ZOOLOGICAL SCIENCE 26: 97–111 (2009) ¤ 2009 Zoological Society of Japan

[ESSAY]

An Advanced Filter-Feeder Hypothesis for

Urochordate Evolution
Nori Satoh*
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502,
Japan and Marine Genomics Unit, Okinawa Institute of Science and Technology
Promotion Corporation, Uruma, Okinawa 904-2234, Japan

Chordates consist of cephalochordates, urochordates, and vertebrates. They originated from a

common ancestor(s) by evolving a novel type of tadpole-like larva characterized by a notochord and
dorsal neural tube. Urochordata or Tunicata comprises three classes, Ascidiacea (ascidians),
Thaliacea (salps), and Appendicularia (larvaceans); are extremely diverse in morphology, life his-
tory, and mode of reproduction; and may include either free-swimming or sessile adults. Recent
molecular phylogenies support the notion that cephalochordates are basal among chordates, with
urochordates the sister group to vertebrates. This revitalized evolutionary scenario compels us to
explain how the huge variety of lifestyles of extant urochordates evolved from a cephalochordate-
like ancestor. Here, an advanced filter-feeder hypothesis is presented, in which, although the
taxonomic position of larvaceans is enigmatic, it is argued that among urochordates, free-living lar-
vaceans are basal, while sessile ascidians are derived. Urochordates might have evolved their traits
in response to strong evolutionary advantages as specialists of suspension filter feeding. Never-
theless, the molecular mechanisms involved in the formation of the notochord and several other
organs in ascidian embryos are more comparable to those of vertebrates than cephalochordates.
Thus, urochordates are close relatives to vertebrates.

Key words: urochordate evolution, appendicularian, ascidian, various lifestyles, advanced filter-feeder
hypothesis

have evolved from a common ancestor shared with the two

INTRODUCTION
The origin and evolution of chordates have been
debated for more than one and a half centuries in conjunc-
tion with the question of the origin of ourselves (vertebrates)
(Darwin, 1859, 1871; Kowalevsky, 1866; Kowalevsky, 1867;
Haeckel, 1868; Garstang, 1928; Berrill, 1955; Romer, 1967;
Satoh and Jeffery, 1995; Gee, 1996; Cameron et al., 2000;
Satoh, 2003; Lacalli, 2005; Gerhart et al., 2005; Swalla,
2006; Swalla and Smith, 2008). The phylum Chordata con-
sists of the subphyla Cephalochordata (lancelets),
Urochordata (tunicates) (Fig. 1) and Vertebrata. The three
groups are characterized by the possession of the noto-
chord, dorsal hollow neural tube, branchial slits, endostyle,
and postanal tail, the former two being hallmarks of the
chordate body plan. The chordates are also deuterostomes,
with a group that also includes echinoderms (starfish and
sea urchins), hemichordates (acorn worms) (Schaeffer, 1987;
Brusca and Brusca, 2003), and the recently characterized
Xenoturbella (Bourlat et al., 2003). Chordates are thought to
* Corresponding author. Phone: +81-75-753-4081;
Fax : +81-75-705-1113;
E-mail: norisky@oist.jp
doi:10.2108/zsj.26.97
major non-chordate deuterostome groups.
Previously, various protostome animal groups, including
mollusks, annelids, and lophophorates, were discussed in
relation to the origin of chordates (e.g., Løvtrup, 1977;
Gutmann, 1981), but it is now the consensus that protos-
tomes are not relevant to the origin of chordates. Of non-
chordate deuterostome groups, hemichordates have been
discussed most frequently in relation to the origin of chor-
dates (Barrington, 1965; Schaeffer, 1987; Cameron et al.,
2000; Tagawa et al., 2001; Gerhart et al., 2005; Swalla,
2006; Swalla and Smith, 2008), although Jefferies (1986)
discussed their relationship to an extinct carpoid echino-
derm. Two major alternative ancestors for the origin of chor-
dates are a sessile ancestor and a free-living ancestor (Fig.
2) (see Lacalli [2005] for a recent discussion). The former
scenario (sessile ancestor) hypothesizes that ancestral deu-
terostomes were sedentary, like modern pterobranch
hemichordates (Fig. 2A) (Garstang, 1928; Berrill, 1955;
Romer, 1967). Then, chordates evolved from the motile lar-
val stage of sessile ascidians by paedomorphosis, in which
the larval stage became sexually mature and replaced the
adult. The ancestral life cycle was thereby truncated, and a
new adult form evolved by incorporating features originally
evolved to serve the larval stage. According to the alterna-
98 N. Satoh
tive scenario of progressive alteration of motile adults, a
free-living, enteropneust hemichordate-like deuterostome
ancestor evolved to a cephalochordate-like chordate ances-
tor (Fig. 2B) (Tokioka, 1971; Presley et al., 1996; Salvini-
Plawen, 1999; Holland, 2000; Cameron et al., 2000). As
opposed to being larvae, the sequence of ancestral forms
would in this case consist of motile, bilaterally symmetrical
animals. Therefore, motile forms such as enteropneusts and
amphioxus would be considered the main lineage, while
tunicates would be more derived.
THE ABORAL-DORSALIZATION HYPOTHESIS FOR THE
ORIGIN OF CHORDATES
In a previous essay, I proposed the aboral-dorsalization
hypothesis to explain how chordates evolved from a com-
mon ancestor(s) shared with non-chordate deuterostomes
(Satoh, 2008). This hypothesis is based on the consider-
ations that (a) extant cephalochordates are closest to the
chordate ancestor, (b) the occurrence of tadpole-type larvae
that possessed a notochord and neural tube was essential
for origination of the chordate body plan, (c) these two
characters were completely novel, without any relation to
structures of extant non-chordate deuterostomes, (d) mor-
phogenesis to form the two characters occurs during
embryogenesis, and due to limited space on the oral side of
the ancestral embryo, the notochord and neural tube
evolved on the aboral side of the embryo, leading the aboral
side to dorsalize. Each of these issues is relevant to uro-
chordate evolution and is discussed in more detail below,
even if it seems repetitious the previous essay.
(a) Cephalochordates are closest to the chordate ances-
tor
Molecular phylogeny has provided a powerful tool for
examining phylogenetic relationships among deuterostomes
and thereby their evolution (e.g., Wada and Satoh, 1994;
Cameron et al., 2000; Winchell et al., 2002). Recent Bayes-
ian analyses that used sequenced genomes and a large
quantity of expressed sequence tags (ESTs) and that com-
pared ~60,000 aligned amino acid positions deduced from
~1,000 nuclear genes, showed clearly that echinoderms and
hemichordates form a clade (Ambulacraria) and that, among
Fig. 1. Urochordates for which evolu-
tionary relationships are discussed in the
present study. (A) A larvacean,
Oikopleura dioica (by Atsuo Nishino).
(B) A solitary ascidian, Ciona intestinalis.
(C) A compound ascidian, Botryllus
schloseri (by Shigeki Fujiwara). (D) A
thaliacian, Doliolum nationalisi, showing
budding (by Atsuo Nishino).
Fig. 2. Two alternative scenarios of chordate evolution. (A) Sce-
nario in which urochordates (U) are basal among chordates, and
cephalochordates (C) and vertebrates (V) form the sister group. In
this scenario, sessile pterobranchian hemichordates (Hp) are
thought to be closest to the chordate ancestor(s); sessile ascidians
(As) are basal among urochordates, and appendicularians (Ap)
evolved later. (B) Another scenario in which cephalochordates (C)
are basal among chordates, and urochordates (U) and vertebrates
(V) form the sister group. In this scenario, free-living enteropneust
hemichordates (He) are closest to the chordate ancestor(s); free-
living appendicularians (Ap) are basal among urochordates, and
ascidians (As) evolved later. D, deuterostome ancestor.
another distinct clade of chordates, cephalochordates repre-
sent the most basal extant chordate lineage, with urochor-
dates the sister group to vertebrates (Fig. 3) (Delsuc et al.,
2006; Bourlat et al., 2006; Putnam et al., 2008; Swalla and
Smith, 2008). The evolutionary relationships shown by
Putnam et al. (2008) and deduced from the sequenced
genomes may provide a final conclusion regarding this
enigmatic problem that has long been debated. My further
discussion of the evolution of urochordates is based on this
evolutionary scenario.
This revised or revitalized phylogenic relationship is sug-
gestive regarding the evolution of deuterostomes and the
origin of chordates. Namely, the fact that cephalochordates
are basal and free living strongly suggests the motile ances-
try of chordates that were derived from enteropneust
hemichordate-like animals (Fig. 2B). The motile ancestry of
chordates also eliminates the embryological problem or gap
between hemichordate enteropneusts and urochordate
ascidians that arises if chordate ancestry is considered to be
sessile and ascidian-like (Fig. 2A). Bilaterally symmetrical
 A Hypothesis for Urochordate Evolution 99

