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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. 1189 1190 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 1191 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- 1192 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. 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