Stach2008Chordate phylogeny

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S P E C I A L L Y COMM I S S I ON ED F E A TUR E
Chordate phylogeny and evolution: a not so simple
three-taxon problem
T. Stach
Fakulta¨t fu¨r Biologie, Zoologie – Systematik und Evolution der Tiere, Freie Universita¨t Berlin, Berlin, Germany
Keywords
chordata; morphology; cladistic analysis;
characters.
Correspondence
Thomas Stach, Fakulta¨t fu¨r Biologie,
Zoologie – Systematik und Evolution der
Tiere, Freie Universita¨t Berlin, Ko¨nigin-
Luise-Straße 1-3, 14195 Berlin, Germany.
Email: tstach@zoosyst-berlin.de
Editor: Gunther Zupanc
Received 4 March 2008; revised 1 July 2008;
accepted 3 July 2008
doi:10.1111/j.1469-7998.2008.00497.x
Abstract
Traditional concepts of chordate phylogeny have recently been in turmoil: in a
large-scale molecular study, the traditional hypothesis that cephalochordates are
sister taxon to craniates was replaced by the hypothesis of a sister group relation-
ship between tunicates and craniates. It was claimed that the morphological
evidence that supported traditional phylogeny was weak and that morphological
characters at least equally strong could be mustered in support of the ‘new
phylogeny.’ In the present review, it is shown that the uncritical use of published
codings of morphological characters in recent phylogenetic analyses is responsible
for this perception. To ameliorate this situation, the main focus of the present
publication is a review of the morphological evidence that has been deemed
relevant in chordate phylogeny. Characters are presented in enough detail to allow
readers to make self-reliant informed decisions on character coding. I then analyze
these characters cladistically, and it is demonstrated that support of the traditional
hypothesis is substantial. I briefly evaluate molecular systematic studies and
criticize ‘evo-devo’ studies for lack of cladistic rigor in the evolutionary interpreta-
tions of their data by (1) failing to formally code their characters (2) failing to
subject their data to the congruence test with other characters, the crucial test in
phylogenetic analyses. Finally, a short and by necessity eclectic discussion of
suggested evolutionary scenarios is presented.
Introduction
Chordata, a taxon consisting of about 50 000 animals,
includes Homo sapiens, our own species, and it might be for
this latter reason that the interest in understanding the
evolution of chordates never ceased. It is probably the
former number that makes this understanding so difficult,
because with this number, although low compared with
some protostomian groups, comes an enormous range of
life styles and corresponding body plans. These body plans
fall into three easily recognized groups: Tunicata, Cephalo-
chordata and Craniata (Different names exist for identical
taxa. For example, Acrania is used instead of Cephalochor-
data, Urochordata instead of Tunicata or Vertebrata in-
stead of Craniata. In German publications, Craniota is used
instead of Craniata. Dohle (2004) presents a tabulation of
different names in use for higher deuterostome taxa.). The
main bulk in sheer number belongs to the Craniata and
therein to the Osteognathostomata, with the primarily
aquatic Osteichthyes (bony fishes) being first in number
and arguably also in diversity. The Cephalochordata com-
prise a small confined group of about 27 species that are very
similar in appearance, anatomy and life style. The Tunicata,
on the other hand, while only mustering about 2500 strictly
marine species, show a formidable range of habits, body
plans and life histories. The most numerous group of
tunicates is ascidians that are sessile filter feeders with
tadpole larvae that can propagate sexually and asexually. A
significant group, the Thaliacea, has mastered and explored
the holoplanktonic realm by evolving a dizzying variety of
adaptations including metagenesis, heteromorphy and bio-
luminescence. Another holoplanktonic life form belonging
to the Tunicata is a small group of about 60 species named
Appendicularia (=Larvacea), for they retain the tadpole
tail for their entire life.
The three chordate taxa, Craniata, Cephalochordata and
Tunicata, are clearly monophyletic. For a proper applica-
tion of phylogenetic methodology, it is important to demon-
strate that the taxa under consideration are indeed
monophyletic. Therefore, the monophyly of the taxa under
consideration will be discussed in the following paragraphs,
before the interrelationship of these taxa is contemplated.
Monophyly of Chordata
Recently, it had been suggested that Chordata is not mono-
phyletic by pointing out that there are similarities between
tunicates, cephalochordates and protostomians regarding
Journal of Zoology
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 117
Journal of Zoology. Print ISSN 0952-8369
gene expression pattern (Raineri, 2006). While such a con-
troversial interpretation might stimulate further research,
there is no reason to assume that the similarities in gene
expression pattern of genes involved in axis specification are
synapomorphies of protochordates and protostomians.
Rather, consistent with current knowledge, it can be con-
cluded that these similarities are plesiomorphic characters
within the Bilateria. Another currently controversial debate
focuses on the taxonomic rank of Chordata and its three
subordinate taxa. Cameron, Garey & Swalla (2000) pro-
posed to elevate the Tunicata to phylum rank because ‘they
have a unique adult body plan, are the only metazoan
subphylum classified by their larvae, and are a monophyletic
group that share specific synapomorphies, including the
tunic and an open circulatory system’ (Cameron et al.,
2000). This suggestion, however, is not helpful, as taxo-
nomic ranks are unimportant human constructs modern
taxonomy tries to abolish (e.g. de Queiroz, 2006; Rieppel,
2006). The Chordata itself is supported as a monophyletic
taxon by numerous autapomorphies.
Notochord
The notochord, or Chorda dorsalis, is a skeletal rod that
serves as a central stiffening element that ensures length
constancy in undulatory locomotion. The notochord
stretches from the posterior tip of the tail to the trunk in
tunicates, from the posterior tip of the tail to the posterior
brain capsule in craniates, and from the posterior tip of the
tail to the anterior tip of the animal in cephalochordates. In
all taxa, it is derived from the dorsal epithelium of the
archenteron during embryogenesis (e.g. Hausen &Riebesell,
1991; Stach, 2000; Munro & Odell, 2002). That the noto-
chord is homologous in all chordates is also corroborated by
the fact that the brachyury gene plays a similar role in
determination of the notochord in all chordates. These
similarities support the hypothesis that the notochords are
homologous in all three chordate taxa, although the cells
that make up the notochord differ drastically in the three
clades (Welsch, 1968b; Welsch & Storch, 1969; Schmitz,
1998; Stach, 1999).
Dorsal neural cord with a neural canal,
Reissner’s fiber and neurenteric canal
Dorsal to the notochord lies a strand of nervous tissue that
comprises the central nervous system in chordates. In all
taxa, it has an anterior swelling that receives input from
anterior sensory structures and stretches over the length of
the entire body during one point in ontogeny (Meinertzha-
gen & Okamura, 2001; Lemaire, Bertrand & Hudson, 2002).
In addition, all neural cords in chordates are similar in that
they possess a central fluid-filled canal with cilia and a gel-
like strand of unknown function, the so-called Reissner’s
fiber (Olsson, 1993). Moreover, the canal is continuous with
the endodermal archenteron at its posterior end. This con-
nection is called the neurenteric canal or the canalis neur-
entericus, and it obliterates during ontogeny in all chordates
(Salvini-Plawen, 1998).
Neurulation
The central nervous system describedabove originates
during ontogeny in a process that is called neurulation in
all chordates (Keller, 1975, 1976; Nicol & Meinertzhagen,
1988a,b; Hirakow & Kajita, 1994; Stach, 2000). The dorsal
ectoderm in the midline of the embryo flattens and either
rolls up and sinks beneath the final ectodermal epithelium or
is overgrown by it. Thus, despite its position inside of the
animal, the central nervous system is ectodermal in origin.
In addition, the cascades determining neural fate (MEK-
pathway) are similar in all chordates (see e.g. Lemaire et al.,
2002; Hudson et al., 2003; Meinertzhagen, Lemaire &
Okamura, 2004).
Pharyngeal branchial complex
All chordate taxa possess a pharyngeal branchial complex
(Ruppert, 1997b). That is, the pharynx in protochordates
and also in all primarily aquatic craniates is perforated and
possesses gill slits. The primitive function of the branchial
complex in chordates was filter feeding. A strong case can be
made that a structure homologous to the chordate branchial
complex can be found in enteropneusts (Pardos & Benito,
Some scenarios of chordate origin
Immediately after Kowalevsky’s (1866) pioneering embryological
study of ascidians, ascidians were no longer considered molluscs
but chordates and entered center stage in considerations of
chordate evolution. They stayed there for good and can still
surprise (Pourquie, 2001; Delsuc et al., 2006). The evolutionary
origin of vertebrates from invertebrates has always fascinated
biologists, and there is no shortage of inventive – sometimes
fantastic – evolutionary scenarios. For example, the idea that
articulates (annelids and arthropods) directly gave rise to
vertebrates by flipping over during evolution found support by
prominent zoologists of its time (Geoffroy Saint-Hilaire, 1822;
Dohrn, 1875; Semper, 1875) and has recently been revived in
modified form (Nu¨bler-Jung & Arendt, 1994; Arendt & Nu¨bler-Jung,
1999). Patten (1890) suggested that vertebrates evolved from
arachnids as did Gaskell (1890). The former preferred scorpions as
ancestral, the latter horseshoe-crabs. Other hypotheses
suggested nemerteans (Hubrecht, 1883; Jensen, 1960) as
possible direct ancestors of vertebrates or even proposed that the
chordate body plan evolved by fusion of numerous individual salps
(Lacalli, 1999). Early on, attention has been directed to other
deuterostomes, with enteropneusts (Bateson, 1886; Barrington,
1965) and echinoderms (Gisle´n, 1930; Jefferies, 1986) being
important in several schemes. However, the most influential,
productive and unrefuted hypothesis to date remains Garstang’s
(Garstang, 1894, 1928) neoteny hypothesis.
