Origin of the Mesozoa Inferred from 18s rRNA Gene Sequences
Jan Pawlowski,” Juan-Ignacio Montoya-Burgos,? Jose’ F. Fahrni,” Jean Wiiest,? and
Louisette Zuninetti”
*Departement de Zoologie et Biologie animale, Universite de Geneve; and tMusCum d’Histoire naturelle de Geneve
The phylum Mesozoa comprises small, simply organized wormlike parasites of marine invertebrates and is composed of two classes, the Rhombozoa and the Orthonectida. The origin of Mesozoa is uncertain; they are classically
considered either as degenerate turbellarians or as primitive multicellular animals related to ciliated protists. In order
to precisely determine the phylogenetic position of this group we sequenced the complete 18s rRNA gene of one
rhombozoid, Dicyema sp., and one orthonectid, Rhopalura ophiocomae. The sequence analysis shows that the
Mesozoa branch early in the animal evolution, closely to nematodes and myxozoans. Our data indicate probably
separate origins of rhombozoids and orthonectids, suggesting that their placement in the same phylum needs to be
revised.
Introduction
The Mesozoa are one of the most enigmatic groups
of living animals. These small ciliated wormlike organisms are parasites of several unrelated groups of marine
invertebrates, including echinoderms, cephalopods,
nemertines, polychaetes, and flatworms. They are bilaterally symmetrical, but have only two cell layers and
lack any recognizable organs, excepting the gonad (Margulis and Schwartz 1988, pp. 186-187). On the basis of
morphological, cytological, and biochemical studies, it
has long been debated whether the Mesozoa were truly
primitive multicellular animals or whether they were degenerated as a result of parasitism (Dodson 1956; Grasse, Poisson, and Tuzet 1970, p. 458; Brusca and Brusca
1990, pp. 178-179). Because of their morphological resemblances with flatworm larvae and complex life cycle, the Mesozoa were considered to be degenerate flatworms (Nouvel 1948; Stunkard 1954, 1972). According
to some authors, however, they may represent a possible
intermediate between protists and more complex metazoans (Hyman 1959, pp. 713-715 ; Lapan and Morowitz
1972). Some others considered them as evolved multicellular protists (Cavalier-Smith 1993).
Some doubts also exist concerning the homogeneity of the phylum Mesozoa, which comprises two classes: the Orthonectida and the Rhombozoa, the latter including the orders Dicyemida and Heterocyemida (Margulis and Schwartz 1988, pp. 186187). According to
Kozloff (1969), the morphological resemblance of
rhombozoids and orthonectids is purely superficial.
Stunkard (1954) noticed important differences in the
sexual stages of both classes. For these and other authors
(Nouvel 1948; Lapan and Morowitz 1972; Barnes 1987,
p. 204), rhombozoids and orthonectids are not at all
closely related to each other and should not be classified
together in a single phylum. Brusca and Brusca (1990,
p. 172) even proposed to replace the phylum Mesozoa
by two new phyla: Rhombozoa and Orthonectida.
Key words: Mesozoa, Dicyema sp., Rhopalura ophiocomae, Amphipholis squamata, 18s rRNA gene, phylogeny.
Address for correspondence and reprints: Jan Pawlowski, D6partement de Zoologie et Biologie animale, Universitk de Genbve, 154,
Rte de Malagnou, CH-1224 ChCne-Bougeries, Switzerland. E-mail:
[email protected].
Mol. Biol. Ed. 13(8):1128-l 132. 1996
0 1996 by the Society for Molecular Biology and Evolution. ISSN: 0737.4038
1128
Recently, the phylogenetic analysis of ribosomal
RNA gene sequences has generated new perspectives for
the study of the evolutionary history of Metazoa. Sequence data have provided new evidence for a monophyletic origin of Metazoa and early divergence of the
diplo- and triploblastic assemblages (Field et al. 1988;
Lake 1990; Christen et al. 1991; Wainright et al. 1993;
Christen 1994). They also allowed revision of the phylogeny of major animal groups, particularly the Lophophorata (Halanych et al. 1995) and the Aschelminthes
(Winnepenninckx et al. 1995); moreover, they revealed
an unexpected animal origin of the Myxozoa (Smothers
et al. 1994), classically considered as multicellular protists. Still little is known, however, about the phylogenetic position of some “lower” animals, including the
phylum Mesozoa (Rieger 1994).
As far as we know there were only two attempts,
both concerning the genus Dicyem, to infer the origin
of mesozoans from molecular data. The analysis of the
5S rRNA sequence of D. misakiense suggested that dicyemids may be the most ancient multicellular animals
(Ohama et al. 1984). However, these results are questionable because only a small number of nucleotides
(about 120 in the 5S rRNA gene) were analyzed. Recently, on the basis of the 18s rDNA sequences of two
dicyemids, D. orientale and D. acuticephalum, Katayama et al. (1995) suggested that they diverged from a
triploblastic ancestor.
