Origin and early evolution of the

vertebrates: new insights from

advances in molecular biology,
anatomy, and palaeontology
Nicholas D. Holland
1
* and Junyuan Chen
2
Summary
Recent advancesinmolecular biologyandmicroanatomy
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, pos-
sibly related to modern lampreys) and Yunnanozoon and
Haikouella (evidently stem-group vertebrates). The ear-
liest vertebrates had an unequivocally marine origin,
probably evolved mineralised pharyngeal denticles be-
fore 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 inbiology andpalaeontology support theidea
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
Livinginvertebratechordates comprisethetunicates (themost
familiar of which are the ascidians) and the cephalochordates
BioEssays 23:142151, 2001 John Wiley & Sons, Inc. BioEssays 23.2 142
1
Division of Marine Biology, Scripps Institution of Oceanography.
2
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 92093-
0202.
Review articles
(amphioxus = lancelets). Within the chordates, tunicates are
considered to be the sister group of the cephalochordate
plus vertebrate clade.
(1012)
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 simpli-
fication 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 cepha-
lochordates, 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 fewcentimeters long,
that spends most of its time in shallowburrows, 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 theanterior end. Thesalient chordatefeatures 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 re-
constructions based on serial transmission electron micro-
scopy. Much of this information has been reviewed
elsewhere,
(1618)
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 systemconsists of a dorsal
hollowtube 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 fromboth
approaches indicate that amphioxus has a diencephalic
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 telence-
phalic forebrain, and there is probably no specialised isthmo-
cerebellar 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
Table 1. Gene expression relevant for body part
homologies between amphioxus and vertebrates
Gene
Body part expression References
1. Brain Otx 23
BF1 24
Hox3 25
Hox1 14
Hox4 26
Islet 27
2. Frontal eye
a
Pax6 28
3. Lamellar organ
b
Pax6 28
4. D/V pattern of nerve cord Msx 29
Hedgehog 30
HNF3 31
Netrin 32
5. Neural crest precursor distal-less 22
snail 33
Pax3/7 34
Msx 29
6. Notochord Brachyury (T) 35
HNF3 31
7. Gill slits Pax1 36
Pax2/5/8 20
8. Hatschek's nephridium
c
Pax2/5/8 20
9. Endostyle
d
Pax2/5/8 20
Nk2-1 37
Thyroid peroxidase 38
10. Gastric endocrine cells
e
Xlox, Ilp 39
Neurogenin 40
a
Likely homolog of vertebrate paired eyes.
b
Likely homolog of vertebrate pineal photoreceptor cells.
c
Likely homolog of vertebrate pronephros.
d
Likely homolog of vertebrate thyroid gland.
e
Likely homolog of vertebrate pancreatic endocrine cell.
Review articles
BioEssays 23.2 143
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 (Table2). Oftenthesequestionsinvolvedeciding
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 heart-
less,
(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.
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.
Review articles
144 BioEssays 23.2
Definite and proposed chordates from
the early Palaeozoic
Euconodont animals
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.
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-termburrowing cannot be
categorically ruled out. Faintly V-shaped myomeres occur all
along the anteroposterior axis, and a putative notochord runs
along thedorsal edgeof theanimal (aninefficient 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 eye-
stalks, while intended to be humerous, runs the risk of being
taken at face value by many readers.
In summary, thecephalochordate affinity of Pikaiais 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 fromvendozoans, which are enigmatic fossils fromthe
late Precambrian)
(54)
or possibly a protostome-like crea-
ture.
(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)
Lower Cambrian fossils from China
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)
Yunnanozoon and Haikouella
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 parti-
cular phylum.
(58)
Subsequent studies of numerous additional
specimens indicated that Yunnanozoon was an inver-
tebrate 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.
Table 2. Outstanding questions
a
about amphioxus
anatomy
Question References
1. Is the presence of myomeres at the
anterior end primitive or derived? 8, 44
2. Is the extension of the notochord to 32
the anterior end primitive or derived?
3. Is the presence of the peripharyngeal 45
space (atrium) primitive or derived?
4. Is the cyclopic frontal eye primitive 19, 32
or derived from originally paired eyes?
5. Does amphioxus have a homolog to at 41
least part of the vertebrate heart?
6. What is the composition of the visceral 18
arch skeleton?
7. Does the circulatory system have an
endothelial lining? 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)
Review articles
BioEssays 23.2 145
(One dissenting view
(67)
admits that Haikouella and Yunna-
nozoon 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 (ap-
proximately 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.
