Download Evolutionary History of Free-Swimming and

Evolutionary History of Free-Swimming and Sessile Lifestyles in
Urochordates as Deduced from 18S rDNA Molecular Phylogeny
Hiroshi Wada1
School of Animal and Microbial Sciences, The University of Reading Whiteknights, Reading, England
Whether the ancestral chordates were free-swimming or sessile is a longstanding question that remains to be settled.
Vertebrates and amphioxi are free-swimming, but the most basal chordate subphylum (the urochordates) includes
both sessile and free-swimming species. Here, I report molecular phylogenetic analyses of 18S rDNA of urochordates to deduce which lifestyle is ancestral. This revealed a close relationship between salps and doliolids and
paraphyly of the ascidians. An early divergence of larvaceans, which show a tadpole-like body plan throughout
life, is also supported by the analyses. Based on this phylogeny, a free-swimming ancestor for chordates is more
parsimonious than a sessile ancestor. The evolutionary history of various lifestyles of chordates from this ancestral
form is proposed.
Introduction
One of the main controversies concerning the origin of vertebrates is about the nature of the chordate
ancestors. In particular, did vertebrates evolve from freeliving ancestors, or did they derive from sessile ancestors, similar to ascidians, via paedomorphosis? Vertebrates are a member of phylum Chordata, which also
includes cephalochordates (represented by amphioxi)
and urochordates. Among them, urochordates are the
most basal group, with cephalochordates and vertebrates
being sister groups (Maisey 1986; Schaeffer 1987; Brusca and Brusca 1990; Wada and Satoh 1994; Turbeville
et al. 1994; Nielsen 1995). Cephalochordates and vertebrates show a highly motile body plan throughout their
lives which is referred to here as the tadpole-like body
plan. In both taxa, the motility is driven by lateral muscles using a notochord or vertebrae as a support, with a
neural tube and a gut lying dorsally and ventrally, respectively. There is little doubt that the common ancestor of the cephalochordates and the vertebrates showed
this tadpole-like body plan throughout life. The controversy centers on the more basal ancestors of all chordates; a problem that has been difficult to resolve due
to the existence of a variety of lifestyles in the urochordates. Urochordates are classified into five groups:
ascidians, salps, doliolids, pyrosomes, and larvaceans.
Three types of lifestyles are found for them. The first is
observed for ascidians, which have a sessile adult; the
second includes the pelagic adult of salps, doliolids, and
pyrosomes. In these two types, a tadpole-like body plan
is seen only in the larval stage, although some species
have lost the tadpole larvae secondarily. In the third type
of lifestyle, observed in larvaceans, the tadpole-like
body plan is retained throughout life, although the motile tail in the adult is used for collecting foods as well
1Present address: Seto Marine Biological Laboratory, Kyoto University, Japan.
Key words: Urochordata, 18S rDNA, molecular phylogeny, chordate evolution, ascidian, larvacean.
Address for correspondence and reprints: Hiroshi Wada, Seto Marine Biological Laboratory, Kyoto University, 459 Shirahama-cho,
Nishimuro-gun, Wakayama 649-2211, Japan. E-mail:
[email protected].
Mol. Biol. Evol. 15(9):1189–1194. 1998
q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
as for locomotion. The points at issue are which of these
lifestyles represents the primitive condition for urochordates, and which lifestyle was possessed by the ancestor
of all chordates. Haeckel (1868) extended his recapitulation theory to this case and proposed that the first
chordates were free-living, retaining a tadpole-like body
plan throughout life, and that the sessile lifestyle of the
ascidian has been acquired secondarily as a terminal addition to development. This idea has been supported by
several authors, including Darwin (1871) and, more recently, Tokioka (1971), Jollie (1973), and Jefferies
(1986). In contrast, Garstang (1928) proposed that the
ancestral chordates had sessile adults and that the tadpole-like body plan evolved in the larval stage of these
ancestors. Cephalochordates and vertebrates then
evolved their fully motile lifestyle by paedomorphosis
of the sessile ancestors. Garstang (1928) also insisted
that the larvaceans evolved by paedomorphosis from the
doliolids. Although Garstang’s views have been accepted and developed by several modern authors (Berrill
1955; Romer 1967), the controversy still remains to be
settled. A reliable phylogeny of the urochordates should
reveal the polarity of changes in the evolution of lifestyles of the urochordates, which, in turn, should allow
implications to be drawn concerning the origin of the
chordates.