Fig. 3. Phylogenetic relationships among deuterostomes and the evolution of chordates. The deuterostomes, consisting of Xenoturbella,
echinoderms, hemichordates, cephalochordates, urochordates (tunicates), and vertebrates, comprise two major clades. One consists of
echinoderms and hemichordates (clade I, the Ambulacraria), and the other consists of the chordates (clade II). The chordates comprise the
cephalochordates, urochordates, and vertebrates, among which the cephalochordates are basal (arrow), and urochordates and vertebrates
form the sister group. However, due to a high rate of DNA sequence substitution, the molecular phylogeny presented here does not necessarily
provide a conclusive result on the phylogenetic position of appendicularians and pterobranchians (dotted lines). From Swalla and Smith (2008).

cleavage, very early fate determination, formation of a tad-
pole larva with a comparatively low number of constituent
cells, and larvae that do not open a mouth are all features
of ascidian embryogenesis that are unique and derived
among deuterostomes, as discussed below (Satoh, 1994;
Satoh, 2003). In contrast, the radially symmetrical cleavage
pattern and formation of hollow blastulae of enteropneusts
closely resemble those of cephalochordates, thus making
the connection between non-chordate and chordate deu-
terostome embryogenesis very easy (Fig. 1B).
(b) The occurrence of tadpole-type larvae with a noto-
chord and neural tube was essential for the origin of the
chordate body plan
Comparison of embryogenesis between chordates and
non-chordate deuterostomes highlights one conspicuous
feature that is seen only in chordates, namely, the formation
of tadpole-type larvae. During the Cambrian explosion,
which occurred 540–520 million years ago, metazoans
evolved in the sea by devising various body plans that are
seen in extinct and extant animals (Conway Morris, 1982;
Brusca and Brusca, 2003). What caused the primary
100 N. Satoh
variation in animal body plans? I conjecture that the most
important factors were feeding and reproduction. It is highly
likely that, to gain niches that provided more efficient
feeding, metazoans had attempted various modes of
locomotion, which is inevitably associated with the evolution
of various forms or morphologies (Willmer, 1990; Nielsen,
2001; Brusca and Brusca, 2003). Competition to gain more
efficient feeding niches might also have affected the life
history of metazoans. Most marine invertebrates show a
biphasic life history, namely, a larval stage and adult stage,
which also are key to considering urochordate evolution
(see below). It has been long argued whether a mode of
direct development from fertilized eggs to juveniles is
primitive or a mode of indirect development with larval stage
and adult stage is basal (e.g., Nielsen, 2001; Callery et al.,
2001; Smith et al., 2007). The biphasic life style has the
advantage of providing the opportunity to twice invade new
niches for feeding. In ascidians, it has been demonstrated
that direct development was derived from indirect
development (Hadfield et al., 1995; Jeffery and Swalla,
1990; Gyoja et al., 2007)
All marine non-chordate deuterostomes develop larvae
that swim with cilia or a band of cilia, for example, the plu-
teus of sea urchins and tornaria of acorn worms (my discus-
sion on this point is restricted to deuterostomes and does
not extend to protostomes; Nielsen, 2001). It is highly likely
that our ancestor abandoned this cilia-propelled style of
swimming and instead developed tadpole-type larvae
(Satoh, 2003). Given that feeding is one of the most impor-
tant activities of living organisms, the origin of a more effi-
cient mode of locomotion increased feeding efficiency. A
tadpole-type larva comprises the trunk and tail regions, with
the former containing a dorsal central nervous system and
the latter a midline notochord, dorsal nerve cord, and bilat-
eral muscle cells. The larva swims by beating the tail with
striated muscles, and this type of larva is thought to be able
to swim faster and thus catch prey more efficiently than lar-
vae with ciliary movement. The notochord is the supporting
organ for the tail and provides the stiffness required for effi-
cient working of the tail muscles. Among the five synapo-
morphies of chordates, the notochord, dorsal neural tube,
and postanal tail originated along with efficient locomotion of
the larval form, while the branchial slits and endostyle devel-
oped along with filter feeding. As discussed in a previous
review, cephalochordates, which are now positioned as the
stem lineage of chordates, show an intermediate mode of
larval swimming, first with ciliary movement and then with a
beating tail (Satoh, 2008).
(c) The notochord and hollow neural tube are completely
novel organs, without any relationship to organs of non-
chordate deuterostomes
If one compares cephalochordate embryogenesis with
enteropneust hemichordate embryogenesis, it is clear that
the embryos and larvae of hemichordates have no
structures (tissues or organs) that are comparable to the
chordate notochord and neural tube. In other words, the
notochord and neural tube are characteristic features of
chordates, and completely novel, without any relationship to
structures in non-chordate deuterostomes.
The early embryogenesis of the cephalochordate
amphioxus, up to the late neurula stage, is suggestive about
chordate origins (e.g., Hatschek, 1893; Conklin, 1932;
Hirakow and Kajita, 1994). The radially symmetrical cleav-
age pattern resulting in the formation of a hollow blastula
resembles the cleavage patterns of sea cucumbers
(Echinodermata) and acorn worms (Hemichordata). The
chordate affinities of amphioxus appear at the gastrula and
neurula stages. In contrast to non-chordate deuterostome
gastrulation, in which the archenteron invaginates into a
wide blastocoelic space, in cephalochordate gastrulation the
flattened endodermal plate ingresses deeply to the animal
side of the embryo, causing the embryo to become cup-
shaped with a deepened archenteron. Ascidians also exhibit
this type of gastrulation (Satoh, 1994). Each cell of the ecto-
derm has developed a single cilium by about the time of late
gastrulation, and after hatching the embryos and larvae
swim first by ciliary movement and then by twitching the
muscular tail. The neural plate is formed as the flattened
dorsal side of the embryo, and as in vertebrates, the neural
tube is formed by the curling up of both ends of the plate to
coalesce at the dorsal midline. As the neural tube forms, the
notochord develops from the adjacent chordamesodermal
plate constituting the roof of the archenteron, by a pouching
off upwards of midline cells along the chordamesodermal
plate. The amphioxus notochord has muscular properties
(Ruppert, 1997; Suzuki and Satoh, 2000).
Relevant to chordate origins, it was emphasized that
morphogenesis to form the dorsal neural tube is completely
different from that to form the apical nervous system of non-
chordate deuterostome embryos. In other words, we no lon-
ger need to consider Garstang’s dipleurula theory that dor-
sal neural tube formation has an affinity to the ciliary-band
system of non-chordate embryos (Garstang, 1928; Nielsen,
1999). In addition to the dorsal neural tube, the notochord is
also a novel organ that originated during chordate evolution.
It is worth mentioning that the neural tube and notochord
form on the dorsal side of the archenteron in the gastrula
and neurula stages, rather than at the stage when the juve-
nile is formed.
(d) Morphogenesis to form the two characters occurs on
the aboral side due to limited space of the oral side of the
ancestral embryo
Given that the evolution of tadpole-type larvae was
critical for chordate origins, it is clear which developmental
stages we should compare between non-chordate and
chordate deuterostomes in order to gain insight into
chordate origins. The notochord and dorsal neural tube,
hallmark chordate characters, are formed by morphogenesis
that occurs during embryogenesis, rather than during the
stage of juvenile formation by metamorphosis of the larva.
Therefore, the mode of embryogenesis should be compared
between chordate and non-chordate deuterostomes. For
example, adult enteropneust hemichordates (acorn worms)
contain a structure, the stomochord, which is a protrusion of
gland-like epithelium into the coelomic space of the probos-
cis. The possible relationship of the stomochord or
hemichord to the origin of the notochord has long been
debated and even led to the name “hemichordates” (half-
chordates) (Bateson, 1886); however, there is no molecular
evidence to support its evolutionary connection to the noto-
 A Hypothesis for Urochordate Evolution 101

