Origin of the Mesozoa inferred from 18S rRNA gene sequences.

Jan Pawlowski Juan-Ignacio Montoya-Burgos ? Jose' F. Fahrni Jean Wiiest Louisette Zuninetti 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. - 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. 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 monoDophyletic origin of Metazoa and early divergence of thwe n diplo- and triploblastic assemblages (Field et al. 198lao8; Lake 1990; Christen et al. 1991; Wainright et al. 199edd3; Christen 1994). They also allowed revision of the phfryo logeny of major animal groups, particularly the Lophohmphorata (Halanych et al. 1995) and the Aschelminthttes p (Winnepenninckx et al. 1995); moreover, they reveal//ed : m an unexpected animal origin of the Myxozoa (Smotheebrs . et al. 1994), classically considered as multicellular prxoof tists. Still little is known, however, about the phylogerdo netic position of some lower animals, including thjuoe phylum Mesozoa (Rieger 1994). lran As far as we know there were only two attempt.os, s r both concerning the genus Dicyem, to infer the orig/bgin of mesozoans from molecular data. The analysis of tgyhe 5S rRNA sequence of D. misakiense suggested that deuis cyemids may be the most ancient multicellular animatols n (Ohama et al. 1984). However, these results are queDse tionable because only a small number of nucleotidcees m (about 120 in the 5S rRNA gene) were analyzed. Rbee cently, on the basis of the 18s rDNA sequences of twr2o 8 dicyemids, D. orientale and D. acuticephalum, Kataya,2 ma et al. (1995) suggested that they diverged from 104a 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). 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 (...truncated)