Wada and Satoh (1994) have reported analyses of
urochordate relationships using 18S rDNA sequences
where only larvaceans, salps, and ascidians were studied. Although the early divergence of larvaceans is supported by those analyses, further studies including pyrosomes and doliolids have been weighted in order to
deduce the evolutionary history of urochordates and to
draw implications for the evolutionary origin of chordates. The relationship between larvaceans and doliolids
is especially crucial in this regard, because several authors have proposed that the larvaceans may have
evolved by paedomorphosis from doliolids (Garstang
1928; Bone 1960; Nielsen 1995). Here, I report molecular phylogenetic analyses of 18S rDNAs from all five
representative groups of urochordates (larvaceans, ascidians, salps, pyrosomes, and doliolids), and, based on
the phylogeny concluded here, I propose an evolutionary history of urochordates, with special emphasis on
lifestyle evolution.
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Wada
FIG. 1.—Molecular phylogenetic trees constructed from urochordate 18S rDNA sequences. The tree topology and branch lengths come
from the NJ analysis. Bootstrap values for internal branches are shown in each node; the upper numbers are bootstrap values by NJ and the
italic numbers under them are those by MP.
Materials and Methods
DNA Isolation and PCR Amplification
Genomic DNA extraction and PCR amplification
were performed as described in Wada and Satoh (1994),
except that for amplification of Ciona intestinalis and
Halocynthia roretzi rDNA, Pfu DNA polymerase (Stratagene) was used. For amplification of Oikopleura dioica, DNA from a genomic DNA library was used as the
template.
Sequences
Sequences were determined after subcloning amplified DNAs into pUC 18 plasmid vector (Pharmacia).
Primers used for sequencing reactions are described in
Wada and Satoh (1994). In order to exclude any sequence errors arising from misamplification by Taq
DNA polymerase (Wada et al., 1992), I determined sequences of three clones from independent PCR amplifications for each species; identical sequence in at least
two clones was taken as representative. In the course of
sequence determination, I found major and minor versions of 18S rDNA in the O. dioica library; the minor
copy (one clone from eight) is very similar to a previously reported sequence from Oikopleura sp. in Wada
and Satoh (1994) (Oikopleura sp. 1 in fig. 1). It is likely,
therefore, that the minor version of 18S rDNA is from
a different species of Oikopleura (Oikopleura sp. 2: accession number AB013015) contaminated during the
preparation of the genomic library. I also found a minor
copy (1 clone from 11) from Doliolum nationalis which
is very divergent but shows a clear affinity to the sequence of the major D. nationalis 18S rDNA. The unusual divergence suggests it is probably a pseudogene,
and it was therefore excluded from the analyses.
Phylogenetic Analyses
An alignment of the sequences was constructed by
eye using SeqApp manual aligner. The alignment is
available upon request. Clustal V (Higgins, Bleasby, and
Fuchs 1992) was used for the neighbor-joining (NJ)
method (Saitou and Nei 1987). Evolutionary distances
were calculated according to Kimura’s (1980) two-parameter method. Gaps and insertions were excluded in
the NJ analyses. The confidence of the tree topology was
assessed by 1,000 bootstrap resamplings (Felsenstein
1985). For maximum-parsimony (MP) analyses, PAUP
3.1.1 branch-and-bound options (Swofford 1993) were
used. The confidence was assessed by 100 bootstrap resamplings. fastDNAml 1.0 (Olsen et al. 1993) was used
for the maximum-likelihood (ML) analyses (Felsenstein
1981). Jumble options were used to find a true ML tree.