Most of these ideas did not stand up well to the scrutiny of cladistic
phylogenetic argumentation and are of little more than historic
interest today. Yet, with the advent of molecular systematics and
phylogenetic analyses carried out by computer programs, the
progress achieved in 150 years of morphological investigations into
the evolution of chordates has been treated contemptuously. The
present review calls for a more considerate treatment of the
morphological evidence at hand.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London118
Chordate phylogeny and evolution T. Stach
1987; Ruppert, Cameron & Frick, 1999; Rychel et al., 2006),
and there is some scientific debate as to whether the homo-
log to the chordate branchial complex in a general sense was
already present in pterobranchs or in fossil echinoderms
(Gilmour, 1978; Jefferies, 1986; Benito & Fernando, 1997;
Dominguez Alonso & Jefferies, 2001; Nielsen, 2001; Camer-
on, 2002; Dominguez, Jacobson & Jefferies, 2002). How-
ever, the specific form of the pharyngeal branchial complex
with a ventral endostyle producing a continuous mucous
net, incorporating iodine, a dorsal food-collecting structure,
anterior lateral peripharyngeal ciliated bands and anterior
tentacle-like structures that prevent larger particles from
entering the branchial basket is uniquely shared by all
chordate taxa (Olsson, 1963; Jorgensen, 1966; Fiala-Me´dioni,
1978a,b; Mallat, 1979, 1981; Riisga˚rd & Svane, 1999).
Epithelium of the pericard forms the
musculature of the heart (Fig. 1)
The craniate heart is a complex organ situated ventrally
immediately behind the branchial basket in primitive forms
(e.g. Starck, 1982; Mickoleit, 2004). While it is known that
the vascular system of craniates, including the heart, is lined
by an endothelium, this is not the case in tunicates and
cephalochordates (Ra¨hr, 1981; Corley, 1995; Burighel &
Cloney, 1997; Ruppert, 1997b). Nevertheless, a ventral
major blood vessel exists in the branchial basket in all
chordate taxa (Ra¨hr, 1981; Starck, 1982; Corley, 1995). In
addition, in all three taxa, a center for the propulsion of the
blood fluid is present just behind the branchial basket.
Moreover, the cells equipped with contractile filaments
providing the contractive force for this propulsion are
similarly derived from a coelomic epithelium, the pericard
in tunicates and craniates (Hirakow, 1989) and the extensive
rostral coelom in cephalochordates (Stach, 1998), which
therefore could be homologous in its medio-ventral part to
the pericard.
Mesodermal tail musculature derived from
dorsolateral pockets of archenteron
During early ontogeny, all chordates go through a neurula
stage during which two processes occur more or less simul-
taneously. While the mid-dorsal part of the epidermis is
becoming the neural system, the dorsolateral flanks of the
archenteron are beginning to build the mesoderm that
eventually becomes the musculature lateral to the notochord
that serves in undulatory swimming in all chordates (Con-
klin, 1905, 1932; Salvini-Plawen, 1989; Hausen & Riebesell,
1991; Swalla, 1993; Stach, 2000; Kuratani, Kuraku &
Murakami, 2002).
Hypophysis – pituitary
In craniates, the ventral nervous tissue of the thalamus
region in the diencephalon is in close contact with the
adenohypohysis, which is ontogenetically derived from the
stomodaeal epithelium (Hartenstein, 2006). In cephalochor-
dates, a similar close positional relationship exists between a
ventral extension of the brain vesicle and a dorsal extension
of the epithelium of the buccal cavity that is named
Hatschek’s pit (Gorbman, 1994, 1999; Gorbman, Nozaki &
Kubokawa, 1999). The ontogeny of Hatschek’s pit is com-
plicated (Hatschek, 1881; Stach, 1996, 2002), but in addition
to the aforementioned topological similarity different ade-
nohypophyseal hormones have been demonstrated to be
present in Hatschek’s pit in cephalochordates (Olsson, 1969;
Gorbman et al., 1983; Gorbman, 1995). In tunicates, a
similar topological relationship exists between the neural
gland and the ciliated funnel that opens into the dorsal
anterior branchial basket (Ruppert, 1990). Again, several
hormones found in craniate adenohypophysis have been
demonstrated using immunocytological techniques (Roma-
nov, 2000). While gene expression studies are less conclu-
sive, with some key pituitary genes not being expressed in
the neural gland or the ciliated funnel of tunicates, homol-
ogy of the pituitary in chordates is supported by these
studies as well (Sherwood, Adams & Tello, 2005; Candiani
et al., 2008). Thus, the presence of a hypophysis system
including a neural and an adenohypohyseal part with
particular hormones has to be interpreted as an autapomor-
phy of Chordata.
Pineal eye
The dorsal wall of the diencephalon of craniates forms a
photoreceptor, the pineal eye that contains cells with stacks
of lamellae modified from cilia that are identical in ultra-
structure to lamellar cells in the dorsal wall of the cerebral
vesicle of cephalochordates (Pu & Dowling, 1981; Lacalli,
1994, 2004). Several authors have argued that the ocellus of
tunicates could also be homologous to the pineal eye (Eakin,
1973; Whittaker, 1997), which seemsto be corroborated by
sequence similarities in the opsin genes (Kusakabe et al.,
2001). However, these similarities also exist to the opsins in
the lateral eyes of vertebrates and to sequences isolated from
cephalochordates (Terakita, 2005). Thus, if the arguments
against the homology of the tunicate ocellus to the paired
ecm
et
pcc
bv
pcc
pc
ecm
pcc
bv
bv
pcpc
ecm
(a) (b) (c)
Figure 1 Schematic cross-sections through the
developing heart in the three higher chordate
taxa. (a) Ciona intestinalis, after Oliphant &
Cavey (1972); (b) Branchiostoma lanceolatum,
after Stach (1998); (c) not further specified
craniate, after Hirakow (1989). bv, blood vessel;
ecm, extracellular material; et, endothelium; pc,
pericard; pcc, pericardial cavity.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 119
Chordate phylogeny and evolutionT. Stach
vertebrate eyes are considered, the hypothesis of homology
to the pineal eye in vertebrates and the lamellar body of
cephalochordates becomes a reasonable, yet controversial
alternative. Thus, given these premonitions, the pineal eye
can be assumed to be another synapomorphy of Chordata.
In addition to these unique morphological autapomor-
phies, Chordata share some gene regulatory pathways that
are equally unique to Chordata. Summaries of the simila-
rities in the expression pattern of homologous genes that
seem to be unique to Chordata abound and can, for
example, be found in Rowe (2004), Lemaire (2006), Schlos-
ser (2007).
Monophyly of higher chordate taxa
Monophyly of Craniata
The Craniata, to begin with, show so many autapomorphies
that only a few of the more complex ones will be mentioned
in this paragraph.
Multilayered epidermis
Whereas in the vast majority of invertebrate taxa, with the
exception of chaetognaths, the epidermis consists of a single
epithelial cell layer, this is not the case in craniates (e.g.
Mickoleit, 2004; Westheide & Rieger, 2007). On the con-
trary, all craniates possess a multilayered epidermis in
intimate functional contact with the mesodermal dermis
(e.g. Starck, 1982; Romer & Parsons, 1986). As a biological
peculiarity, the more apical epidermal cells store increasing
amounts of ceratin within the craniate taxa (Flaxman, 1972;
Karabinos, Zimek & Weber, 2004).
Endothelially lined blood vessels
Blood vessels in the invertebrate taxa are, as a rule, spaces
within the extracellular matrix (Ruppert & Carle, 1983;
Westheide & Rieger, 2007), with the exception of nemer-
tines. While they can be either vast and more or less
undefined as in the molluscs or anatomically highly stable
and precisely organized as in many annelids and cephalo-
chordates, they always lack a cellular endothelial lining
(Ruppert & Carle, 1983; Goldschmid, 2007).
Craniate-type heart
The craniate-type heart is, in the plesiomorphic condition,
situated on the ventral side immediately behind the gill-
bearing pharynx. Thus, in its primitive craniate condition
the heart has inherited its position from the last common
ancestor of the Chordata. It carries only unoxygenated
blood. The ground plan of the plesiomorphic condition of
the craniate heart can be stated more precisely. It is
muscular and consists of distinct parts: an atrium, a ven-
tricle and the conus arteriosus (Starck, 1982; Mickoleit,
2004).
Anterior sensory organs
This short phrase summarizes countless detailed autapo-
morphies of the Craniata. It comprises paired lateral eyes,
organs with a complicated ontogeny that involves an evagi-
nation of the diencephalon and the induction of an epider-
mal lens (Starck, 1982; Hall, 1995; Mickoleit, 2004). It also
comprises the labyrinth system that allows the fast-moving
craniates to orientate in three-dimensional (3D) space
(Starck, 1982; Streit, 2001; Mickoleit, 2004). Anterior sense
organs present in the last common ancestor of the recent
craniates also include the olfactory organs associated with
the telencephalon, and the diencephalic dorsal parietal and
pineal eyes (Starck, 1982; Mickoleit, 2004; Janvier, 2008).