Thetwodozenbilaterallyarrangedmyomeresof Haikouella
are located between the fifth visceral arch and the base of the
caudal projection. Because successive myomeres are sepa-
rated by relatively straight myosepta, instead of the more
familiar chordate V- or W-shaped myosepta, some have
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.
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.
Review articles
146 BioEssays 23.2
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 endof thebody. Thenotochordruns alongthe
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
thebody, thenotochordis lightly bandedsegmentally, 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.
Althoughnothingdefiniteis knownabout thediet 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
thevertebratethyroid). Eachof theproposedendostylar 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 fewof 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.
Thepharynx ispresumably enclosedinanatriumasthereis
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 cepha-
lochordates 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 stem-
and crown-groups,
(70)
Haikouella can also be considered a
stem-group vertebrate.
Cathaymyrus
This species, whichis only knownfromasinglefossil, 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, anddiscovery of additional
specimens will be needed to distinguish between these two
possibilities.
Myllokunmingia and Haikouichthys
These Lower Cambrian species are each known froma 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
regionof conspicuous Z-shapedmyomeres. Thereis adefinite
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
Review articles
BioEssays 23.2 147
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 cartila-
ginous, 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 fromadditional 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 phylogene-
tic 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.
Review articles
148 BioEssays 23.2
Impact of new data on ideas about early
vertebrate evolution
Marine versus fresh water origin of the vertebrates
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.
The evolutionary origin of vertebrate paired fins
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 osteos-
tracans with pectoral appendages.
(79)
In a given ostracoderm
group, such appendages were apparently an independently
derived feature, and only the pectoral appendages of osteo-
stracans 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 Hai-
kouichthys 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.
Mineralised teeth and dermal skeleton evolution
It has long been thought that vertebrate teeth evolved from
modified parts of themineralised 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.
Insights into feeding of early vertebrates
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,
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
mouthby aciliary current and/or by amorepowerful 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.
Controversy over anterior segmentation
in chordates
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 segmenta-
tion, if present at all, is a derived feature and they tend to favor
scenarios deriving chordates from non-metameric or oligo-
meric 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 branchio-
meric musculature (not demonstrable in available fossils). In
vertebrates, the branchiomeric muscles and the somitic
myomeres shareacommonembryological originfromparaxial
mesoderm.
(84)
Moreover, developmental expression domains
of amphioxus Hox genes
(8)
are also consistent with aspects of
the segmentalist model of head segmentation. This under-
scores 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
Review articles
BioEssays 23.2 149
most successful animal groups. Althoughneural 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 skele-
tons) 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 designa-
tion 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
NieuwkoopcentreandtheSpemannorganiser. Finally, studies
of developmental gene expression should provide additional
insights into chordate body part homologies, especially from
more detailed work on phylogenetically important deuteros-
tome 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
1. Gee H. Before the Backbone: Views on the Origin of the Vertebrates.
London: Chapman & Hall. 1996.
2. Janvier P. Early Vertebrates. Oxford: Clarendon Press. 1996.
3. Hay-Schmidt A. The evolution of the serotonergic nervous system. Proc
Roy Soc Lond B 2000;267:10711079.
4. De Robertis EM. The ancestry of segmentation. Nature 1997;387:2526.
5. Gans C, Northcutt RG. Neural crest and the origin of vertebrates: a new
head. Science 1983;220:268274.
6. Hall B. The Neural Crest in Development and Evolution. New York:
Springer. 1999.
7. Dohrn A. The Origin of Vertebrates and the Principle of Succession of
Functions: Genealogical Sketches (MT Ghiselin translation). Hist Phil Life
Sci 1994;16:396.
8. Holland PWH. Embryonic development of heads, skeleton and
amphioxus: Goodrich revisited. Int J Dev Biol 2000;44:2934.
9. Ruvinsky I, Silver LM, Gibson-Brown JJ. Phylogenetic analysis of T-box
genes demonstrates the importance of amphioxus for understanding
evolution of the vertebrate genome. Genetics 2000;156:12491257.
10. Maisey JG. Heads and tails: a chordate phylogeny. Cladistics
1986;44:201256.
11. Schaffer B. Deuterostome monophyly and phylogeny. Evol Biol
1987;21:179235.
12. Turbeville JM, Schulz JR, Raff RA. Deuterostome phylogeny and the
sister group of chordates: evidence from molecules and morphology.