chord (Tagawa et al., 1998; Peterson et al., 1999).

During the evolution of chordates, the notochord and
hollow neural tube came to be formed on the dorsal side of
the ancestral form of the embryo. Bilaterian bodies are char-
acterized by three major axes, namely, the anterior-posterior
(A-P) axis, dorsal-ventral (D-V) axis, and right-left (L-R) axis.
Early embryos of most non-chordate deuterostomes lack the
D-V axis; instead, they have an oral-aboral axis (Davidson
et al., 2002; Gerhart, 2006). One might imagine that during
the embryogenesis of ancestral chordates, the notochord
and neural tube could have formed in any side of the
embryo. However, formation of the notochord and neural
tube in the anterior or posterior regions of the ancestral
embryo would have hindered proper formation of the diges-
tive system. Formation of the organs on either the right or
left side would have made the body asymmetric along the
A-P axis, which probably would have been deleterious for
the efficient head-forward movement of larvae. The last
alternative position for notochord and neural tube formation
was oral or aboral. This choice might have been determined
by the space available within the embryo. In non-chordate
deuterostome embryos, the anterior edge of the archenteron
bends ventrally and then connects with the newly formed
mouth invagination or stomadium to form a complete diges-
tive tract. It is natural to consider that this mode of digestive
gut formation might have been retained in embryos of chor-
date ancestor(s). This means that the oral side of the
embryo contains very limited space, while the aboral side
provides more space to develop novel structures. Accord-
ingly, the neural tube and notochord were formed on the
aboral side of the ancestral embryo. In other words, the abo-
ral side of non-chordate deuterostome embryos became the
dorsal side of the chordate embryos to create the tadpole-
type larva as the result of necessity. This hypothesis of abo-
ral dorsalization for the origin of chordates appears to be
much simpler and more natural than hypotheses previously
proposed (Satoh, 2008).
Although this hypothesis must be discussed further
together with data from molecular developmental biology
(Satoh, in preparation), the content of the hypothesis is very
suggestive for discussing the origin and evolution of uro-
chordates. Recent molecular developmental studies of the
hemichordate Saccoglossus kowalevskii compared the
UROCHORDATES AND THE EVOLUTION OF THEIR
EXTREMELY DIVERSE LIFESTYLES
Urochordates are sometimes called tunicates. The
name Tunicata was coined by Lamarck (1816) based on the
fact that all urochordates are covered with a “tunic” that
contains a cellulose-like polysaccaride called tunicin, a
unique characteristic that strongly suggests their
monophyly. A bacterial gene for cellulose synthase was
likely introduced by horizontal transfer into the genome of
the urochordate ancestor (Mathysse et al., 2004; Nakashima
et al., 2004). The tunic functions as an exoskeleton, provid-
ing support and maintaining body shape, and thus might
have helped in the evolution of sessile ascidians. Several
zoologists prefer Tunicata to the term Urochordata, because
the tunic is a synapomorphy for urochordates and because
urochordates are highly divergent from Cephalochordata
and Vertebrata (e.g., Zeng and Swalla, 2005). I prefer the
term Urochordata because of the importance of keeping in
mind the phylogenetic relationship of urochordates to the
two other chordate groups. All urochordates have bilateral
symmetry and have incurrent (branchial) and excurrent
(atrial) siphons that control the flow of seawater through the
pharynx. The ventral pharynx bears the endostyle, a thyroid-
gland homolog (e.g., Ogasawara et al., 1999). Adults have
a large, solid neural ganglion that controls muscle activity in
the siphons as well as the mantle.
Urochordata consists of three classes, Appendicularia,
Ascidiacea, and Thaliacea (Table 1; Fig. 1). Planktonic
appendicularians (larvaceans) are solitary and comprise ~65
species (Fig. 1A; Alldredge, 1976; Galt and Fenaux, 1990).
Thaliaceans are all planktonic and colonial and comprise
three orders, Pyrosomida, Doliolida (Fig. 1D), and Salpida,
the last two of which are joined into Myosomata (Bone,
1998; Van Soest, 1998; Godeaux, 1998). So far, ~75 spe-
cies have been described as members of Thaliacea. In con-
trast to these two classes, Class Ascidiacea contains ~2300
Table 1. Comparison of characters among groups of urochor-
dates.
sperm test
sexuality Life style motility
morphology cells

expression profiles of genes involved in the formation of the Outgroup

antero-posterior axis (Lowe et al., 2003) and dorso-ventral Hemichordates ‫ۃ‬,‫ۅ‬ solitary buried primitive –
(Enteropneusts)
axis (Lowe et al., 2006). Lowe et al. (2006) showed that the
Cephalochordates ‫ۃ‬,‫ۅ‬ solitary half-buried primitive –
Bmp-chordin axis is present in the hemichordate embryo
Urochordates
and that hemichordates develop the mouth on the non-Bmp
side, like arthropods but opposite to chordates. Signaling by Appendicularia (‫ۅ‬,‫)ۃ‬ solitary planktonic primitive –

Bmp and its antagonist chordin govern the ventral and dor- Ascidiacea

sal side, respectively, of cephalochordate (Yu et al., 2007) Enterogona

and vertebrate embryos (De Robertis and Sasai, 1996), Phlebobranchia solitary/social sessile modified +
strongly suggesting that the dorso-ventral axis was rear- Aplousobranchia colonial sessile modified +
ranged and inverted during the evolution of chordates (Low Pleurogona
et al., 2006; Gerhart, 2006). Here I just wish to point out that Stolidobranchia solitary/social/colonial sessile modified +
the dorso-ventral axis inversion does not necessarily explain
Aspiraculata solitary/social sessile modified +
the occurrence of the notochord and the hollow neural tube
Thaliacia
on the dorsal side of chordate embryos.
Pyrosomida colonial planktonic modified ?

