouichthys represent vertebrates on the basis of visceral arch skeletons (and more equivocal cartilaginous skulls), which imply development from neural crest. Myllokunmingia and Haikouichthys have been grouped within the vertebrates, closer to lampreys than hagfish.(72) However, due to the preponderance of equivocal characters in the phylogenetic analysis, this conclusion must be considered highly provisional.(73)

Figure 4. Cladogram placing Haikouella (arrow) within the context of the same set of extinct and extant chordates analysed in a recent study of conodonts.(49) For this analysis, we used the same method (PAUP 3.1.1) and the same 103 characters listed on p. 28 of Donoghue et al.(49) The character states for Haikouella were as follows: 1,1,0,1,1 ?,?,?,?,? ?,?,?,0,0 0,0,0,0,? ?,?,?,?,? ?,?,1,1,0 0,0,?,?,1 ?,0,0,?,1 0,?,?,0,0 0,0,0,?,1 0,0,?,?,? 0,?,0,0,0 1,0,0,0,0 0,0,0,0,0 0,0,?,0,0 0,1,0,0,0 0,0,?,?,? ?,?,?,?,? ?,?,?,?,? ?,?,?,?,? 0,?,?. The illustrated tree is a strict consensus of three equally parsimonious trees (182 steps; consistency index excluding non-informative characters 0.63). The analysis indicates that Haikouella is a stem-group vertebrate (sensu lato).

Figure 5. Soft body fossil of Haikouichthys in side view with the anterior toward the left. CS, fragmented cartilaginous skull; DF, dorsal fin; MM, myomere; VLF?, possible ventrolateral fin; VS, visceral arch skeleton. 5 mm scale bar. Myllokunmingia is similar, although no visceral arch skeletal elements are visible; instead the pharynx bears a series of possible gill-like structures and corresponding branchial pouches.

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Impact of new data on ideas about early vertebrate evolution and the water exits via the pharyngeal slits. Haikouella and Yunnanozoon also evidently had an endostyle, indicating that they too could filter feed. Water may have been drawn into the mouth by a ciliary current and/or by a more powerful contractile pumping by the pharynx. The latter mode of pumping is suggested by the putative elasticity of the pharyngeal arch skeleton. Suction could have been effected by elastic recoil of the visceral arch skeleton alternating with contraction of pharyngeal constrictor muscles (not preserved in the fossils). Pumping by pharyngeal contraction also implies that there were anti-reflux valves somewhere in the flow system, but these have not been demonstrated in the fossils. It was heretofore widely assumed that the earliest vertebrates pumped water through their pharynx with a robust muscular velum.(2) Although Yunnanozoon has an inconspicuous velum,(60) it resembles the amphioxus velum,(18) which is not used for pumping water through the gut. Thus, suction by pharyngeal contraction may have preceded velar bailing in early vertebrate evolution, although both methods could have coexisted.

There is an old controversy over whether the invertebrate vertebrate transition took place in freshwater(76,77) or the sea.(78) Because it is now known that Lower Cambrian vertebrates lived in unequivocal marine environments(57,58) long before the advent of any known freshwater animals, the old argument is conclusively settled in favor of a marine origin for the vertebrates.

Marine versus fresh water origin of the vertebrates

Until recently, vertebrate fossils with lateral appendages were known only for the gnathostomes and for several ostracoderm groups among the agnathans. Ostracoderms with lateral appendages include heterostracans with pectoral extensions of the head shield, anaspids with lateral folds, and osteostracans with pectoral appendages.(79) In a given ostracoderm group, such appendages were apparently an independently derived feature, and only the pectoral appendages of osteostracans and gnathostomes might have had a common derivation. It has recently been argued that gnathostome pelvic fins arose as novel structures following posterior co-option of part of the genetic program originally specifying the pectoral fins.(80) This modern view is at odds with classical derivations of lateral fins either from gill arches or from continuous ventrolateral folds of the body wall. Although neither of these classical hypothesis is currently in favor,(81) the possible presence of ventrolateral fins in Myllokunmingia and Haikouichthys might justify revival of the lateral fin-fold theory in some form. Even if additional fossil material eventually validates these ventrolateral fins, however, it remains possible that they represent independent derivations that do not share a common ancestry with the lateral appendages of gnathostomes.