Mol Biol Evol 1994;11:648655.
13. Corbo JC, Levine M, Zeller RW. Characterization of a notochord-specific
enhancer from the Brachyury promoter region of the ascidian, Ciona
intestinalis. Development 1997;124:589602.
14. Holland PWH, Garcia-Ferna ndez J. Hox genes and chordate evolution.
Dev Biol 1996;173:382394.
15. Stokes MD, Holland ND. The lancelet. Amer Sci 1998;86:552560.
16. Holland PWH. Molecular biology of lancelets: insights into development
and evolution. Israel J Zool 1996;42 Suppl:247272.
17. Holland LZ, Holland ND. Developmental gene expression in amphioxus:
new insights into the evolutionary origin of vertebrate brain regions,
neural crest, and rostrocaudal segmentation. Amer Zool 1998;38:647
658.
18. Ruppert EE. Cephalochordata (Acrania). In Harrison FW, Ruppert EE,
editors. Microscopic Anatomy of Invertebrates, Vol. 15: Hemichordata,
Chaetognatha, and the Invertebrate Chordates. New York: Wiley. 1997.
p. 349504.
19. Lacalli TC, Holland ND, West JE. Landmarks in the anterior central
nervous system of amphioxus larvae. Phil Trans Roy Soc Lond B
1994;351:243263.
20. Kozmik Z, Holland ND, Kalousova A, Paces J, Schubert M, Holland LZ.
Characterization of an amphioxus paired box gene, AmphiPax2/5/8:
developmental expression in optic support cells, nephridium, thyroid-like
structures and pharyngeal gill slits, but not in the midbrain-hindbrain
boundary region. Development 1999;126:12951304.
21. Holland LZ, Holland ND. Chordate origins of the vertebrate central
nervous system. Curr Opin Neurobiol 1999;9:596602.
22. Holland ND, Panganiban G, Henyey EL, Holland LZ. Sequence and
expression of AmphiDll, an amphioxus Distal-less gene transcribed in
the ectoderm, epidermis and nervous system: insights into evolution of
craniate forebrain and neural crest. Development 1996;122:29112920.
23. Williams NA, Holland PWH. Old head on young shoulders. Nature
1996;383:490.
24. Torreson NA, Martinez-Barbera JP, Beardsley A, Caubit X, Kraus S.
Conservation of BF-1 expression in amphioxus and zebrafish suggests
evolutionary ancestry of anterior cell types that contribute to the
vertebrate telencephalon. Dev Genes Evol 1998;208:431439.
25. Holland PWH, Holland LZ, Williams NA, Holland ND. An amphioxus
homeobox gene: sequence conservation, spatial expression during
development and insights into vertebrate evolution. Development
1992;116:653661.
26. Wada H, Garcia-Ferna ndez J, Holland PWH. Colinear and segmental
expression of amphioxus Hox genes. Dev Biol 1999;213:131141.
27. Jackman WR, Langeland JA, Kimmel CB. islet reveals segmentation in
the amphioxus hindbrain homolog. Dev Biol 2000;230:1626.
28. Glardon S, Holland LZ, Gehring WJ, Holland ND. Isolation and
developmental expression of the amphioxus Pax-6 gene (AmphiPax-6):
insights into eye and photoreceptor evolution. Development
1998;125:27012710.
29. Sharman AC, Shimeld SM, Holland PWH. An amphioxus Msx gene
expressed predominantly in the dorsal neural tube. Dev Genes Evol
1999;209:260263.
30. Shimeld SM. The evolution of the hedgehog gene family in chordates:
insights from amphioxus hedgehog. Dev Genes Evol 1999;209:40-47.
31. Shimeld SM. Characterization of amphioxus HNF-3 genes: conserved
expression in the notochord and floor plate. Dev Biol 1997;183:7485.
Review articles
150 BioEssays 23.2
32. Shimeld SM. An amphioxus netrin gene is expressed in midline
structures during embryonic and larval development. Dev Genes Evol
2000;210:337344.
33. Langeland JA, Tomsa JM, Jackman WR, Kimmel CB. An amphioxus snail
gene: expression in paraxial mesoderm and neural plate suggests a
conserved role in patterning the embryo. Dev Genes Evol 1998;208:569
577.
34. Holland LZ, Schubert M, Kozmik Z, Holland ND. AmphiPax3/7, an
amphioxus paired box gene: insights into chordate myogenesis,
neurogenesis, and the possible evolutionary precursor of definitive
vertebrate neural crest. Evol Dev 1999;1:153165.