Doliolida colonial planktonic modified +

Salpia colonial planktonic modified ?

102
species, some of which are solitary (Fig. 1B) and some colo-
nial (Fig. 1C), and which may exhibit enormously diverged
lifestyles (Berrill, 1950; Satoh, 1994). It comprises two
orders, Enterogona and Pleurogona. Enterogona consists of
Aplousobranchia and Phlebobranchia; all members of the
former are colonial, while the latter includes Ciona intestinalis
(Fig. 1B), an emerging model organism whose genome has
been decoded (Dehal et al., 2002; Satoh et al., 2003; Satoh
and Levine, 2005; Shi et al., 2005; Nishida, 2005;
Passamaneck and Di Gregorio, 2005). Pleurogona consists
of Stolidobranchia and Aspiraculata, and Stolidobranchia
consists of Molgulidae, Styelidae, and Pyuridae (Table 1).
(a) Appendicularians, a highly derived group among uro-
chordates
Appendicularians, or larvaceans, are highly derived
compared with the other urochordate groups, which also
show different modes of lifestyle (Alldredge, 1976; Galt and
Fenaux, 1990; Nishino and Satoh, 2001). Appendicularians
retain the tail as adults, using it to beat water through a
secreted cellulose “house” to filter feed. Filtering of food is
accomplished by meshes in the house cellulose and by
mucous nets secreted by the animal. Appendicularians are
found in the surface water of all oceans and are sometimes
extremely abundant.
Oikopleura dioica is a larvacean that has recently been
used for molecular embryological studies (e.g., Nishida,
2008; see below). Its life cycle is extremely short, only about
5 days. In accord with its very rapid life cycle, its genome
became reduced to 50–65 Mb in length, the smallest
genome yet found in a chordate (Seo et al., 2001). Although
reduction of the genome is also evident in the ascidian
Ciona intestinalis (Dehal et al., 2002), this tendency is more
conspicuous in larvaceans, and the chromosome number of
O. dioica is reduced to only four. This tendency for genome
reduction in appendicularians and ascidians caused several
lineage-specific reorganizations of genes and genomes
(Satoh et al., 2006). One example is breakage of the Hox
gene cluster. Hox genes are characterized by a clustered
organization and by colinarity between gene order within the
cluster and sequential pattern of expression during develop-
ment. In contrast to a typical organization of 13 paralogy
groups in cephalochordates (Garcia-Fernandez and Holland,
1994), with the number now extended to 15 (Holland et al.,
2008), the Ciona intestinalis genome lacks Hox7, -8, and -9
(Dehal et al., 2002), and the Hox cluster is split between
two chromosomes (Ikuta et al., 2004; Shoguchi et al.,
2008). The Oikopleura genome also appears to have lost
Hox3, -5, -6, -7, and -8, and the remaining Hox genes are
scattered in the genome, showing a rather severe break-
down of the Hox cluster (Seo et al., 2004). The loss of
classical retinoic acid signaling in Oikopleura is likely an
evolutionary alteration associated with the rupture of the
Hox cluster (Canestro and Postlethwait, 2007).
(b) Ascidians
Ascidians also exhibit a variety of lifestyles: some are
solitary, while others are social or colonial (Satoh, 1994).
The embryological mode of ascidians is unique among chor-
dates, with features such as bilaterally symmetrical cleav-
age, blastula formation without a real blastocoel, very early
N. Satoh
determination of the developmental fate of embryonic cells,
gastrulation that starts around the 120-cell stage, formation
of a tadpole-type larva composed of only ~2500 cells, and a
very short period of larval life during which the mouth is
never opened (Satoh, 1994; Jeffery and Swalla, 1997;
Nishida, 2005). The larvae of solitary ascidians are plank-
tonic, but for only about 2 days or less, and metamorphose
quickly to sessile juveniles. In contrast to this fully indirect
development, many compound ascidians composed of tiny
zooids produce relatively few eggs with high yolk content.
These eggs are fertilized and subsequently brooded within
the cloacal chamber. They are not released until the swim-
ming tadpole larvae develop. Compound or colonial ascidi-
ans show high capability and various modes of asexual
reproduction (e.g., Manni et al., 2007; Kawamura et al.,
2008). In addition, various degrees of larval suppression
occur among ascidians with this mixed life-history strategy,
and some Molgula species actually display fully direct devel-
opment (Jeffery and Swalla, 1990; Gyoja et al., 2007).
(c) Thaliaceans are also derived
All thaliaceans lack a free-swimming larval stage, and
they differ markedly in their approaches to direct
development (Bone, 1998; Van Soest, 1998; Godeaux,
1998). In pyrosomes, each zygote develops directly to an
oozooid, with no evidence of a larval stage. The oozooid
then buds to produce a colony. Doliolids produce tailed
larvae with approximately 40 notochord cells, but each larva
is encased in a cuticular capsule and does not swim. The
larva metamorphoses to an oozooid. Salps undergo internal
fertilization within the oviduct. The zygotes implant and form
a placenta-like association with the parent in a uterine
chamber in the oviduct. The embryos develop directly to the
adult form.
PHYLOGENETIC RELATIONSHIPS AMONG
UROCHORDATES
As discussed in the Introduction, the phylogenetic
position of urochordates among chordates and the
phylogenetic relationship among the three classes of
urochordates are quite different according to the two
alternative scenarios of the origin of chordate. For example,
in the traditional sessile-ancestors scenario, urochordates
are placed most basal, having an evolutionary relationship
to sessile pterobranchian hemichordates, and therefore ses-
sile ascidians represent the stem group among
urochordates (Fig. 2A). Cephalochordates evolved from the
ascidian larval stage, and subsequently vertebrates evolved
(Fig. 2A), whereas among urochordates, larvaceans evolved
from the ascidian larval stage by paedomorphism (Fig. 2A).
In the alternative and more reliable scenario of free-living
chordate ancestors, cephalochordates represent the stem
group among chordates, having an evolutionary relationship
to free-living enteropneust hemichordates, while urochor-
dates are more derived (Fig. 2B). Among the three urochor-
date classes, solitary, free-living appendicularians become
the stem group (Fig. 2B).
As discussed above, it is now evident that the cephalo-
chordates are basal among chordates and that urochordates
evolved from cephalochordate-like ancestors. Nevertheless,
there are still at least three possibilities for the evolution of
 A Hypothesis for Urochordate Evolution
urochordates (Fig. 4). Although it has been suggested that
Thaliacea are polyphyletic, with doliolidas having arisen
early from the ascidian lineage and salps having arisen later
(Holland, 1989), the present essay considers thaliacians as
a single clade in order to avoid further confusion caused by
the complexity of this urochordate group. The first scenario
hypothesizes that ascidians arose earlier and that
larvaceans evolved from the ascidian larval stage by
paedomorphism (Fig. 4A). This view is supported by recent
studies of Oikopleura dioica (Stach, 2007; Stach et al.,
2008). During the metamorphosis of ascidians from free-
living tadpole larvae to sessile juveniles, the body axis is
rotated around 90°. Recently, Stach et al. (2008) followed
precisely the lineage of Oikopleura embryonic cells and
found that embryonic epidermal cells in the trunk migrate
through 90°, although larvaceans never show the mode of
metamorphosis seen in ascidians. They regarded this and
other traits as common to urochordates and insisted that the
last common ancestor of tunicates had a sessile adult like
ascidians. However, these similarities in embryonic
development between larvaceans and ascidians do not
provide a conclusive answer as to which originated first
during urochordate evolution.