The evolutionary origin of vertebrate paired fins

Controversy over anterior segmentation in chordates

It has long been thought that vertebrate teeth evolved from modified parts of the mineralised dermal skeleton,(82) although some have claimed that the earliest mineralised skeletons of vertebrates were denticles in the oropharyngeal region.(45,83) The presence of teeth in euconodont animals and possibly mineralised pharyngeal denticles in Yunnanozoon(59) and Haikouella(65) is consistent with the revisionist view that teeth evolved before the mineralised dermal skeletons during vertebrate evolution.

Mineralised teeth and dermal skeleton evolution

Invertebrate chordates are filter feeders, drawing water with food particles into the pharynx by beating cilia. The food particles are retained on mucus produced by the endostyle,

Insights into feeding of early vertebrates

The controversial topic of vertebrate head segmentation can be resolved into several questions about the anterior skeletal, muscular, and nervous systems (e.g., which, if any, of these tissues is segmented; is one in some way more fundamentally segmented than the others; and how many head segments are present?). If such segmentation was present in basal vertebrates, as claimed by segmentalists,(8) evolutionary scenarios deriving chordates from metameric ancestors (e.g., an annelid-like creature) are strengthened. Conversely, anti-segmentalists(44) believe that vertebrate head segmentation, if present at all, is a derived feature and they tend to favor scenarios deriving chordates from non-metameric or oligomeric ancestors. At first glance, the absence of demonstrable myomeres from the anterior region of Yunnanozoon and Haikouella appears to favor the anti-segmentalist view. It is likely that some muscular segmentation was present in the head, however, because an elastic visceral arch skeleton implies the presence of an opposing segmented branchiomeric musculature (not demonstrable in available fossils). In vertebrates, the branchiomeric muscles and the somitic myomeres share a common embryological origin from paraxial mesoderm.(84) Moreover, developmental expression domains of amphioxus Hox genes(8) are also consistent with aspects of the segmentalist model of head segmentation. This underscores the complexity of the problem of vertebrate head segmentation, which is far from being resolved.

Neural crest as a key feature of vertebrate evolution

Definitive neural crest is the quintessential feature of vertebrates(5,6,85) and enabled their evolution as one of the

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most successful animal groups. Although neural crest, as seen in extant vertebrates, is a complex character that probably arose through several steps,(69) there is good evidence for its presence in a simple but definitive form in Haikouichthys and Myllokunmingia (i.e., the visceral and possibly cranial skeletons) and in Yunnanozoon and Haikouella (i.e., the visceral skeletons and pharyngeal denticles). The likely presence of neural crest in the last two species is particularly interesting because they have been identified as stem-group vertebrates lacking a full suite of vertebrate features. Indeed, the ``invention'' of neural crest was so important for the origin and early evolution of the vertebrates that the name ``cristozoa'' (crest animals) would be an appropriate designation for stem- plus crown-group vertebrates. Short-term prospects for further advances in understanding early vertebrate evolution It is likely that the Lower Cambrian mudstones of south China will continue to yield early vertebrate fossils that will be highly significant, but in ways that cannot be clearly predicted in advance. In contrast to palaeontology, biological advances in understanding the origin and early evolution of the vertebrates are more easily foreseen, at least for the near future. For example, continuing microanatomical studies of the larval nervous system of amphioxus are beginning to hint at the presence of a limbic system(86) and can be expected to reveal the detailed structure of the hindbrain. In comparative molecular genetics, more attention to the gastrula and neurula stages of higher deuterostomes should provide insights into the evolutionary origin of key developmental features like the Nieuwkoop centre and the Spemann organiser. Finally, studies of developmental gene expression should provide additional insights into chordate body part homologies, especially from more detailed work on phylogenetically important deuterostome groups like hemichordates, appendicularian tunicates, hagfishes and lampreys. Acknowledgments Phil Hastings provided invaluable help with the cladistic analysis. Our manuscript was improved by the critical comments of Jeremy Gibson-Brown, Linda Holland, and Michael Schubert. Figures 2 and 5 are reprinted by permission fom Nature (402:4246; 518522) copyright (1999) Nature Magazines Ltd. References
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