Brain
Probably the most impressive autapomorphic trait of the
craniates is the brain encased in a skeletal neurocranium. In
all craniates, it consists of distinct parts that can be recog-
nized as corresponding, and whose ontogeny is highly
similar as well. Just to allude to the substance behind this
claim, I mention the Telencephalon, Diencephalon, Mesen-
cephalon and Rhombencephalon as easily recognized
homologous parts of the craniate brain (Starck, 1975a,
1982; Romer & Parsons, 1986; Mickoleit, 2004; Murakami
et al., 2005).
Spinal nerves
In craniates, nerves leave the spinal cord in each myomere
on both sides to innervate musculature and connect to
sensory cells (Starck, 1982; Romer & Parsons, 1986; Kelly
Kuan et al., 2004; Mickoleit, 2004). They show a regular
pattern in as much as in the primitive craniate condition, the
dorsal nerves are connected to the peripheral nerves in a
ganglion that is situated close to the spinal cord in the so-
called spinal ganglion. While the dorsal counterparts of
nerve roots are also found in cephalochordates, the cell
bodies reside within the nerve cord and do not form a spinal
ganglion (Bone, 1960a,b; Wicht & Lacalli, 2005).
There are countless more morphological (and molecular)
characters that attest to the monophyly of Craniata, in fact
too many to list here. The reader is kindly referred to the
excellent accounts that are available on this subject, for
example, Starck (1975a), Romer & Parsons (1986) and
Mickoleit (2004).
Monophyly of Cephalochordata
Compared with craniates, the 27 species of cephalochor-
dates are rather uniform and relatively simple organisms,
and yet there are also too many autapomorphic traits that
establish the monophyly of this clade to list them all in the
present review.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London120
Chordate phylogeny and evolution T. Stach
Hesse ocelli
Numerous simple pigment cup eyes, the Hesse ocelli, are
situated in the neural cord of cephalochordates. Most of
these ocelli consist of only two cells (Eakin, 1979; Ruppert,
1997a; Wicht & Lacalli, 2005; Castro et al., 2006): a photo-
receptor cell that sinks its microvillar photosensitive mem-
branes into the concavity of a pigment cell.
Corpuscules of de Quatrefage
These sensory structures consist of a central ciliated primary
sensory cell that is surrounded by sheath cells. The corpus-
cules of de Quatrefage are situated in the rostral part of the
animals. They seem to be epidermal in origin, and yet extend
with their basal part into the subepidermal connective tissue
(Schulte & Riehl, 1977; Baatrup, 1981; Fritzsch, 1996;
Ruppert, 1997a; Wicht & Lacalli, 2005).
Cyrtopodocytes (Fig. 2)
This unique cell type that obviously serves an excretory
function is unique in the animal kingdom. The coelomic
cyrtopodocyte consists of a podocytic part, in which
cytoplasmic extensions cover a blood vessel. These exten-
sions interdigitate with similar extensions of the neighboring
cells and a slit membrane is formed between the cells
and above the blood vessel. The second part of the cyrtopo-
docyte resembles a protonephridic solenocyte; a central
apical cilium is surrounded by 10 long, slender and stiff
microvilli. These microvilli project into the excretory canal
that leads to the outside, and they are covered by a sheath of
extracellular material that forms a second filtration barrier
(Brandenburg & Ku¨mmel, 1961; Ruppert, 1996; Stach &
Eisler, 1998).
Oral cirri
A ring of oral cirri, each of which contains a skeletal rod,
surrounds the anterior entrance to the preoral cavity. These
oral cirri can be moved by way of an associated muscle
system (Franz, 1923, 1927; Schulte & Riehl, 1977; Ruppert,
1997a).
Notochord
Unique among the chordates,the main part of the noto-
chord consists of cells that contain contractile fibers. These
paramyosin fibers are arranged in a striated fashion and can
contract the cells laterally, thus stiffening the notochord
locally. The notochord cells send cytoplasmic extensions
through pores in the strong notochordal sheath to the nerve
cord, obviously contacting the nerve cord for innervation.
In addition to this unique notochord cell type, the noto-
chord contains at least one more cell type dorsally and
ventrally, the Mu¨ller cells. Such a differentiation of noto-
chord cells is also unique within the chordates (Welsch,
1968b; Flood, 1975; Ruppert, 1997a; Stach, 1999).
Larval asymmetries (Fig. 2)
This phrase alludes to many unique features in the ontogeny
of cephalochordates. Here, I want to mention three of them
as they are specific, easy to distinguish characters: the left-
sided oral papilla, the right-sided, obliquely v-shaped rudi-
ment of the endostyle and the club-shaped gland situated
immediately behind the larval endostyle in the pharynx
(Conklin, 1932; Olsson, 1983; Stach, 2000).
Again, as for the craniates, there are too many morpho-
logical characters that attest to the monophyly of Cephalo-
chordata to list here. The reader is kindly referred to the
excellent accounts that are available on this subject, for
example, Franz (1927), Ruppert (1997a) and Stach (2000).
an
ap
hc
nc
nt
pa pp
csg
en
(a)
gs
bv
cc
ci
co
ec
(b)
nu
mv
pc
ci
mv
pk
Figure 2 (a) A highly asymmetric cephalochor-
date larva, about a week old (after Stach, 2000).
Note the left-sided mouth opening and the
single gill slit in the ventral midline. The latter
will eventually become incorporated into the
series of gill slits on the right side of the body.
an, anus; ap, anterior pigment spot; csg, club-
shaped gland; en, endostyle; gs, gill slit; hc,
head coelom; nc, notochord; nt, nerve tube; pa,
preoral papilla; pp, preorap pit. (b) Schematic
drawing of the excretory cells of cephalochor-
dates, the so-called cyrtopodocytes (after Bran-
denburg & Ku¨mmel, 1961). Foot-like processes
cover a blood vessel, similar to the podocytes in
craniate kidneys. A cilium surrounded by 10
slender microvilli supports the drainage of excre-
tory fluid through the excretory canal. bv, blood
vessel; cc, coelomic cell; ci, cilium; co, coelom;
ec, excretory canal; mv, microvilli; nu, nucleus;
pc, podocytic extensions; pk, perikaryon.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 121
Chordate phylogeny and evolutionT. Stach
Monophyly of the Tunicata
If one considers the diversity of life styles and anatomies in
Tunicata, it is hardly surprising that the monophyly of this
taxon is difficult to ascertain. Yet, even in the case of the
Tunicata, there are unique, uncontroverted (Wenzel, 2002)
autapomorphies that can be listed to back the hypothesis
that this taxon is monophyletic.
Tunic
The monolayered epidermis of tunicates is covered by a
sheath of extracellular material, the so-called tunic. It can
contain cells that seem to contribute to the integrity of the
tunic in several ways. In addition, the tunic contains
proteins, but more significantly cellulose fibers. This is the
only case in the animal kingdom that cellulose is produced,
and similarities in cellulose-synthetase genes suggest that
this ability was inherited by lateral gene transfer from
bacteria. It has been demonstrated in over 180 species from
all major tunicate groups (Hirose et al., 1997; Kimura et al.,
2001; Nakashima et al., 2004; Davison & Blaxter, 2005;
Stach, 2007).
Heartbeat reversal
This is a convenient and at the same time informative
apomorphic character for the Tunicata, because it can be
seen in most species with the help of a dissecting microscope
alone. All species examined so far show this phenomenon,
where the blood is pumped anteriorly toward the pharynx for
several minutes and, after a short pause, pumped posteriorly
toward the intestinal organs (Skramlik, 1938; Kriebel, 1967).
Hermaphroditism
If one considers the potential phylogenetic neighborhood of
the Tunicata, that is, the deuterostome taxa, it becomes clear
that hermaphroditism is the derived state. However, her-
maphroditism as a character state is prone to homoplasy, as
it highly depends on the ecology of the animals. For
example, sessile organisms tend to be hermaphroditic, in
order to increase their chances of successful reproduction,
and it is quite probable that the last common ancestor of
Tunicata was a sessile organism (Huus & Knudsen, 1950;
Stach & Turbeville, 2002). While most planktonic appendi-
cularians are hermaphroditic, the well-known species Oiko-
pleura dioica is dioecious.
Pylorus gland
The pylorus gland seems to be present in all tunicates.
However, the minute, probably progenetically derived
appendicularians (Stach et al., 2008) possess only isolated
glandular cells that might be homologous to the organ
named pylorus gland in other tunicates (Seeliger & Hart-
meyer, 1911; Neumann, 1933; Berrill, 1947; Brien, 1948;
Huus & Knudsen, 1950; Ihle, 1958).
Larval visceral ganglion
A morphologically distinct visceral ganglion is present in all
tunicate tadpole larvae that have been examined so far,
including the probably progenetic appendicularians (Bur-
ighel & Cloney, 1997; Søviknes, Chourrout & Glover, 2005).