The second scenario suggests that there are two clades
among urochordates, one consisting of larvaceans and
Pleurogona ascidians and the other of Enterogona ascidians
and thaliacians (Fig. 4B). The third scenario emphasizes
that larvaceans arose earlier as common ancestors of
ascidians and thaliacians (Fig. 4C). According to this last
scenario, ascidians evolved a lifestyle with a sessile adult
stage in their own lineage.
Fig. 4. Three alternative scenarios for urochordate evolution. (A)
Scenario in which three groups of urochordates [appendicularians
(Ap), ascidians (As), and thaliacians (Th)] evolved almost simultane-
ously. (B) Scenario showing two lineages of urochordates, one
including appendicularians and Enterogona ascidians (Ase). and the
other Pluerogona ascidians (Asp) and thaliacians. (C) Scenario in
which appendicularians (Ap) evolved first, and then a large group of
ascidians (As) emerged, from which thaliacians (Th) arose later.
103
As mentioned earlier, molecular phylogenetic analyses
have contributed enormously to solving complicated
problems in metazoan phylogeny. Recent molecular
phylogenetic studies using the 18S and 28S rRNA genes
demonstrated that urochordates are monophyletic, and that
among them, appendicularians originated first and then
diverged into the large group of ascidians and its sister
group, the thaliaceans, although the taxonomic position of
larvaceans remains enigmatic (Fig. 3) (Wada, 1998;
Cameron et al., 2000; Zeng and Swalla, 2005). This
supports the evolutionary scenario shown in Fig. 4C.
Although the monophyly of Stolidobranchia (including
Molgulidae, Pyuridae and Styelidae) is strongly supported
by 18S rRNA gene sequences, the molecular phylogeny of
urochordates is inconclusive, mainly because of a high rate
of change in DNA sequences (B.J. Swalla, personal
communication). Additional molecular phylogenetic studies
are required to understand the relationships among
thaliaceans.
THE ADVACED FILTER-FEEDER HYPOTHESIS FOR
UROCHORDATE EVOLUTION
Here I propose the advanced filter-feeder hypothesis to
explain the evolution and resultant diverged characters of
urochordates, especially of ascidians. As mentioned above,
given that cephalochordates are the extant lineage closest
to chordate ancestors, the most primitive urochordates must
have similarities with cephalochordates and also with non-
chordate deuterostomes. First, I argue that appendicularians
exhibit the characters required of basal urochordates, and
then explain the essence of the advanced filter-feeder
hypothesis.
(a) Appendicularians are basal, and ascidians and thalia-
cians are derived
As discussed above, the evolution of urochordates
remains enigmatic. Circumferential evidence mentioned
below, however, compels me to deduce that appendiculari-
ans are basal among urochordates, and that ascidians and
thaliacians are more derived from the appendicularian-like
ancestor.
Solitary lifestyle. As shown in Table 1, the colonial
and/or social lifestyle exhibited by various species of ascid-
ians and thaliacians is not always common in other deu-
terostome groups, except in pterobranch hemichordates. In
hemichordates, molecular phylogeny supports the notion
that solitary enteropneusts are basal while colonial ptero-
branchians are derived (Cameron, 2005). Therefore, it is
reasonable to assume that solitary appendicularians are
close to the urochordate ancestor, which is shared with
cephalochordates (Wada, 1998). In this scenario, solitary
ascidians are basal and colonial ascidians are derived
(Wada et al., 1994). The evolution of sessile ascidians from
planktonic appendicularians will be discussed later.
Sexuality. Gonochorism is common in most echino-
derms, hemichordates, and cephalochordates (Table 1).
Among urochordates, Oikopleura dioica exhibits gonocho-
rism, although all other appendicularians are hermaphroditic
(Table 1). All ascidians and thaliaceans are hermaphroditic.
Therefore, at least one species of appendicularian retains
this trait of non-chordate deuterostomes and cephalochor-
104 N. Satoh
dates. The evolution of hermaphroditism required that ascid-
ians undergo various changes in mode of reproduction, as
discussed below.
Morphology of gametes. Strong support for appendic-
ularians being basal among urochordates comes from the
morphology of sperm and eggs. The morphology of sper-
mtozoa and mode of acrosomal reaction are criteria that
dechotomize ascidians and the other non-chordate and
chordate deuterostomes. Spermatozoa are morphologically
divided into primitive spermatozoa and modified spermtozoa
(Franzen, 1956), and the modification of sperm morphology
has been discussed in the context of metazoan evolution
and/or speciation. Echinoderms, hemichordates, cephalo-
chordates, larvaceans, and lower vertebrates exhibit exter-
nal fertilization, and they all have primitive spermatozoa
(Table 1). In contrast, ascidians and thaliacians show mod-
ified spermtozoa. Holland et al. (1988) and Holland (1989)
reported that, among urochordates, appendicularians have
sperm that are plesiomorphic to sperm of non-chordate deu-
terostomes and cephalochordates. Among tunicates, sper-
miogenesis in doliolids is most like that in solitary ascidians.
On this basis, Holland et al. (1988) argued that appendicu-
larians are the stem group of urochordates, and that ascidi-
ans could not have given rise to appendicularians either
directly or via doliolids (Holland et al., 1988). Reflecting the
sperm morphology, and egg morphology as well (see
below), the mode of acrosome reaction differs between
ascidians and the other chordate groups (cephalochordates,
appendicularians, lower vertebrates) (Fig. 5). In contrast to
the usual acrosome reaction seen in most deuterostomes
and marine invertebrates (e.g., Holland et al., 1988;
Morisawa, 1999; Morisawa et al., 2004), the acrosomal
Fig. 5. A plausible scenario of urochordate evolution deduced from
morphology of sperm and mode of acrosome reaction. Spermtozoa
of hemichordate acorn worms (H), the cephalochordate amphioxus
(C), and the lower vertebrate lamprey (V) have so-called primitive
spermatozoa, and in the acrosome reaction sperm project an
acrosomal process (asterisks). Among urochordates (U), apppen-
dicularians (Ap) have primitive sperm, while ascidians (As) have
modified sperm, which do not project an acrosomal process during
the acrosome reaction. These facts strongly suggest that appendic-
ularians are basal among urochordates and that ascidians arose
later. D, deuterostome ancestor. Modified from Morisawa et al.
(2004).
reaction in ascidians is highly a modified one in which sperm
do not project an acrosomal process (Fukumoto, 1990).
In morphology and the presence of test cells within the
perivitelline space, the eggs of ascidians and thaliacians are
completely unique (Table 1). Although the function of test
cells remains to be examined (Satoh, 1994; Aoyama et al.,
2008), the production of these cells required changes in the
gene regulatory networks responsible for gamete formation.
Test cells may be uniquely fitted to the ascidian strategy of
reproduction. In contrast to ascidian/thaliacean eggs,
appendicularian eggs have no test cells, nor do the eggs of
any other non-chordate or chordate deuterostome groups. If
ascidians were basal, it is rather hard to imagine that appen-
dicularians would have happened to lose this unique
character that was once established by elaborate genetic
networks.
Paedomorphism. An opposing argument to
appendicularians being basal comes from the evolutionary
scenario in which a sessile ascidian-like ancestor gave rise
to motile cephalochordates via paedomorphism. Appendicu-
larians are solitary, planktonic creatures that resemble in
certain ways the larval stages of ascidians, hence the name
larvaceans. Their retention of larval features, including a
notochord and a dorsal nerve cord, suggests that they arose