Results
I determined almost-full-length 18S rDNA sequences from a larvacean, O. dioica; a pyrosome, Pyrosoma atlanticum; a doliolid, D. nationalis; and two
species of ascidian, C. intestinalis and H. roretzi. Previously reported 18S rDNA sequences of the ascidian
Styela plicata and the salp Thalia democratica are also
included for molecular phylogenetic analyses, together
with those of Balanoglossus carnosus (hemichordate
acornworm) and Asterias amurensis (echinoderm starfish) as outgroups. These outgroup taxa were chosen because of their slower substitution rate of 18S rDNAs,
which is reflected in the shorter branch lengths of the
phylogenetic tree in Wada and Satoh (1994). Accession
numbers of the sequences studied here are listed in table
1. The full sequence of 18S rDNA from the ascidian
Herdmania momus has been reported by Degnan et al.
(1990). However, the substitution rate of the Herdmania
Molecular Phylogeny of Urochordates
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Table 1
Taxa Examined in the Present Study and Accession Numbers of their 18S rDNA Sequences
Classification
Urochordata
Enterogona ascidian . . . . . . . . .
Pleurogona ascidian . . . . . . . . .
Salp . . . . . . . . . . . . . . . . . . . . . .
Pyrosoma . . . . . . . . . . . . . . . . . .
Larvacea . . . . . . . . . . . . . . . . . .
Doliolum . . . . . . . . . . . . . . . . . .
Cephalochrodata . . . . . . . . . . . . . .
Hemichordata . . . . . . . . . . . . . . . .
Echinodermata . . . . . . . . . . . . . . .
a
Species
Ciona intestinalis
Halocynthia roretzi
Thalia democratica
Pyrosoma atlanticum
Oikopleura dioica
Oikopleura sp. 1a
Oikopleura sp. 2a
Doliolum natinalis
Branchiostoma floridae
Balanoglossus carnosus
Asterias amurensis
Accession Number
AB013017
AB013016
D14366
AB013011
AB013014
D14360
AB013015
AB013012
M97571
D14359
D14358
References
Present study
Present study
Wada and Satoh
Present study
Present study
Wada and Satoh
Present study
Present study
Stock and Whitt
Wada and Satoh
Wada and Satoh
(1994)
(1994)
(1992)
(1994)
(1994)
See Materials and Methods.
18S rDNA sequence is relatively high. Thus, in order to
avoid the effect of a sequence with a high substitution
rate on tree topology, I did not include Herdmania in
the analyses. Phylogenetic analyses were performed on
1,461 confidently aligned sites by the NJ (Saitou and
Nei 1987), ML (Felsenstein 1981), and MP methods.
The topologies of phylogenetic trees obtained by
NJ and ML are identical (fig. 1). The phylogenetic tree
obtained by MP is also identical to those obtained by
NJ and ML except that the former supports the sister
grouping of Pyrosome with [Thalia 1 Doliolum] (bootstrap value of 53%) instead of Ciona. That the larvaceans are the earliest lineage to diverge within the urochordates is supported by all methods and is supported
by relatively high bootstrap values (68.6% by NJ, 71%
by MP; note that bootstrap values higher than 70% correspond to a probability higher than 95%; Hillis and
Bull 1993). This finding is consistent with previous
analyses (Wada and Satoh 1994). The early divergence
of larvaceans is also supported in analyses where amphioxus (Branchiostoma floridae) is added as an outgroup, although the bootstrap values are slightly lower
(66.7% by NJ and 55% by MP). These lower bootstrap
values are probably due to the relatively higher substitution rate of the amphioxus 18S rDNA sequence.
The most strongly supported relationship in the
present analyses is the monophyly of [Ciona 1 Pyrosoma 1 Doliolum 1 Thalia], excluding the larvaceans
and two other ascidians (Styela and Halocynthia). This
relationship is supported by all methods and with 100%
bootstrap values by NJ and MP. Thus, monophyly of the
ascidians is strongly contradicted. Traditionally, the
class Ascidiacea is divided into two orders: Enterogona,
which includes C. intestinalis, and Pleurogona, which
includes S. plicata and H. roretzi (Berrill 1950). Therefore, this molecular phylogeny indicates that the Enterogona ascidians are more closely related to pyrosomes,
doliolids, and salps than to the Pleurogona ascidians.