35. Holland PWH, Koschorz B, Holland LZ, Herrmann BG. Conservation of
Brachyury (Y) genes in amphioxus and vertebrates: developmental and
evolutionary implications. Development 1995;121:42834291.
36. Holland ND, Holland LZ, Kozmik Z. An amphioxus Pax gene,
AmphiPax1, expressed in embryonic endoderm, but not in mesoderm:
implications for evolution of Class I paired box genes. Mol Mar Biol Evol
1995;35:206214.
37. Venkatesh TV, Holland ND, Holland LZ, Su MT, Bodmer R. Sequence
and developmental expression of amphioxus AmphiNK2-1: insights into
the evolutionary origin of the vertebrate thyroid gland and forebrain. Dev
Genes Evol 1999;209:254259.
38. Ogasawara M. Overlapping expression of amphioxus homologs of the
thyroid transcription factor-1 gene and thyroid peroxidase gene in the
endostyle: insights into evolution of the thyroid gland. Dev Genes Evol
2000;210:231242.
39. Holland PWH, Patton SJ, Brooke NM, Garcia-Ferna ndez J. Genetic
patterning of ectoderm and endoderm in amphioxus: from homeobox
genes to hormones. In Kawashima S, Kikuyama S, editors. Advances in
Comparative Endocrinology: XII International Congress of Comparative
Endocrinology, Yokohama, Japan, Vol. I. Bologna, Monduzzi. 1997.
p 247252.
40. Holland LZ, Schubert M, Holland ND, Neuman T. Evolutionary
conservation of the presumptive neural plate markers AmphiSox1/2/3
and AmphiNeurogenin in the invertebrate chordate amphioxus. Dev Biol
2000;226:1833.
41. Hirakow R. The vertebrate heart in relation to the protochordates.
Fortschr Zool 1985;30:367369.
42. Sannasi A, Hermann HR. Chitin in the cephalochordate, Branchiostoma
floridae. Experientia 1970;26:351352.
43. Briggs DEG, Kear AJ. Decay of Branchiostoma: implications for soft
tissue preservation in conodonts and other primitive chordates. Lethaia
1994;26:275287.
44. Romer AS. The vertebrate as a dual animalsomitic and visceral. Evol
Biol 1972;6:121156.
45. Simonetta AM, Pucci A, Dzik J. Hypotheses on the origin and early
evolution of chordates in the light of recent zoological and palaeonto-
logical evidence. Ital J Zool 1999;66:99119.
46. Stach T, Eisler K. The ontogeny of the nephridial system of the
larval amphioxus (Branchiostoma lanceolatum). Acta Zool Stockh
1998;79:113118.
47. Flood PR. Ciliary rootlet-fibers as tail fin-rays in larval amphioxus
(Branchiostoma lanceolatum, Pallas). J Ultrastr Res 1975;51:218225.
48. Holland ND, Holland LZ. The histochemistry and fine structure of the
nutritional reserves in the fin rays of a lancelet, Branchiostoma lance-
olatum (Cephalochordata=Acrania). Acta Zool Stockh 1991;72:203207.
49. Donoghue PCJ, Forey PL, Aldridge RJ. Conodont affinity and chordate
phylogeny. Biol Rev 2000;75:191251.
50. Gould SJ. Wonderful Life. New York: Norton. 1989.
51. Shu DG, Conway Morris S, Zhang XL. A Pikaia-like chordate from the
Lower Cambrian of China. Nature 1996;384:157158.
52. Conway Morris S. The Crucible of Creation. Oxford: Oxford University
Press. 1998.
53. Riddell C. [Cover illustration for] Wonderful Life. London: Penguin Books.
1991.
54. Dzik J. Yunnanozoon and the ancestry of chordates. Acta Paleontol
Polon 1995;40:341360.
55. Butterfield NJ. Organic preservation of non-mineralizing organisms and
the taphonomy of the Burgess Shale. Paleobiology 1990;16:272286.
56. Janvier P. Les vertebre s avant le Silurien. Geobios 1998;30:931950.
57. Chen JY, Erdtmann BD. Lower Cambrian fossil Lagersta tte from
Chengjiang, Yunnan, China: insights for reconstructing early metazoan
life. In: Simonetta M, Conway Morris S, eds; The Early Evolution of
Metazoa and the Significance of Problematic Taxa. Cambridge: Cam-
bridge University Press. 1991. p 5776.