by paedomorphosis (e.g., Stach, 2007), but this view may
be a complete misunderstanding. Even though the adult
appears to retain the tadpole form, this does not necessarily
represent paedomorphism. In general, the mode of develop-
ment of appendicularians is comparable with that of the
cephalochordate amphioxus. That is, cephalochordate
embryos develop into tiny fish-like larvae that, after meta-
morphosis, develop into adults morphologically very similar
to the larvae. Similarly, appendicularian embryos first
develop into tadpole-like larvae that, after metamorphosis,
further develop into adults of similar morphology. Likewise,
fish embryos develop first into larvae and then adults, with
comparable external morphology in both stages. It is worth
mentioning here that this type of metamorphosis is common
to chordate stem groups, including cephalochordates,
appendicularians, and lower vertebrates. Among urochor-
dates, dramatic changes in adult morphology occurred in the
ascidian/thaliacean lineage, where the free-living larval form
was abandoned to produce sessile adults with incurrent and
outcurrent siphons.
Thaliaceans. Thaliaceans are highly diverse, and their
phylogenetic relationship to ascidians appears enigmatic.
Molecular phylogeny and sperm morphology suggest that
thaliaceans originated together with Enterogona ascidians
but not with the clade of all ascidians including Pleurogona
(Wada, 1998; Holland et al., 1988; Winchell et al., 2002;
Zeng and Swalla, 2005). That is, thaliaceans are likely to
have evolved from a particular group of ascidians; therefore,
the most plausible scenario for the evolution of urochordates
is that appendicularians first evolved from lancelet-like
ancestors; then a large group of ascidians evolved; and
finally thaliaceans diverged from a group of ascidians. It is
generally thought that members of the Enterogona (adults
with one gonad) are more primitive than members of the
Pleurogona (adults with two gonads), because a change in
sexuality from gonochorism to hermaphroditism might pro-
duce a descendant with a gonad consisting of an ovary and
 A Hypothesis for Urochordate Evolution
a testis (Enterogona). Ascidians with a pair of gonads
(Pleurogona) are more advanced and originated from an
ancestor with a single gonad. Extending this view further,
thaliaceans may have originated early in the evolutionary
history of ascidians (Fig. 3).
(b) Urochordate evolution towards advanced filter feed-
ers
In line with the urochordate evolutionary scenario
described above, the advanced filter-feeder hypothesis
might explain the variety of urochordate lifestyles as follows.
The net result of their evolutionary strategy was to give rise
as quickly as possible to an adult provided with two siphons
(mouth and anus) and filter-feeding digestive machinery.
The basal chordate amphioxus is also a filter feeder with
branchial slits and an endostyle. In spite of its fish-like body,
amphioxus usually exhibits a half-buried lifestyle. From the
amphioxus-like ancestor, appendicularians evolved to be
more actively motile filter feeders (see previous section,
“Appendicularians, a highly derived group among urochor-
dates”). The broad distribution and occasional extreme
abundance of appendicularians indicate that they have suc-
ceeded markedly as specialists of filter feeding. In general,
urochordates tend to have a precocious lifestyle in which
they become filter feeders as early as possible. This ten-
dency is more conspicuous in larvaceans, as was discussed
above in the section on appendicularians.
In contrast to the success of appendicularians as plank-
tonic filter feeders, ascidians adopted another way to fit into
a niche as sessile filter feeders. The suspension filter-
feeding system, in which seawater flows from the incurrent
siphon through the branchial slits to the outcurrent siphon
and prey is trapped with mucus substances secreted by the
endostyle, might be the ideal machinery to match its objec-
tive. The sessile form, by attaching to substrates, becomes
able to live with much lower energy consumption than motile
forms. The primitive lifestyle of ascidians was solitary. Their
very rapid embryogenesis to form tadpole larvae, in which
the mouth and anus never open, is a strategy for finding
substrates to which they can attach and commence meta-
morphosis to form sessile juveniles. Even though a short lar-
val life allows dispersal over only small distances, the larval
period is probably sufficient for the selection of suitable sub-
strata. Therefore, ascidians skip a developmental event that
is common to most marine invertebrate larvae, namely,
opening the mouth and anus to achieve a functional diges-
tive tract. Furthermore, some species of Molgulidae and
Styelidae adopted direct development by skipping the larval
stage (Jeffery and Swalla, 1990; Gyoja et al., 2007). The
advanced filter-feeder hypothesis explains this as their
acquiring a further mode to give rise even more quickly to
adults as sessile filter-feeder specialists. Thaliaceans might
have somehow evolved as planktonic filter feeders from ses-
sile filter feeders.
One of the tradeoffs of becoming sessile vs motile dur-
ing the evolution of urochordates was the change in sexual-
ity from gonochorism to hermaphroditism. For immotile
ascidians, hermaphroditism doubled the opportunity for
sperm and eggs to meet. However, this advantage was
accompanied by a disadvantage, namely, higher risk of
accumulation of mutations in the genome. To avoid this risk,
105
ascidian eggs might select non-self sperm. Special molecu-
lar mechanisms responsible for the recognition of self- and
non-self gametes have been established to escape this dis-
advantage (Morgan, 1939; Sawada et al., 2004; Kurn et al.,
2007; Harada et al., 2008). The creation of test cells within
the perivitelline space may also have been related to avoid-
ance of this disadvantage of hermaphroditism.
Another evolutionary strategy of sessile ascidians was
to make colonies by asexual reproduction or budding (e.g.,
Manni et al., 2007; Kawamura et al., 2008). Because
asexual reproduction or colony formation is seen in various
ascidian species, it is likely that its acquisition occurred inde-
pendently among different families of ascidians (Wada et al.,
1992; Zeng and Swalla, 2005). Once a colony was estab-
lished, the significance of the development of free-swimming
larvae might have been reduced. Colonial ascidians thus
became less dependent on the production of a large quantity
of comparatively yolkless zygotes. Instead, they developed
another way of embryogenesis, namely, brooding yolky eggs
within the cloacal chamber. The tendency for precocious
production of the juvenile form within the trunk region of
larvae is also a common feature of colonial ascidians.
One problem in extending the strategy of the colonial
lifestyle is that expanding colonies would meet and
consequently compete for their feeding niche. Histocompat-
ibility is another strategy promoting asexual reproduction in
colonial ascidians. Colonies sharing a given common
genetic background are able to fuse, while those without a
common genetic background are not able to fuse (Oka and
Watanabe, 1960). The complex molecular mechanisms of
this histocompatibility are now being elucidated (De Tomaso
et al., 2005).
In summary, the huge variety of lifestyles exhibited by
urochordates can be accounted for by the idea that they
adopted strategies to develop into filter-feeder specialists.
EVOLUTION OF UROCHORDATES AS THE SISTER
GROUP OF VERTEBRATES
As discussed above, the great variety of lifestyles exhib-
ited by urochordates can be accounted for by the advanced
filter-feeder hypothesis. Vertebrates are another animal
group with extensive evolution, beginning with the hagfish
and lamprey, continuing through fish, amphibians, reptiles,
birds, and mammals, and including 50,000 extant species.