The paraphyly of ascidians is also supported by analyses
of shorter 18S rDNA sequences from a greater number
of species (10 Pleurogona ascidian species and 7 Enterogona species, 917 sites; data not shown).
A close relationship between Thalia and Doliolum
is supported by all methods, and the bootstrap values
for this relationship are 65.8% by NJ and 68% by MP.
The phylogenetic relationships deduced from the present
results are summarized in figure 2 as a strict consensus
tree of the NJ, MP, and ML trees.
Discussion
Wada and Satoh (1994) reported the molecular phylogenetic analyses of 18S rDNAs from the larvaceans,
the salps, and the pleurogona ascidians, and concluded
that the larvacean is the first diverged taxa among them.
The present analyses extended the previous analyses by
including the doliolid, the pyrosome, and the enterogona
ascidian. The relationship between doliolids and larvaceans is of particular interest, because several authors
have suggested the close relationship between the doliolids and the larvaceans. Garstang (1928) pointed out
the similarities in the structures of the intestine and the
pharynx between larvaceans and doliolids and proposed
that the larvaceans have paedomorphically evolved from
the doliolids. In his words, ‘‘We believe we can satisfy
any scrutator that anatomy, house and pharyngeal rotator
are pure Doliolid in all their relations, with highly original specializations’’ (Garstang 1985). Nielsen (1995)
also suggested a close relationship between the larvaceans and the doliolids based on the absence of mesodermal cells and cellulose in the tunic, and the periodic
shedding of the tunic. However, the close relationship
between the doliolids and the larvaceans is strongly refuted in the present analyses. The early divergence of
the larvaceans is supported by bootstrap values of 68.6%
by NJ and 71% by MP. Considering that bootstrap values more than 70% correspond to a probability of more
than 95% (Hillis and Bull 1993), these bootstrap values
can be regarded as significantly high. However, there are
several cases where a wrong tree topology is supported
by a high bootstrap value (Maley and Marshall 1998).
Therefore, we may not be able to reject the possibility
that larvaceans are not the earliest diverged group solely
from the present result. Further analyses of other molecules would help to resolve this problem.
The paraphyletic nature of the ascidians is the second point concluded in the present analyses. Ascidians
were at first classified into three groups, Aplousobran-
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Wada
FIG. 2.—Schematic diagrams of chordate evolution hypothesized based on the phylogenetic relationships deduced from the present analyses.
Phylogenetic tree is constructed as a strict consensus tree of NJ, ML, and MP trees.
chia, Phlebobranchia, and Stolidobranchia, with the pyrosomes and the doliolids being included in the Aplousobranchia (Lahille 1886, 1890; quoted in Berrill 1950).
Therefore, Lahille (1886, 1890) regarded the ascidians
as paraphyletic. However, this classification has been reexamined by several authors, and at present, the classification by Garstang (1896, 1928) is generally accepted, with the pyrosomes and the doliolids being removed
from the Aplousobranchia and the monophyletic ascidians being classified into two orders: the Enterogona
(Aplousobranchia and Phlebobranchia), in which Ciona
is included, and the Pleurogona (Stolidobranchia), in
which Styela and Halocynthia are included. However,
Garstang (1928) admitted that the pyrosomes, the doliolids, and the salps have some features in common
with enterogona ascidians, especially in the mode of
budding.
It is worth pointing out that the conclusions of the
present molecular phylogenetic analyses of urochordate
18S rDNA are very consistent with the phylogeny based
on sperm morphology (Holland, Gorsky, and Fenaux
1988; Holland 1989, 1991). From the observation that
the sperm of the larvaceans is the least derived of all
tunicate sperm both in form and function, it was concluded that the larvaceans diverged first in urochordates.