58. Hou XG, Ramsko ld L, Bergstro m J. Composition and preservation of the
Chengjiang faunaa Lower Cambrian soft-bodied biota. Zool Skr
1991;20:395411.
59. Chen JY, Dzik J, Edgecombe GD, Ramskold L, Zhou GQ. A possible
Early Cambrian chordate. Nature 1995;377:720722.
60. Chen JY, Li CW. Early Cambrian chordate from Chengjiang, China. Bull
Natl Mus Nat Sci Taichung 1997;10:257273.
61. Gould SJ. Of it, not above it. Nature 1995;377:681682.
62. Shu D, Zhang X, Chen L. Reinterpretation of Yunnanozoon as the earliest
known hemichordate. Nature 1996;380:428-430.
63. Dewell RA. Colonial origin for Eumetazoa: major morphological transi-
tions and the origin of bilaterian complexity. J Morphol 2000;243:3574.
64. Bergstro m J, Naumann WW, Viehweg J, Marti-Mus M. Conodonts,
calcichordates and the origin of vertebrates. Mitt Mus Naturkund Berl
Geowiss Reihe 1998;1:8192.
65. Chen JY, Huang DY, Li CW. An early Cambrian craniate-like chordate.
Nature 1999;402:518522.
66. Chen JY, Li CW. Distant ancestor of mankind unearthed: 520 million
year-old fish-like fossils reveal early history of vertebrates. Sci Prog
2000;83:123133.
67. Conway Morris SC. The Cambrian ``explosion'': slow fuse or mega-
tonnage? Proc Natl Acad Sci USA 2000;97:44264429.
68. Stokes MD. Larval locomotion of the lancelet Branchiostoma floridae. J
Exp Biol 1997;200:16611680.
69. Shimeld SM, Holland PWH. Vertebrate innovations. Proc Natl Acad Sci
USA 2000;97:44494452.
70. Jefferies RPS. The origin of chordates: a methodological essay. In House
MR, ed; The Origin of Major Invertebrate Groups. London: Systematics
Association. 1979. p.1515.
71. Shu DG, Conway Morris S, Zhang XL. A Pikaia-like chordate from the
Lower Cambrian of China. Nature 1996;384:157158.
72. Shu DG, Luo HL, Conway Morris S, Zhang XL, Hu SX, Chen L, Han J, Zhu
M, Li Y, Chen LZ. Lower Cambrian vertebrates from south China. Nature
1999;402:4246.
73. Janvier P. Catching the first fish. Nature 1999;402:2122.
74. Monastersky R. 1999. Waking up to the dawn of vertebrates. Sci News
1999;156(19):292.
75. Zimmer C. Fossils give glimpse of old mother lamprey. Science
1999;286:10641065.
76. Smith HW. From Fish to Philosopher. Boston: Little Brown. 1953.
77. Berrill NJ. The Origin of Vertebrates. Oxford: Clarendon Press. 1955.
78. Halstead LB. The vertebrate invasion of freshwater. Phil Trans Roy Soc
Lond B 1985;309:243258.
79. Forey P, Janvier P. Agnathans and the origin of jawed vertebrates.
Nature 1993;361:129134.
80. Gibson-Brown JJ, Agulnik SI, Silver LM, Niswander L, Papaioannou VE.
Involvement of T-box genes Tbx2-Tnx5 in vertebrate limb specification
and development. Development 1998;124:24992509.
81. Bemis WE, Grande L. Development of the median fins of the North
American Paddlefish (Polyodon spathula), and a reevaluation of the
lateral fin-fold hypothesis. In Arratia G, Schulze HP, eds; Mesozoic
Fishes, Vol. 2, Systematics and Fossil Record. Mu nchen: Pfeil. 1999.
p 4168.
82. Romer AS. The Vertebrate Body, Third Edition. Philadelphia: Saunders.
1962.
83. Smith MM, Coates MI. Evolutionary origins of the vertebrate dentition:
phylogenetic patterns and developmental evolution. Eur J Oral Sci
1998;106(Suppl 1):482500.
84. Noden DM. The embryonic origins of avian cephalic and cervical
muscles and associated connective tissues. Am J Anat 1983;168:257
276.
85. Butler AB. Chordate evolution and the origin of craniates: an old brain in
a new head. Anat Rec 2000;261:111125.
86. Zimmer C. In search of vertebrate origins: beyond blood and bone.
Science 2000;287:15761579.
Review articles
BioEssays 23.2 151