While at first glance, the unique presence of a distinct
ganglion immediately anterior to the tail complex seems to
be a clear autapomorphy of the Tunicata, this does not
preclude that this part of the nervous system has a corre-
sponding homologous part in other chordates, which is not
morphologically distinct (Dufour et al., 2006).
With the monophyly of the three taxa established by
arguments, I will proceed to the main task of this paper, that
is, to review the possible interrelationships of these three
taxa as can be inferred from the morphological evidence.
The competing hypotheses
With only three monophyletic groups, there are in theory
only three possible ways to draw a rooted phylogenetic tree
for the Chordata. These possibilities can be seen in Fig. 3. In
the remainder of this paper, I will call the hypothesis that
Tunicata is sister taxon to Cephalochordata ‘Atriozoa
hypothesis,’ the hypothesis that Tunicata is sister taxon to
Craniata is called ‘Olfactores hypothesis,’ while the
hypothesis that Cephalochordata is sister taxon to Craniata
is called ‘Notochordata hypothesis.’ For long, the ‘Noto-
chordata hypothesis’ seemed to be widely accepted, with the
‘Atriozoa hypothesis’ being of merely historical interest and
the ‘Olfactores hypothesis’ being only invoked because of
the controversial interpretation of unusual fossils. In sup-
porting the ‘Notochordata hypothesis,’ Ax even nicknamed
the cephalochordates ‘honorary vertebrates’ (Ax, 2001).
This picture has been upset recently, because of the analysis
of large sequence datasets supporting the ‘Olfactores
hypothesis’ (Delsuc et al., 2006; Dunn et al., 2008; Putnam
et al., 2008; Swalla & Smith, 2008). Delsuc and coleagues
suggested that the morphological evidence that led to
the ‘Notochordata hypothesis’ was meager and at least
rivaled by morphological data supporting the ‘Olfactores
Ce
ph
alo
cho
rda
ta
Cra
nia
ta
Tu
nic
ata
Ce
ph
alo
cho
rda
ta
Cra
nia
ta
Tu
nic
ata
Cra
nia
ta
Ce
ph
alo
cor
da
ta
Tu
nic
ata
(a) (b) (c)
Figure 3 With three higher monophyletic taxa, only three rooted
cladogramms are logically possible. These are seen here. (a) Cepha-
lochordata (Tunicata, Craniata): Olfactores hypothesis. (b) Craniata
(Cephalochordata, Tunicata): Atriozoa hypothesis. (c) Tunicata (Crania-
ta, Cephalochordata): Notochordata hypothesis.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London122
Chordate phylogeny and evolutionT. Stach
hypothesis.’ In order to give a more complete picture, I will
review the morphological evidence available in some detail
by explaining the morphological reasoning behind the three
competing hypotheses. I will then proceed to compile and
analyze a matrix, amenable for cladistic analysis including
the characters advanced from proponents of all three
hypotheses. Then, I will take a cursory look at the molecular
evidence and the evolutionary interpretations of this com-
bined evidence.
The Atriozoa hypothesis
The Atriozoa hypothesis suggests that Cephalochordata
and Tunicata are sister taxa to the exclusion of Craniata.
This hypothesis derives its name from the fact that it was
originally based on the shared common possession of a
water jacket around the branchial basket, that is, the atrium.
The hypothesis was suggested by Minot (1897) and sup-
ported by Masterman (1898), but is also reflected in the
name ‘Protochordata’ that, as a taxon, would also comprise
Tunicata and Cephalochordata. However, the term Proto-
chordata is more often used as a colloquial name and
etymologically also hints at the possibility that it might
describe a paraphyletic grouping. While this hypothesis
hardly finds any support in recent scientific publications,
proponents of this hypothesis suggested some potential
synapomorphies that will be discussed in the following
paragraphs.
Atrium (Lankester, 1890; Minot, 1897)
In stark contrast to the craniates, the enteropneusts and the
pterobranchs, tunicates and cephalochordates possess a
water jacket around their branchial basket that is called an
atrium or a peribranchial chamber (Dohle, 2004). This space
is delimited by the branchial basket on the interior face and
by a double layer of monolayered epithelium with mesoder-
mal muscle cells in between on the exterior face. In both
taxa, there is a single atrial opening that lies on the ventral
side in cephalochordates, but dorsal in most tunicates.
However, the atrial opening is posteriorly placed in Thalia-
cea, and an atrium is entirely lacking in Appendicularia. The
atrium originates as a paired dorsal rudiment in Aplouso-
branchiata and part of the Phlebobranchiata, but as an
unpaired dorsal rudiment in Stolidobranchiata and as an
unpaired ventral room delimited by epidermal folds in
cephalochordates. Therefore, the homology of the atria of
Tunicata and Cephalochordata has been doubted (Pietsch-
mann, 1962; Dohle, 2004).
Discoidal notochord cells arranged in single
file
Ruppert (1997b) suggested that the arrangement of cells that
comprise the notochord in a single row (‘stack of coins’) is
common only to Tunicata and Cephalochordata to the
exclusion of Craniata. In most tunicates, this ‘stack of coin’
stage is present only as a temporary stage during larval
development and is lost in the swimming larva and is absent
in adult tunicates. It remains present in adult cephalochor-
dates, where the notochord cells are differentiated as muscle
cells. In craniates, as Stach (1999) pointed out, the arrange-
ment of the notochord cells in single file is also present
during embryogenesis, but is lost later on. Thus, in the
general form as suggested by Ruppert (1997b), this character
is not supporting the closer relationship of Tunicata and
Cephalochordata but instead is another autapomorphy of
Chordata.
Intercellular spaces between notochordal
cells
Another character suggested by Ruppert (1997b) as suppor-
tive of the ‘Atriozoa hyothesis’ is the presence of intercel-
lular spaces between notochordal cells. In tunicates, such
intercellular spaces can be limited to small pockets between
subsequent cells of the ‘stack of coin,’ but in many species,
these spaces enlarge, become confluent and form a fluid-
filled canal. In this way, the cytoplasms of notochordal
cells become restricted to the walls of the aforementioned
canal and in character become epithelial, fulfilling the
definition of a coelom as given by Bartolomaeus (1993). In
Distal Intermediate Proximal
Figure 4 Schematic drawings of the interpreta-
tion of the fossil mitrate Peltocystis cornuta
after Jefferies (1986). Top: dorsal view, bottom:
lateral view. The three-partite tail is the autapo-
morphy of the Olfactores.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 123
Chordate phylogeny and evolutionT. Stach
cephalochordates, the intercellular spaces originate in a
similar way as small pockets between subsequent cells.
These pockets expand, but do not form a continuous central
canal (Stach, 1999, 2000). However, they are confluent
by way of two narrow canals dorsally and ventrally of
the notochord cells. The notochord cells of craniates
are large, numerous and ‘turgescent.’ In the electron
microscopic aspect, the turgor-providing vacuoles contain
flocculent material and are membrane bound, with inter-
mediate filaments strengthening the membrane (Schmitz,
1998).
The Olfactores hypothesis
Three-partition of the hindtail (Fig. 4)
This character is the main synapomorphy on which the
‘Olfactores hypothesis’ was originally founded. It is based
on the detailed interpretation of the enigmatic fossils named
carpoids in the hypothesized stem lineages of tunicates and
craniates (Jefferies, 1986). The character is not present in
recent chordates; therefore, independent reduction is
required for tunicates and craniates. While I have not
examined these fossils myself, I want to point out that their
interpretation is controversial (Jollie, 1982; Peterson, 1995;
Jefferies, 1997; Ruta, 1999). Accepting the interpretation of
Jefferies (Jefferies, 1986, 1997; Jefferies, Brown & Daley,
1996; Dominguez Alonso & Jefferies, 2001) also makes it
necessary to suppose the independent loss of calcareous
mesodermal skeletal structures in tunicates, cephalochordates
and craniates alike. In addition, it also follows that the peculiar
stereome structure of calcitic hard parts of the skeleton, which
is found only in Echinodermata in extant fauna, evolved in the
last common ancestor of all Deuterostomia.
Loss of preoral kidney (Ruppert, 2005)
Hatschek’s nephridium is a single large nephridium that is
situated in the dorsal roof of the preoral cavity in am-
phioxus. Cephalochordates, in addition, possess segmental
nephridial structures situated along the dorsolateral sides of
the branchial basket. This situation is similar to the one
found in enteropneusts, where a single large excretory organ
is present anterior to the mouth and repeated coelomic units
with parts of their epithelium differentiated as podocytes are
present along the dorsolateral sides of the brancial basket
(Stach, 2002). In the primitive craniate condition, segmental
nephridial structures are present along the most part of the
body, but no single excretory organ can be found anterior to
the mouth (Starck, 1975b, 1982; Kluge & Fischer, 1990,
1991). In tunicates, mesodermal excretory structures invol-
ving ultrafiltration through basement membranes held by
podocytic cell extensions are not present (Burighel & Clo-
ney, 1997). Thus, reduction in the entire typical deuteros-
tome excretory system has to be assumed in Tunicata and
the loss of the anterior preoral kidney can be inferred for
Craniata.