Holland (1991) also suggests the paraphyly of the ascidians; the Enterogona ascidians are suggested to be
more closely related to the pyrosomes and the salps than
to the Pleurogona ascidians. These two conclusions are
consistent with the present study. However, Holland
(1989) described the sperm of doliolids as less derived
than those of the salps and the ascidians, because its
acrosome vesicle undergoes exocytosis, but those of
salps and ascidians do not. Thus, the divergence of doliolids is suggested to have occurred after that of larvaceans, which is not consistent with the present result.
Loss of acrosome reactions may have occurred independently in the salps and the ascidians.
Based on the phylogenetic framework concluded
here, I propose the following most-parsimonious scenario for the evolution of the chordates. The early divergence of the larvaceans suggests that the ancestors
in node 1 and node 2 (fig. 2) retain motility, with a
tadpole-like body plan, throughout their lives. This idea
is consistent with molecular developmental data of ascidians, revealing that, despite its simplicity, the ascidian
tadpole larva (like cephalochordates 1 vertebrates) possesses a highly organized neural tube with traces of segmentation (Wada, Holland, and Satoh 1996), dorsoventral differentiation (Wada, Holland, and Satoh 1996;
Corbo et al. 1997; Wada et al. 1997), and subdivision
into regions homologous to fore- and midbrain, anterior
hindbrain, and posterior hindbrain plus spinal cord, respectively (Wada et al. 1998). Together with the fact that
larvacean neural tube is also segmentally organized
(Flood 1973; Bone 1989), it is likely that the ancestral
chordates already possessed a complicatedly organized
neural tube. It is unlikely that such a highly organized
Molecular Phylogeny of Urochordates
neural tube evolved solely for use during larval stages
(especially considering that the main function of extant
ascidian larvae is simply to find a place to settle and
metamorphose), lending further support to the existence
of a motile chordate ancestor.
Determinative cleavages, gastrulation with a small
cell number, and larvae with a small cell number are
shared characters of larvaceans (Delsman 1910, 1912;
Berrill 1950), the Enterogona, and the Pleurogona ascidians (Berrill 1950; Satoh 1994), but not of cephalochordates, vertebrates, or other deuterostomes. This suggests that acceleration of development accompanied by
a transition to a determinative development and simplification of the tadpole-like body plan occurred at node 2.
From this urochordate ancestor, the larvaceans
evolved by acquiring a highly specified feeding method
using secreted house. In another lineage from the urochordate ancestors, the tadpole-like body plan was lost
in the adult stage, and pharyngeal regions were enlarged
(node 3). The individual zooids of the ascidians and the
pyrosomes are similar in terms of the structure of pharynx and their way of creating a water current for feeding
(Berrill 1950). The ancestors at nodes 3 and 4 must have
possessed structures similar to those of ascidians and
pyrosomes, although we cannot say whether they were
sessile or pelagic. Compared with the ascidians and the
pyrosomes, the salps and the doliolids have developed
muscle bands. Based on the deduced phylogeny, it is
parsimonious to deduce that this feature was acquired at
node 5.
In conclusion, 18S rDNA molecular phylogeny
supports the early divergence of the larvaceans, the
paraphyletic nature of the ascidians, and a close relationship between the salps and the doliolids. Based on
these results, together with other lines of evidence, I
propose that ancestral chordates possessed a tadpole-like
body plan into the adult stage; this was also retained in
the adult stage of ancestral urochordates. From this ancestor, an ascidian-like or a pyrosome-like ancestor
evolved by enlarging the pharyngeal region of the body
and losing the tadpole-like body plan in the adult. The
salps and the doliolids have evolved by developing muscle bands.
Acknowledgments
I would like to express my special thanks to Peter
W. H. Holland, in whose laboratory this work was undertaken, for numerous discussions on the evolution of
chordates. I also thank Quentin Bone for the specimens
of D. nationalis and P. atlanticum, Mike Levine for the
genomic library of O. dioica, and Teruaki Nishikawa,
Seb Shimeld, and Mari Kobayashi for critical reading of
the manuscript. This research was supported by the Human Frontier Long-Term Fellowship.
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Accepted June 2, 1998