In this study we present the first complete 18s
rDNA sequence of the orthonectid Rhopalura ophiocomae. We compare it to other eukaryotic sequences, including the new sequences of the dicyemid Dicyema sp.
and the ophiuroid Amphipholis squamata (sequenced for
control reasons), in order to establish the phylogenetic
position of both classes of the phylum Mesozoa.
Materials and Methods
DNA Isolation, PCR Amplification, Cloning, and
Sequencing
Living specimens of Dicyema sp. were isolated
from the kidney of the cuttlefish Sepia oficinalis, while
specimens of Rhopalura ophiocomae (fig. 1) were isolated from gonads of the bristle star Amphipholis squam&z. The cuttlefish and bristle stars were collected near
the Marine Biology Laboratory in Luc sur Mer (France).
Origin of Mesozoa 1131
with long branches, i.e., having an unusually high nucleotide substitution rate, and therefore the position of
dicyemids cannot be firmly established.
It is well known that differences in evolutionary
rate may lead to significant errors in the estimation of
the sequence divergence and, in consequence, may result in biased topologies of the phylogenetic trees (Olsen
1987). However, the phenomenon of long branch attraction does not explain entirely why the phylogenetic position of the Mesozoa is so difficult to establish. As
shown in the NJ tree (fig. 2~) and the MP tree (fig. 2b),
none of the internal branches within the triploblastic assemblage is strongly supported. This confirms that the
phylogeny of triploblastic animals is difficult to resolve
using only the 18s rDNA sequences. This may be due
to the fact that the diversification of the major metazoan
phyla occurred in a short geological time (Erwin 199 1;
Philippe, Chenuil, and Adoutte 1994). Indeed, the paleontologists estimate that the Cambrian explosion of
the Metazoa lasted less than 20 Myr, while it has been
suggested that the 18s rDNA sequences cannot resolve
cladogenetic events separated by less than 40 Myr (Philippe, Chenuil, and Adoutte 1994). According to these
authors, more than 2,000 variable nucleotides would be
required to resolve the multifttrcation of the animal phyla.
In spite of these limitations, the analysis of 18s
rDNA sequences provides a sufficient basis to discuss
the different hypotheses concerning the mesozoan origin. In view of our data, their divergence within the
assemblage of unicellular eukaryotes, either as the most
evolved multicellular protist (Cavalier-Smith 1993) or as
an intermediate group between protists and animals
(Margulis and Schwartz 1988, pp. 186-187), can hardly
be retained. The firmly established position of the Mesozoa within the animal kingdom suggests that their
“primitive” cellular structure is not an ancestral situation, but results from secondary regression probably
linked to their parasitic mode of life. A close relationship between Mesozoa and ciliated protists seems to be
purely speculative. The principal argument for this relationship, other than the abundance of cilia on the mesozoan surface, is a very low G+C content measured in
total DNA (23%) and ribosomal RNA (40%) (Lapan and
Morowitz 1972). According to our sequences, however,
the G+C content in the 18s rRNA gene of Dicyema sp.
(45.6%) and R. ophiocomae (47.3%) is only slightly
lower than the average G+C content (48.1%) in other
invertebrates.
Our results do not confirm the hypothesis that the
Mesozoa are closely related to some parasitic flatworms.
In all analyses the position of mesozoans is clearly separated from that of trematodes and cestodes. The simplicity of structure in the mesozoans may result from
parasitism, but it does not necessarily indicate a common origin with other parasites. The morphological similarity between mesozoan larvae and the miracidial larva
of digenetic trematodes, considered as the principal argument for the common evolutionary origin of these
groups, is probably superficial. We cannot exclude, however, that Mesozoa derive from a turbellarian-like an-
cestor. Indeed, Katayama et al. (1995) showed that the
acoel turbellarian Convolufu nuikaiensis branches closely to myxozoans, dicyemids, and nematodes in the 18s
rDNA tree. However, the sequences of other acoel turbellarians are needed before we can affirm that the turbellarians are polyphyletic and some of them may be
the ancestors of myxozoans, mesozoans, and other Metazoa.
In all our trees, the sequences of Dicyema sp. and
R. ophiocomue branch separately from each other, suggesting that the Mesozoa are polyphyletic. This confirms
the results of comparative studies of mesozoan morphology and life cycle, showing that, although rhombozoids and orthonectids have many common features,
they are probably not closely related (Stunkard 1954;
Kozloff 1969; Lapan and Morowitz 1972). Particularly,
the resemblance of some stages in the life cycle of
rhombozdids, the nematogens and rhombogens, to adult
orthonectids is purely superficial; both forms show a
layer of ciliated cells enclosing an axial mass, but the
characteristics of the cells are different (Kozloff 1969).
By strengthening the evidence of multiple origins of
Mesozoa, our results support the proposition to replace
the phylum Mesozoa by two new phyla Rhombozoa and
Orthonectida, as suggested by Brusca and Brusca (1990,
p. 172).
Acknowledgments
The authors are grateful to Jean Mariaux for critical
comments. This work was partially supported by Swiss
National Science Foundation grant 3 l-3963293 (JP).
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Accepted June 25, 1996