Septate junctions replaced by tight
junctions (Ruppert, 2005)
Septate junctions are present as an apical barrier within the
apical junctional complexes of epidermis cells in almost all
animals. However, they are occasionally found in craniates
(Gobel, 1971; Friend & Gilula, 1972; Sotelo & Llinas, 1972;
Altorfer & Hedinger, 1975) but have never been found in
tunicates. On the other hand, there are several cases of cell
junctions that are ultrastructurally indiscernible from tight
junctions in other invertebrates such as amphipods, bivalves
or crayfish (Lane & Chandler, 1980). Thus, while there are
several homologous genes necessary for the correctexpres-
sion of septate junctions in Drosophila melanogaster and
tight junctions in craniates (Willot et al., 1993; Stevenson &
Keon, 1998), this is no proof for homology of the structure
itself, especially because both types do occur together in the
same species.
Swimming musculature, partly a functional
syncytium (Ruppert, 2005)
This is a peculiar character. Historically, it had been discussed
whether the myofibrils in the tail of ascidian larvae were
continuous (Berrill & Sheldon, 1964) between adjoining cells.
However, detailed electron microscopic investigations have
shown that the fibrils (=cells) were indeed separate, but that
the cells are connected by special cell junctions (Cavey &
Cloney, 1976; Cavey, 1982; Cavey & Strecker, 1985; Burighel
& Cloney, 1997). In vertebrates, the lateral muscle cells of the
myotome fuse to form a true syncytium (Starck, 1975b;
Sachidamamdam & Dhawan, 2003; Kielbowna & Daczews-
ka, 2005). Thus, while it is correct to state that the myofibrils
are functionally connected in tunicates and craniates, a
syncytium is only found in craniates. The character state with
separate, mononucleatemuscle cells present in ascidian larvae
is the plesiomorphic condition in Chordata and is present in
cephalochordates as well as other deuterostomes. Whether
the connection by special cell junctions in ascidian larvae is
homologous to the fact that myofibrils are formed inside of
syncytia in craniates is doubtful. At least, this character
therefore should be coded as two separate ones: (1) muscle
cells forming a syncytium and (2) myofibrils of originally
separate cells functionally coupled.
Brain with coronet cells (Saccus vasculosus)
(Ruppert, 2005)
The homology of coronet cells found in the brain of
O. dioica with cells from the Saccus vasculosus in craniates
has been originally suggested by Olsson (1975). As similarity
between the two cell types, this author mentions that the
vertebrate cells possess numerous apical globuli, whereas the
tunicate cell possesses a single globus. Both types of cells are
rich in endoplasmic reticulum, Golgi complexes and vesicles
and seem to function in secretion. Despite these similarities,
there are differences as well. The globus in O. dioica cells
possesses a rudimentary cilium. No such structure exists in
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London124
Chordate phylogeny and evolution T. Stach
the globuli of the craniate cells. In addition, the coronet cells
in craniates are embedded in a tall epithelium and connected
by an apical junction with the neighboring cells. The
neighboring cells in O. dioica coronet cells, on the other
hand, are extremely flat compared with the coronet cells,
and no cell junctions are depicted in Olsson’s publication.
Neuromast cells with a cupula (Ruppert,
2005) (Fig. 5)
The suggested homology of sensory cells found in tunicates
with the neuromast cells of craniates was suggested by Bone
& Ryan (1978). These authors found primary sensory cells
in the atrium of Ciona intestinalis, which showed a deeply
inserted apical cilium surrounded by microvilli and were
covered by a mucus dome. Sensory cilia with an apical
cilium surrounded by microvilli are common among deuter-
ostomes, but a cupula is uniquely present in the atrium of
tunicates and in the acustico-lateralis neuromast organs in
craniates. However, the case for homology of the two
systems is not straightforward. The neuromasts in craniates
are secondary sensory cells and are innervated by a separate
nerve, whereas the sensory cells in the cupular organs of
C. intestinalis are primary sensory cells with their own
efferent axon. To complicate matters even more, Burighel
et al. (2003) suggested the homology of the acustico-lateralis
sensory structures in craniates to another group of cells
present on the oral tentacles in Botryllus schlosseri, another
tunicate species. These possible homologs to the neuromast
cells also possess apical cilia surrounded by microvilli, and
are secondary sensory cells. However, they do not possess a
mucus cupula. In Branchiostoma sp., there are several
sensory cell types (primary and secondary) that possess
apical cilia surrounded by microvilli, but no mucus cupula
has been described (Bone & Best, 1978; Baatrup, 1981;
Stokes & Holland, 1995; Lacalli & Hou, 1999).
Mesodermal mesenchyme forms novel
structures (Ruppert, 2005)
The mesoderm consists of two distinct parts in tunicate
larvae: the locomotory larval musculature and the larval
mesenchyme that develops into heart, blood cells and body
musculature in the adult (Nishida, 1987; Hirano & Nishida,
1997). It is not known whether the mesenchyme in tunicates
forms an epithelium at any stage of development. In
cephalochordates, true dorsolateral somites are formed,
embedded in a continuous extracellular matrix and posses-
sing a myocoel that separates the myotome from the
dermatome (Stach, 2000). The medial ventral blood vessel
is accompanied by a contractile coelomic ‘vessel’ that is
probably homologous to the tunicate and craniate heart. A
ventral portion of the mesoderm extends in a segmental
fashion ventrally around the intestine and later during
ontogeny forms the transverse muscle. Dorsal of the ventral
mesoderm and at the ventral border of the dorsolateral
somites, the mesoderm forms segmental nephridia in am-
phioxus (Ruppert, 1996; Stach & Eisler, 1998). The meso-
derm in craniates also forms true dorsolateral somites that,
besides myocoel, myotome and dermatome, possess a scler-
ocoel and a sclerotome, structures involved in forming the
skeletal system. Thus, the cells that produce novel structures
in craniates undergo the well-known epithelial–mesenchy-
mal transition (Thiery & Sleeman, 2006). No such transition
has so far been documented in tunicates, although Katz
(1983), in the only complete anatomical study of a tunicate
larva based on serial transmission electron microscopy,
suggests that the trunk mesoderm consists of two ‘pockets’
rather than a mesenchyme. In addition, the ‘novel struc-
tures’ that, according to Ruppert, should develop from the
larval mesenchyme in tunicates and craniates are not speci-
fied. If he refers to the pericardium/heart this structure is not
novel in an evolutionary sense, because it was already
present in the last common ancestor of the Chordata (and
maybe before). If he refers to the body wall musculature and
musculature of the siphons in the adult sessile ascidians, it is
not clear to what these muscles can be homologized in
craniates. If he refers to the hemocytes in tunicates as being
homologous to blood cells of craniates, the cells found in the
blood vessels of cephalochordates (Welsch, 1975; Ra¨hr,
1981) should also be considered. Thus, the homology and
the level of possible homology of this character implied by
Ruppert’s coding is unclear.
Migratory neural crest (Jeffery, Strickler &
Yamamoto, 2004)
Many features that are craniate apomorphies are derived
ontogenetically from the neural crest (Gans, 1989; North-
cutt, 1996). A peculiarity of neural crest cells in craniates is
that they actively migrate away from their original position
close to the ventro-lateral lines of the early nervous system to
form different tissues and structures in adult craniates. One
population of migratory neural crest cells forms melanocytes
in the epidermis of craniates and is thus responsible for the
sc
ax
ci
cu
sc
ci
cu
sp
sp
(a) (b)
Figure 5 (a) Cupular organ found in the ascidian tunicate Botryllus
schlosseri. Note that the sensory cells are primary sensory cells that
possess their own axonic extensions (after Burighel & Cloney, 1997).
(b) Cupular organ of an osteichthyan craniate in the lateral line system.
Note that the sensory cells do not possess axons, but that branches of
the Nervus lateralis receive the input of these secondary sensoryhair
cells (after Romer & Parsons, 1986).
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 125
Chordate phylogeny and evolutionT. Stach
color pattern visible in many primitively aquatic craniates. In
a phlebobranch ascidian, Ecteinascidia turbinata, Jeffery and
colleagues (Jeffery et al., 2004; Jeffery, 2007) demonstrated
that migratory cells are responsible for the bright orange
coloration observed in larval and adult individuals of this
species. These authors suggested that the cells are homolo-
gous to migratory neural crest cells in craniates. It has to be
noted that based on the similar expression of Pax genes, a
population of cells in the ventro-lateral area of the neural
system of cephalochordates can probably be homologized
with neural crest cells in craniates (Holland & Holland,
2001). However, these cells are not reported to migrate away
from their position in the neural system.
Non-epithelial musculature (Ruppert, 2005)
Ruppert suggested that the musculature in craniates and
tunicates is not epithelial and a plesiomorphic epithelial
condition is retained in cephalochordates. As mentioned
above, the locomotory musculature of all three major
chordate clades develops from dorsolateral archenteron
cells. While in cephalochordates, the medial cells of the
developing somites differentiate into muscle cells, the epithe-
lial somites in craniates go through an epithelial–mesenchy-
mal transition, after which the medial cells differentiate into
muscle cells (Thiery & Sleeman, 2006). In both taxa, a
myocoel separating the medial (prospective) muscle cells
from a lateral cell population exists. In tunicates, there is no
somitic stage with medial and lateral epithelial cells sepa-
rated by a myocoel. The differentiated muscle cells in
craniates and tunicates are apolar and fulfill the definition
of a mesenchyme (Bartolomaeus, 1993). The situation found
in cephalochordates, however, is not the classical epithelial
one. The plate-like muscle cells are in contact with the
extracellular material covering the nerve tube and with the
myocoel. However, each cell spans through the entire length
of the somite, and the cells are not connected by apical
adherens junctions. Thus, coding the character
according to Ruppert’s suggestions leads to conflict with
the ontogenetic similarities seen in the development of
craniates and cephalochordates, and the distinction of the
character states is rather unconventional.