The evolutionary pathways of vertebrates differ in various
ways from those of urochordates. Vertebrates are not filter
feeders, and therefore the morphologies of vertebrates are
in some respects different from those of urochordates. A
general comparison of various features of the three chordate
groups suggests that the origin and evolution of vertebrates
can be much more easily considered in relation to cephalo-
chordates than to urochordates. Since studies of molecular
phylogeny using sequenced genomes demonstrate that uro-
chordates, not cephalochordates, are the sister group to ver-
tebrates (e.g., Putnam et al., 2008), how do we explain this
apparent discrepancy? The following are a few examples of
evidence that urochordates are more relevant than cephal-
ochordates to vertebrates at the molecular level.
(a) Notochord
The notochord is the most prominent feature of chor-
106 N. Satoh
dates. Urochordates are named for the notochord forming in
the tail (uro-), and cephalochordates for the notochord
extending to the tip of the head (cephalo-). We, vertebrates,
are named for replacement of the notochord by vertebrae
during embryogenesis. The notochord of cephalochordates
is primitive, while that of urochordates and vertebrates is
more advanced.
The cephalochordate notochord is formed by pouching-
off from the dorsal region of the archenteron (see Satoh,
2008). In addition, the cephalochordate notochord displays
muscle properties. The notochord cells are occupied by
myofibrils (Ruppert, 1997), and an expressed sequence tag
(EST) analysis indicated that approximately 11% of the
genes expressed in the notochord of Branchiostoma
belcheri adults are muscle-component genes, including
actin, tropomyosin, troponin I, and creatine kinase (Suzuki
and Satoh, 2000). Further examination of the expression
patterns of muscle structural genes in B. belcheri embryos
revealed three types of gene expression: some genes are
expressed exclusively in the notochord, some in both the
notochord and muscle, and some in muscle only (Urano et
al., 2003).
In more advanced urochordates and vertebrates, the
notochord is formed by a convergent extension movement
of presumptive precursor cells that are bilaterally positioned
in gastrulae. These cells do not possess any muscle prop-
erties. Vacuolation within the cells provides an increase in
cell volume and stiffness in both ascidians and amphibians
(Cloney, 1982; Jiang and Smith, 2007). It is worth mention-
ing here that the convergence, intercalation, and extension
of notochord cells comprise one of the most significant mor-
phogenetic processes by which structures that characterize
urochordates and vertebrates are formed (e.g., Keller,
2002).
A T-box transcription factor gene, Brachyury (Bra), plays
a pivotal role in notochord formation (Satoh, 2003). This
gene appeared upon the initial emergence of metazoans
(King et al., 2008), and is thus common to all metazoans.
Analyses of Bra expression and function in a wide variety of
animals suggest that its primary function is involved in
invagination movements of the embryonic cell layer (Smith,
1997; Technau, 2001; Showell et al., 2004). For example,
an acorn worm Bra is expressed in the region of the arch-
enteron invagination and that of the stomodium invagination
(Tagawa et al., 1998). During the evolution of chordates, the
gene was further recruited in the formation of the notochord,
and thus Bra is expressed in both the region of archenteron
invagination and the developing notochord in cephalochor-
date embryos (Terazawa and Satoh. 1995; Holland et al.,
1995) and Xenopus (Smith et al., 1991). In urochordates,
presumably due to the precocious mode of embryogenesis,
the expression in the blastopore disappeared, while the
expression in the developing notochord has been retained
(Yasuo and Satoh, 1993; Corbo et al., 1997; Satoh, 2003).
The notochord cells also communicate with cells of the
surrounding organs to accomplish their proper functions.
Recent studies of Ci-Bra downstream genes in Ciona
embryos revealed that several of the notochord genes are
components of signaling pathways such as Wnt/PCP, NFkB,
and TGF-beta signaling (Hotta et al., 2008).
(b) The central nervous system (CNS)
The identification of genes, including Otx, Pax6, Pax2/5/8,
Engrailed, Fgf8/17/18, and Hox1, which are involved in the
formation and/or patterning of the CNS in chordate embryos
has revealed a basic tripartite ground plan along the antero-
posterior axis of the dorsal nerve cord (Wada et al., 1998;
Wada and Satoh, 2001; Reichert, 2002; Canestro et al.,
2005). The tripartite ground plan comprises an anterior
region expressing Otx family homeobox genes (marking the
forebrain and midbrain of vertebrates), a central region
expressing Pax2/5/8 and Engrailed genes (marking the mid-
brain of vertebrates), a boundary region expressing Fgf8/17/
18 (marking the midbrain-hindbrain boundary of verte-
brates), and a Hox-expressing region (hindbrain and spinal
cord of vertebrates). These regions are not always compa-
rable to the classically defined anatomical divisions of fore-,
mid- and hindbrain, but instead reveal the probable genetic
blueprint upon which chordate brain development is based.
Although the expression profiles of the CNS-blueprint
genes change temporally in embryos of different chordates,
comparison of their profiles is suggestive regarding scenar-
ios of chordate evolution (e.g., Takahashi and Holland,
2004, Canestro et al., 2005). Cephalochordates are some-
times called acraniates, that is, animals without a head.
Expression of their CNS patterning genes looks more prim-
itive or derived than expression in the two other chordate
groups. During formation of the CNS, cephalochordtes show
Otx and Pax6 expression in the anterior region and Hox
expression in the posterior region, but no evident expression
of Pax2/5/8, En, or Dmbx in the intermediate region (Holland
et al., 1997; Kozmik et al., 1999; Takahashi and Holland,
2004).
Interestingly, urochordate ascidians have developed this
basic tripartite ground plan (Wada et al., 1998). In addition
to the expression profiles of Otx, Pax6, Pax2/5/8, and Hox
genes, the expression of Fgf8/17/18, Engrailed, and Dmbx
in the CNS is conserved between vertebrates and urochor-
dates (Imai et al., 2002; Takahashi and Holland, 2004),
although the details of the expression regions are slightly
different. Canestro et al. (2005) examined in detail the
expression patterns of orthologs of vertebrate CNS markers
(Otx, Pax6, Pax2/5/8, Engrailed, and Hox1) in Oikopleura
dioica. Their work revealed that the Oikopleura CNS pos-
sesses homologs of the vertebrate forebrain, hindbrain, and
spinal cord, but not the midbrain. After careful comparison
of the gene expression patterns among cephalochordates,
larvaceans, ascidians, and vertebrates, they suggested that
(1) absence of the midbrain is a urochordate synapomorphy,
(2) there is no convincing evidence for a homolog of a mid-
brain-hindbrain boundary (MHB) organizer in urochordates,
and (3) the expression pattern of MHB-genes in the urochor-
date hindbrain suggests that these function in different ways
from an MHB organizer. Nevertheless, it is evident that the
expression profiles of orthologs of vertebrate CNS markers
became more complicated in ascidians than in cephalochor-
dates and in appendicularians.
A recent fascinating finding in relation to the evolution of
the chordate CNS was the anteroposterior patterning in
developing adults of the hemichordate Saccoglossus
kowalevskii (Lowe et al., 2003). Genes involved in chordate
CNS patterning showed a discrete expression profile along
 A Hypothesis for Urochordate Evolution
the A-P axis of this animal. However, this expression pattern
is not entirely comparable to that for the chordate CNS pat-
terning, because the hemichordate genes are expressed in
the ectodermal epithelial cells, not in the nervous system