Notochord differentiates beyond the stack
of coin stage (Ruppert, 2005)
Again, it is necessary to describe the respective characters in
the three taxa, in order to judge the primary homology
hypothesis implied by the short description given by Rup-
pert. In cephalochordates, the notochordal cells that are
arranged like a stack of coin during early ontogeny roughly
remain in this arrangement. However, the cells become more
irregular in shape, vacuolated and differentiate into muscle
cells. In addition, several intercellular spaces develop and
additional cells with different characteristics are present as
part of the cephalochordate notochord (Welsch, 1968b;
Ruppert, 1997a; Stach, 1999). In primarily aquatic crani-
ates, the notochord is surrounded by a tough sheath and the
cells that are originally arranged like a stack of coins
continue to proliferate until an irregular arrangement is
found (Boeke, 1908; Schinko et al., 1992; Grotmol et al.,
2006). It is interesting to note that the cells become vacuo-
lated like the cells in cephalochordates. In tunicates, the
stack of coin stage in many (although not all) species
develops into a situation that fulfills the definition of a
coelom (Bartolomaeus, 1993). However, the ontogenetic
mechanism is peculiar, as it seems likely that the mesenchy-
mal cells become fenestrated and a central canal is formed
(Burighel & Cloney, 1997). The suggested homology of a
notochord differentiating beyond a stack of coin stage seems
to be purely linguistic.
Hemal system with functionally distinct
circulating corpuscles (Ruppert, 2005)
While the overall anatomy of the hemal system of cephalo-
chordates is strikingly similar in cephalochordates
and craniates (Ra¨hr, 1979), only a single cell type has
been demonstrated from the hemal system of cephalochor-
dates (Welsch, 1975; Ra¨hr, 1981), whereas several types of
cells are present in the blood vessels of tunicates and
craniates (Sawada, Zhang & Cooper, 1993; Cima et al.,
2001). However, it is difficult to pinpoint more detailed
similarities besides the mere differentness of the cell types
that could be interpreted as evidence for homology of
specific cell types.
Multiciliated epithelial cells (Ruppert, 2005)
Several epithelia, such as the ependymal cells in the central
nervous system, the epithelium of the lungs or the epidermis
of some early ontogenetic stages, possess multiciliated cells
in craniates. In tunicates, multiciliated cells can be found,
for example, in the branchial basket, the intestine and in the
ciliated funnel that is associated with the neural gland
(Burighel & Cloney, 1997). Multiciliated cells, however,
have also been observed in the margin of the larval
mouth (Lacalli, Gilmour & Kelly, 1999; Wicht & Lacalli,
2005) of cephalochordates. Also, even structures that are
supposed to be homologous among the three chordate taxa
show different character states in this respect. For example,
Hatschek’s pit is most probably homologous to the ciliated
funnel in tunicates and the adenohypophysis of craniates.
Yet, Hatschek’s pit consists of monociliated cells, the
ciliated funnel of multiciliated cells and the adenohypo-
physis in craniates can be without cilia [Myxine glutinosa
(Fernholm, 1972); Petromyzon marinus (Wright, 1989)],
multiciliated [Canis lupus (Nunez & Gershon, 1976)] or
monociliated cells [Scyllium canicula (Alluchon-Ge´rard,
1978); Carassius auratus (Yamamoto et al., 1982)]. Besides
the heterogenous distribution of the character in chordates,
it has to be kept in mind that one of the potential sister
taxa of Chordata, the Enteropneusta, has several multi-
ciliated epithelia as well. Thus, a complicated character
distribution plus the possibility that the presence of multi-
ciliar and of monociliar epithelia could be the plesiomorphic
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London126
Chordate phylogeny and evolution T. Stach
condition in Chordata makes this character difficult to
evaluate.
Adenohypophysis consisting of an ectoderm
and an endoderm (Ruppert, 2005)
The ontogenetic derivation of the adenohypophysis is not
uniform in craniates. While it seemed to be established
textbook knowledge that the adenohypophysis in craniates
derives from the endoderm and the ectoderm (Romer &
Parsons, 1986), it is not easy to decide whether Rathke’s
pouch, the ontogenetic rudiment of the adenohypophysis,
derives from the endoderm or the ectoderm, because no
clear distinguishing morphological (or other) criteria exist to
characterize the early germ layer affiliation. More modern
investigations seem to suggest that adenohypophysis cells
derive from ectodermal placodes in Gnathostomata (Toro &
Varga, 2007) and Hyperoartia (Uchida et al., 2003), leaving
the basally branching Hyperotreta as sole taxon, in which
the adenohypophysis probably derives from the endoderm
(Gorbman, 1983). In addition, while the neural gland
complex, the structure considered homologous to the
adenohypophysis in Tunicata, seems to possess endodermal
and ectodermal portions (Ruppert, 1990), this can also be
said about the preoral pit/Hatschek’s pit in cephalochor-
dates (Stach, 1996). Thus, the suggested coding of character
states as synapomorphic for Tunicata and Craniata can
be refuted based on alternative hypotheses of primary
homology.
Pax1/9 (Urochordata) or Pax1 and Pax9(Vertebrata) expression in developing pharynx
and musculature (somites) (Ruppert, 2005)
There are two corresponding genes in craniates to the
orthologous Pax1/9 gene that is found in tunicates and
cephalochordates. These genes are expressed in the pharynx
of all chordate taxa and in enteropneusts as well (Ogasawara
et al., 1999; Lowe et al., 2003). In addition, Pax1 and Pax9
are expressed in the mesodermal somites of Gnathostomata
(Brent & Tabin, 2002) but not Hyperoartia (Ogasawara
et al., 2000). I could not find published evidence that Pax1/
9 is expressed in the tail musculature of tunicates, and
although the caption of this paragraph is taken unaltered
from the caption of the cladogram in Ruppert (2005),
Ruppert neither describes nor cites evidence for this char-
acter in the text.
The Notochordata hypothesis
Phosphocreatine as the sole phosphogen in
muscle cells (Hennig, 1984)
Watts (1975) demonstrated that the energy required for
muscle contraction in chordates is stored in different mole-
cules. In tunicates, these are phosphoarginine and phospho-
creatine. In cephalochordates and craniates, on the other
hand, phosphocreatine is the only molecule used to store
nt
nc
dt
mt
ep
(a)
nt
nc
dmt
mtst
sg
ep
(d)
aodDc
vphaov
vca
vh
vcp
(e)
aodDc
vphaov
vca
vh
vcp
(b)
smc
(f)
sc
(c)
sc
sc
mnc
mc
ntc
smc
Figure 6 Comparison of selected organ sys-
tems of cephalochordates (left side: a, b, c)
and craniates (right side: d, e, f). (a) Somite in
the early developmental stage of Branchiosto-
ma lanceolatum (after Stach, 2000). (b) Vascular
system in a cephalochordate (after Romer &
Parsons, 1986). (c) Cross-sections through
early larva of a cephalochordate (after Wicht &
Lacalli, 2005). (d) Somite in a craniate embryo
(after Buckingham et al., 2003). (e) Vascular
system in a craniate chondrichthyan (after
Romer & Parsons, 1986). (f) Cross-section
through the developmental stage of the neural
tube in a craniate (after Romer & Parsons,
1986). aod, aorta dorsalis; aov, aorta ventralis;
Dc, ductus cuvieri; dmt, dermomyotome; dt,
dermatome; ep, epidermis; mc, muscle cells;
mnc, motor nerve cells; mt, myotome; nc,
notochord; nt, neural tube; ntc, notochord cell;
sc, sensory nerve cells; sg, spinal ganglion;
smc, somatomotoric nerve cells; st, sclero-
tome; vca, vena cava anterior; vcp, vena cava
posterior; vh, vena hepatica; vph, vena portae
hepaticae.
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 127
Chordate phylogeny and evolutionT. Stach
energy (Watts, 1975; Livingstone, 1991). Because phosphoar-
ginine and phosphocreatine are also used for storage of
energy in many other marine invertebrates (Rockstein,
1971), it can be inferred that the situation with two different
molecules is plesiomorphic within chordates. The specializa-
tion to the usage of only phosphocreatine in cephalochor-
dates and craniates can thus be interpreted as a shared,
derived character state in notochordates. This interpretation
is consistent with more recent studies of the evolution of the
molecules involved in energy transfer in the respective groups,
the phosphoargininekinases and phosphocreatinekinases,
respectively (Ellington, 2001; Bertin, 2006; Uda et al., 2006).
Dermatome–myotome–myocoel (Fig. 6)
Somites are complex structures consisting not just of muscle
cells (see e.g. Starck, 1975b; Brent & Tabin, 2002), and as
such are only present in craniates and cephalochordates.
Some of the characters can occur independently in the two
groups. For example, the sclerotome is only found in
craniates (Mahadevan, Horton & Gibson-Brown, 2004). A
lateral group of cells that is ultrastructurally different from
the medial muscle cells is present in both taxa. This group of
cells is similar in its lateral position and is separated by a
coelomic compartment, the myocoel from the muscle cells.
The entire arrangement can therefore be homologized as
lateral dermatome, medial myotome and separating myo-
coel (Ruppert, 1997a; Stach, 2000).
Liver caecum
The liver caecum in cephalochordates is a ventral anteriorly
pointing extension of the intestinal tract behind the bran-
chial complex. The cells in the epithelially organized caecum
are probably responsible for the production of digestive
enzymes but also for the storage of energy-rich components
(Welsch, 1975). While the liver in adult craniates is still
connected with the intestinal tract, its function is more
diverse (Romer & Parsons, 1986). However, the ontogeny
of the liver in most craniates, but especially in the larval
lamprey, demonstrates the origin as a ventral epithelial
extension of the intestine behind the branchial basket
(Damas, 1944). These similarities are unique to craniates
and cephalochordates, and the liver caecum can therefore be
interpreted as a homologous structure, a conclusion in
agreement with recent gene expression studies (Jiang et al.,
2007; Tian et al., 2007).
Notochord along major part of body
This character was suggested by Nielsen (2001) and is the
character after which the taxon Notochordata was named
by the same author for the group comprising craniates and
cephalochordates. While a notochord is present in the tails
of all chordates, such a skeletal element is only present in the
anterior trunk of craniates and cephalochordates.
Blood vessels: Aorta dorsalis, ductus cuvieri,
vena portae hepatica (Fig. 6)
In an elegant study, Ra¨hr (1979) visualized the organization
of the circulatory system of cephalochordates. While there
are differences in the organization of the circulatory systems
of primarily aquatic craniates (Romer & Parsons, 1986;
Mickoleit, 2004), the overall similarity is remarkable. In
order to acknowledge these similarities, three major blood
vessels are chosen as representative for detailed homologies
supported by Ra¨hr’s study: the dorsal posterior aorta
dorsalis, which transports oxygenated blood to the caudal
fin; the portal liver vein, vena portae hepatica, which
connects the two capillary systems around the intestine and
the liver caecum; the main vessel that collects anoxic blood
before it is transported to the endostylar artery in the ventral
midline in the branchial basket, the ductus cuvieri (Fig. 6).
Myomeres
While all chordates possess muscles that function together
with fins and a notochord to propel the animal by undula-
tory movement, only in cephalochordates and craniates are
consecutive muscle blocks separated by an extracellular
material containing mainly collagen and proteoglycans
(Stach, 2000; Gemballa, Weitbrecht & Sanche´z-Villagra,
2003). These muscle blocks contain several phylogenetic
informative characters, as they are composed of recogniz-
able subunits, such as dermatome, myotome or myocoel. As
lucidly discussed by Ruppert (1997a), these complex muscle
blocks should be called myomeres.
Shape of myomere with an anterior dorsal
point
3D-reconstructions of the shape of the myosepta in Bran-
chiostoma lanceolatum (Gemballa et al., 2003) and in hagfish
and lamprey (Vogel & Gemballa, 2000) differ in many
details, but are similar in that they possess a prominent
anterior-pointing cone in the dorsal third of the animals.
Red and white muscle cells
Two different types of muscle cells differentiate during
ontogeny in the somatic musculature in craniates and
cephalochordates but not in tunicates (Starck, 1975b; Bur-
ighel & Cloney, 1997; Ruppert, 1997a). These types of
muscle cells are known as fast or white and enduring or red
fibers in craniates (e.g. Devoto et al., 1996). They corre-
spond to the deep (white) and superficial (red) lamellae in
cephalochordates (Flood, 1968). Only a single type of
muscle cells is present in tunicates (Burighel & Cloney,
1997) that probably corresponds to the deep (white) muscle
cells in notochordates.
Myocoels
On its lateral side, the myotome in the myomeres is sepa-rated by a coelomic space from the lateral dermatome in
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London128
Chordate phylogeny and evolution T. Stach
cephalochordates and craniates. This space is called myo-
coel (Ruppert, 1997a; Stach, 2000). No such space exists in
tunicates, even in animals with many muscle cells (Burighel
& Cloney, 1997; Stach & Kirbach, in press). While there are
also reports that a sclerocoel is present in cephalochordates
(Franz, 1925; Prenant, 1936; Ruppert, 1997a), based on
ultrastructural evidence, these reports were phrased very
carefully as preliminary (Ruppert, 1997a) and could not be
detected in the electron microscopic investigation of earlier
development (Hirakow & Kajita, 1994; Stach, 1999). Thus,
the presence of sclerocoels will be considered as an autapo-
morphy of craniates here.
Segmental excretory organs
During ontogeny, the ventral cells of a somite differentiate
to form the excretory system in craniates (e.g. Starck, 1975b;
Romer & Parsons, 1986; Kluge & Fischer, 1990) and in
cephalochordates as well (Ruppert, 1994a,b, 1996; Stach &
Eisler, 1998). In craniates, this area is the well-known
‘Somitenstiel.’ In both groups, the original arrangement of
these structures is segmental and is rearranged later during
ontogeny. The homology of the excretory structures in the
two taxa has also been supported by similarities in gene
expression patterns (Holland et al., 2001)
Repeated mesodermal coelomic podocytes
with apical cilia covering certain blood
vessels
The segmental excretory organs in cephalochordates and
craniates are complex structures that consist of several
subunits that are recognizable and have been formalized as
a character in the preceding paragraph. In cephalochordates
as well as in craniates, each of the segmental excretory
organs consists of podocytic cells that cover the extracellular
matrix surrounding a blood vessel and that possess apical
cilia (Lacy, Castellucci & Reale, 1987; Kluge & Fischer,
1990, 1991). While all these structures are clearly recogniz-
able in cephalochordates, the apical cilium is, in addition,
surrounded by 10 slender microvilli (Ruppert, 1994a,b,
1996; Stach & Eisler, 1998; Westheide & Rieger, 2007). This
similarity to protonephridia in lophotrochozoan taxa is
clearly due to convergence and the peculiar microvilli are
not present in similar form in any other deuterostome
species.
Segmented ventro-lateral trunk mesoderm
Ventral to the ‘Somitenstiel,’ the somites extend most
probably in the form of segmented mesodermal bands in
cephalochordates (Hatschek, 1881; Stach, 2000). A similar
condition can also be seen in the early ontogenetic stages of
lampreys (Damas, 1944; Shimeld & Holland, 2000; Kusa-
kabe & Kuratani, 2005). In lamprey, later during ontogeny,
the ventral mesoderm becomes an unsegmented mesodermal
cell mass (Damas, 1944). This is the case in other craniates
from the beginning where the ventral mesodermal cell mass
differentiates into the lateral plate (‘Seitenplatte’) and forms
the extensive body coeloms (Funayama et al., 1999).
Unpaired dorsal and ventral fins
Unpaired dorsal and ventral fins that are made up of the
epidermis and repeated mesodermal elements are common
to larval and adult craniates and cephalochordates (Starck,
1975b; Romer & Parsons, 1986; Ruppert, 1997a; Stach,
2000; Iwamatsu, 2004). The usually dorsal and ventral fins
in tunicate larvae, on the other hand, are made up of
extracellular material, the tunic, without repetitive structur-
al mesodermal elements (Burighel & Cloney, 1997; Stach,
2007). Adult tunicates lack fins, with the exception of
appendicularians, where fins are horizontally oriented,
representing a derived feature within Tunicata (Stach, 2007).
Fibroblasts
The extracellular matrix in craniates and cephalochordates
is extensive and contains a high amount of collagen, whereas
the extracellular matrix in tunicates is more feeble. In
addition, fibroblasts, a specific cell type situated within the
extracellular matrix, can be found in craniates (Romer,
1972) and cephalochordates (Welsch, 1968a; Ra¨hr, 1981).
In both taxa, fibroblasts produce collagen molecules. While
tunicates possess orthologous FGF genes, they lack the
special cell type, fibroblasts, within the extracellular matrix,
although it might correspond to an extra-zooidal cell type in
the tunic (Swift & Robertson, 1991).
Dorsal segmental nerve roots carrying
somatosensitive, viscerosensitive and
visceromotoric nerve fibers (Fig. 6)
While a dorsal neural cord is common to all chordates,
segmental nerve roots leaving the neural cord in between
consecutive mesodermal muscle blocks are unique to crani-
ates and cephalochordates (Bone, 1960a,b; Starck, 1982;
Romer & Parsons, 1986; Wicht & Lacalli, 2005). The
similarities are detailed, as the nerve fibers in both taxa
contain somatosensitive, viscerosensitive and viseromotoric
fibers. No dorsal segmental nerve roots exist in tunicates
(Burighel & Cloney, 1997; Okada et al., 2001, 2002).
Frontal anterior eye
Larval cephalochordates possess a photoreceptor consisting
of rows of ciliated receptors and pigment cells that are
situated at the anterior tip of the neural cord, with the
receptor cells situated ventrally and the pigment cells dor-
sally (Lacalli, Holland & West, 1994; Lacalli, 1996). This is
also the initial location of the embryonic eye rudiment in
craniates (Starck, 1975b; Li et al., 1997). Tunicate ocelli, on
the other hand, are situated at the posterior wall of the
cerebral vesicle and they possess only a single, ventrally
located cup-shaped pigment cell (Burighel & Cloney, 1997).