(Holland, 2003; Gerhart et al., 2005). Further studies of CNS
gene expression, especially in the indirect-developing
hemichordate Ptychodera flava, are required to understand
the evolution of the chordate CNS.
(c) Cadherins
Strong qualitative evidence showing that urochordates
and vertebrates comprise a clade while cephalochordates
are basal among chordates came from studies of the struc-
ture of cadherin molecules. As shown in Fig. 6, the classic
cadherin family of proteins of vertebrates is characterized as
Ca2+-dependent cell-cell adhesion molecules with Ca2+-
binding extracellular repeats (ECs) in the extracellular region
and a cytoplasmic domain (CP) with an evolutionarily
conserved β -catenin-biding sequence (Takeichi, 1995).
Urochordate cadherins have the domain composition identi-
cal to vertebrate cadherins (Oda and Tsukita, 1999) (Fig. 6).
On the other hand, classic cadherins of non-chordate deu-
terostomes contain ECs, a domain unique to non-chordate
classic cadherins (NC), a cysteine-rich epidermal growth
factor-like domain (CE), a laminine G domain (LG), and a
CP (Oda and Tsukita, 1999) (Fig. 6). In contrast, the cad-
herin of the amphioxus Branchiostoma belcheri is unique in
containing LG, CE, and CP domains (Oda et al., 2002). Oda
et al. (2002) discussed that cephalochordates are basal to
the clade comprising urochordates and vertebrates.
(d) Muscle actins
Vandekerckhove and Weber (1978, 1984) found that
mammalian skeletal muscle actins are distinguishable from
cytoplasmic actins by comparing at least 25 diagnostic posi-
107
tions in the amino acid sequences. In addition, they further
noticed that vertebrate muscle actins differ distinctly from
invertebrate muscle actins, which show cytoplasmic actin
properties (Vandekerckhove and Weber, 1984). Later, T.
Kusakabe et al., (1997) discovered that vertebrate muscle-
type actins occur in urochordates and cephalochordates
(Kusakabe et al., 1999). Molecular phylogenetic analyses of
the chordate muscle-type actin genes demonstrated that
amphioxus muscle actins are basal, while urochordate and
vertebrate muscle actins are more comparable with each
other (R. Kusakabe et al., 1997; Kusakabe et al., 1999).
(e) Opsins
Invertebrates and vertebrates use photoreceptor cells
with different morphology and electrophysiological response.
Eyes of insects and molluscs use rhabdomeric photoreceptor
cells, which depolarize in response to light. On the other
hand, vertebrate eyes use ciliary photoreceptor cells, which
hyperpolarize in response to light. Photoreceptor cells of
ascidian larvae are ciliary and hyperpolarize in response to
light, indicating that urochordate photoreceptor cells are of
the vertebrate type (Eakin and Kuda, 1971; Gorman et al.,
1971; Kusakabe and Tsuda, 2007). Opsins are about 30–50
kDa G-protein-coupled receptor proteins. More than 1000
members of the opsin family have been categorized into
seven subfamilies corresponding to functional classifications
within the family (Terakita, 2005). Ci-opsin1, an opsin gene
of Ciona intestinalis, is expressed in the larval ocellus
(Kusakabe et al., 2001). Ci-opsin is homologous to verte-
brate opsins. In contrast, cephalochordates contain rhabdo-
meric photoreceptor-like opsins but not true vertebrate-type
opsins. This suggests that after diversification of the cephal-
ochordate and urochordate/vertebrate lineages, the clade
comprising urochordates and vertebrates developed opsins
specific to this lineage (Kusakabe and Tsuda, 2007).
Fig. 6. Comparison of the primary structures of
sea urchin LvG-cadherin, hemichordate Pf1,
cephalochordate BbC, urochordate BS and Ci,
and vertebrate (mouse) E-cadherin. Signal sequ-
ences and transmembrane segments are indi-
cated by filled black boxes. The arrowhead shown
for mouse E-cadherin indicates a proteolytic clea-
vage site that is utilized in the maturation of this
protein. Domain abbreviations: EC, cadherin ext-
racellular repeat; NC, non-chordate classic cad-
herin-specific domain; CE, cysteine-rich EGF-like
domain; LG, laminin G-like domain; CP, cytoplas-
mic domain; PCCD complex, primitive classic cad-
herin domain complex. (Modified from Oda et al.,
2002).
108 N. Satoh
CONCLUSIONS AND PERSPECTIVES
One of the three groups of chordates, urochordates
comprise appendicularians, ascidians, and thaliaceans.
Their modes of reproduction, embryogenesis, and lifestyle
are extremely divergent. The present review proposes an
advanced filter-feeder hypothesis to explain the various
modes of urochordate development and lifestyle. The
hypothesis proposes that (1) cephalochordates are basal
among chordate groups, and thus urochordates evolved
from a lancelet-like ancestor, (2) among the three classes of
urochordates, free-living appendicularians originated first
and then sessile ascidians diverged, while thaliaceans orig-
inated from a group of ascidians, and (3) all urochordate
traits may have evolved in accord with strong evolutionary
advantages or constraints due to their being specialists as
suspension filter feeders. In addition to similarities in DNA
sequences revealed by molecular phylogenetic analyses,
the molecular mechanisms involved in the formation of the
notochord and dorsal neural tube in ascidian embryos are
more comparable to those of vertebrates than to those of
cephalochordates. Thus, urochordates are seen to be a
close relative of vertebrates.
The aboral-dorsalization hypothesis for the origin of
chordates (Satoh, 2008) and the advanced filter-feeder
hypothesis for urochordate evolution (the present review)
raise at least two other important questions about the evo-
lution of chordates: first, the origin and evolution of verte-
brates, and second, the molecular mechanisms and/or gene
regulatory networks involved in evolutionary constraints on
chordates. As to the first question of the origin of verte-
brates, the striking similarity of the vertebrate Bauplan to
cephalochordates has been discussed. The fish-like body of
cephalochordates, with vertebrate-like myotomes, closely
resembles the vertebrate body. However, cephalochordates
lack a brain and jaws, and are therefore sometimes called
acraniates. Vertebrates may have evolved rather directly
from a lancelet-like chordate ancestor by developing a head
and jaws (e.g., Gans and Northcutt, 1983; Kuratani, 2004;
Shigetani et al., 2005; Kuratani and Ohta, 2008). The details
of this scenario for vertebrate evolution will be discussed
elsewhere.
The second question may highlight another, so far inten-
sively discussed but not so well understood core problem of
evo-devo, namely, the relationship between morphological
evolution and genetic evolution. As was discussed in the last
section of this review, although urochordates and verte-
brates originated from a lancelet-like common ancestor, they
evolved in rather different ways. The former became
advanced filter feeders, and the latter advanced head-
forward motile animals. They differ in morphology and life-
style in accord with their evolutionary pathways. Acquisition
of the ability to synthesize cellulose by the urochordate
ancestor might have allowed the huge divergence of ascid-
ians and thaliaceans. Nevertheless, gene regulatory
networks are quite similar between urochordates and verte-
brates. More molecular data are required to clarify the evo-
lution of chordates and the origin of vertebrates, especially
data from decoding the genomes of lampreys and hemichor-
dates.
ACKNOWLEDGMENTS
The research in my laboratories was supported by grants from
MEXT, Japan. I acknowledge Take Kusakabe for helpful comments
on the section “Evolution of urochordates as a sister group of ver-
tebrates”, Dr. Elizabeth Nakajima for critical reading of the
manuscript, Kazuko Hirayama for help in preparation of the
manuscript, and SKY for helpful discussion.
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(Received August 20, 2008 / Accepted October 27, 2008)