Tunicate ocelli are also unique in the possession of glycogen-
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London 129
Chordate phylogeny and evolutionT. Stach
filled lens cells (Eakin & Kuda, 1972), while in some species,
receptor cells are similar to the ones in the remaining
chordates in that they are primary receptor cells with apical
ciliary structures (Gorman, McReynolds & Barnes, 1971;
Barnes, 1974; Eakin, 1979). Recent investigations into the
gene expression pattern of Rx genes have been taken as
evidence for the homology of the larval ascidian ocellus to
vertebrate eyes (D’Aniello et al., 2006). This would render
the frontal anterior eye a synapomorphy of Chordata.
However, orthologs of Rx genes are known from many
invertebrates, including Ecdysozoa (Eggert et al., 1998) and
Lophotrochozoa (Arendt et al., 2004) and even plants
(Bendahmane, Kanyuka & Baulcombe, 1999). Morevover,
Rx-gene expression has also been reported from the pineal
eye in vertebrates (Mathers & Jamrich, 2000), supporting
the hypothesis of primary homology between the tunicate
ocellus and the pineal eye in vertebrates suggested above.
Chiasma opticum
The nerve tracts that leave the primary sensory photorecep-
tive cells of the frontal anterior eye cross the ventral midline
of the brain floor to the contralateral side, forming a
commissure or a chiasma in craniates (Starck, 1982; Romer
& Parsons, 1986) and cephalochordates (Lacalli et al., 1994).
No such fiber crossing has been detected in tunicates
(Olsson, Holmberg & Lilliemarck, 1990; Takamura, 1998).
Gene expression studies
In the current review, recourse has been made to gene
expression studies on several occasions in order to substanti-
ate hypotheses of primary homology. While an exhaustive
review of gene expression studies relating to chordate evolu-
tion is beyond the scope of this review, a few comments are
necessary, because the results using this technique are
extensively cited by morphologists and are utilized to sup-
port molecular phylogenies (Nielsen, 2003; Ruppert, 2005;
Lacalli, 2006). Whereas gene expression studies have added
an exciting insight and new levels to our understandingof
embryological processes and as such are relevant for phylo-
genetic considerations, these data have rarely been subjected
to an established cladistic methodology even where used in a
phylogenetic context (reviewed e.g. in Svensson, 2004;
Jenner, 2006). For example, it is largely agreed upon that
homology is a relationship of two characters in different
individuals that is based on the reality of a shared historical
process (e.g. Hall, 1995; Bolker & Raff, 1996; Nielsen &
Martinez, 2003; Svensson, 2004; Cracraft, 2005). This rea-
lity is discovered and suggested as a scientific hypothesis in a
two-step process. Based on similarities, the hypothesis of
primary homology is proposed and is subsequently sub-
jected to the congruence test, where it is evaluated under an
optimality criterion (e.g. parsimony or maximum likeli-
hood; de Pinna, 1991; Kitching et al., 1998; Rieppel &
Kearney, 2002; Richter, 2005; Rieppel, 2005). This is not
done in most evo-devo studies as has been rightfully criti-
cized in earlier publications (Nielsen & Martinez, 2003;
Svensson, 2004; Jenner, 2006). Nevertheless, gene expres-
sion studies can be incorporated into phylogenetic analyses
as evidence for primary homology hypotheses, if gene
expression similarities are observed. It must be emphasized
here that the reverse conclusion is not true: if no similarity is
observed, this is no evidence against homology, especially
not if other (e.g. morphological) similarities exist. The basis
for this logical asymmetry has been noted early on (Darwin,
1859) and has been lucidly discussed by Jenner (2006). Thus,
while gene expression studies can be informative in a
phylogenetic context, if they are supportive of primary
homology hypotheses, Chordata might be the taxon, in
which the second step – coding and analyzing gene expres-
sion patterns as characters – could also be gone. This
optimistic expectation derives from the fact that within
Chordata, a relatively high taxon sampling in gene expres-
sion studies exists. This fortunate situation has already led
to the observation that molecular developmental pathways
can dramatically differ even in the specification of homo-
logous structures (Lemaire, 2006) and should be explored
further for phylogenetic purposes.
Molecular phylogenetic studies
Phylogenetic analyses of molecular sequence data have
become standard during the last decades and have chal-
lenged traditional views of animal relationships (Aguinaldo
et al., 1997; Winnepenninckx, Backeljau &Kristensen, 1998;
Zrzavy et al., 1998; Halanych, 2004). While the interrela-
tionships of higher deuterostome taxa have been addressed
specifically in several molecular studies (Turbeville, Schulz
& Raff, 1994; Winchell et al., 2002; Bourlat et al., 2006),
until today only a few have been specifically designed to
address the relationships of the three monophyletic chordate
taxa (Delsuc et al., 2006; Putnam et al., 2008). However,
several other studies have included members of the three
chordate taxa and are thus suitable to resolve their phylo-
geny. These and some related studies will be briefly reviewed
here.
Most molecular phylogenetic studies utilized 18S rDNA
sequences, and these studies date back to the beginning of
the 1990s. From these early studies, it could be inferred that
at least for chordates molecular sequences do not deliver
only one answer. While Turbeville et al. (1994) found
support for the traditional Notochordata hypothesis, other
authors recovered quite unexpected groupings. In the ana-
lysis of Halanych (1998), for example, Tunicata was a sister
taxon to the remaining triploblasts to the exclusion of
flatworms and nematodes, and the study by Swalla et al.
(2000) found Notochordata monophyletic, but Tunicata
sister taxon to Ambulacraria, a taxon consisting of Echino-
dermata and Hemichordata, a result also found by Bourlat
et al. (2003). Analyzing also 18S rDNA data, Giribet et al.
(2000) found support for the Olfactores hypothesis, whereas
Zeng, Jacobs & Swalla (2006) recovered an unresolved
trichotomy and yet favored the traditional Notochordata
hypothesis. Based on the very results of Zeng and collea-
gues, Swalla & Smith (2008) supported the Olfactores
Journal of Zoology 276 (2008) 117–141 c� 2008 The Author. Journal compilation c� 2008 The Zoological Society of London130
Chordate phylogeny and evolution T. Stach
hypothesis. Stach & Turbeville (2002) also favored the
Notochordata hypothesis; however, these authors, primar-
ily concerned with Tunicata as the ingroup, did not address
this question, but rooted their optimal trees after unordered
and unrestricted analyses. In the first spectral analysis of a
deuterostome dataset of 18S rDNA sequences, Wa¨gele
(2001) concluded that these data contain an exceptionally
low signal-to-noise ratio and these data therefore became a
textbook example for missing phylogenetic signal. Several
studies expanded on the 18S rDNA datasets in terms of
adding additional molecular sequences, while sacrificing
taxon sampling at the same time. Winchell et al. (2002)
added 28S RNA sequences and did not recover a mono-
phyletic Chordata in their analyses. Instead, these authors
recovered a sister-group relationship between Notochordata
and Ambulacraria, and Tunicata as the sister taxon to this
latter grouping. Naylor & Brown (1998), who phylogeneti-
cally analyzed the first complete mitochondrial genome of a
cephalochordate, were also unable to support a monophy-
letic Chordata and suggested that the widely held opinion
that adding longer sequences to converge on the true
phylogeny might not always be justified. The final twist in
results frommolecular studies came recently, when the study
of Delsuc et al. (2006), based on limited taxon sampling (14
deuterostome taxa) but an impressive 33 800 amino acid
positions (c. 14 000 informative sites), supported the Olfac-
tores hypothesis. These authors were also unable to recover
a monophyletic Chordata and found the cephalochordate to
be related to the sea urchin in their analysis, a finding that
was considered unreliable in subsequent analyses (Bourlat
et al., 2006). While the analysis by Delsuc and colleagues is
based on a high amount of sequence data, it has to be kept in
mind that the authors used concatenated operational taxo-
nomic units, which might be problematic (Bapteste et al.,
2008). Moreover, the conclusions are primarily drawn from
model-based analyses, which are known to be sensitive to
model selection (Pol & Siddall, 2001; Kolaczkowski &
Thornton, 2004; Piontkivska, 2004), which is not satisfacto-
rily explored for amino acid alignments (see e.g. (Abascal,
Zardoya & Posada, 2005). In general, we can only judge
which of comparatively few models best fits a given align-
ment, but we have no indication how close this model is to
reality, which, after all, comprises several hundreds of
million years of evolution in millions of taxa. In this context,
the bon mot of Mishler (2005) should also be recalled, who,
with respect to genomic scale phylogenetic studies, stated
that complicated models for tree building can be seen as
attempts to compensate for marginal data.
Phylogenetic analysis: a matrix based
on all combined arguments proposed
by adherents of the competing
hypotheses
For the present review, I have combined all characters that
have been discussed above in favor of one or the other
hypothesis with the intention to make them accessible for a
formal cladistic analysis. The matrix can be found as
Supporting Information Appendix S1, while coding follows
the arguments just presented. When primary character
homology was not refuted outright, the character coding
suggested by other authors was included, in order to test
these homology hypotheses, even if doubts have been
expressed in the presentation above. It contains the 34
characters discussed above, coded as binary characters (four
are parsimony uninformative)