Phylogenetic position of the dicyemid mesozoa inferred from 18s rDNA sequences.

Introduction

The dicyemids are simply organized multicellular animals consisting of an outer layer of 20-40 ciliated somatic cells and an inner core of one long axial cell. They are obligate symbionts in the kidney of cephalopods. Their life cycle is complex; the asexually produced vermiform embryos increase the population in the host, while the infusoriform embryos arising from fertilized eggs pass out of the host body with the urine and are thought to infect another cephalopod host (Brusca and Brusca, 1990).

The dicyemid mesozoans have long been the subject of a phylogenetic controversy (Brusca and Brusca, 1990; Willmer, 1990). They were at first considered to be an extant link between the Protozoa and the Metazoa (Hyman, 1959). However, the resemblance of their complex life cycles to those of parasitic trematodes has led some authors to propose that the dicyemids are descended from an established metazoan group and that their simple body organization results from degeneration attributable to parasitism (Nouvel, 1948; McConnaughey, 1951; Stunkard. 1954; Ginetsinskaya. 1988). Others still view the simple body construction of dicyemids as truly primitive and hold that the group represents an offshoot from early divergent metazoa (Dodson. 1956; Hyman, 1959; Lapan and Morowitz, 1974).

The phylogenetic relationships of eukaryotes have recently come under intense scrutiny in the light of new molecular data. Phylogenetic analyses using nucleotide sequences of 5S rRNA suggested that a dicyemid (Dicyema misakiense) diverged early among such lower metazoa as sponges, cnidarians, and flatworms (Ohama et al., 1984; Hori and Osawa, 1987). But phylogenetic trees based upon comparisons of about 120 sites in the nucleotide sequences of 5S rRNA were different from those inferred from longer nucleotide sequences of 18S or 28S ribosomal RNA (Field et al., 1988; Christen et al., 1991; Katayama et al., 1993; Wainright et al., 1993; Kobayashi et al., 1993). We have sequenced 18S ribosomal RNA genes (18S rDNA) in two species of the dicyemid mesozoa. Our comparison of the nucleotide sequences of small-subunit rDNA for a variety of organisms indicates that the dicyemids belong among the triploblastic animals.

Materials and Methods

Biological materials

We determined almost the entire sequence of 18S rDNA in two species of dicyemid mesozoans and three species of turbellarians (Platyhelminthes). Pure samples of the dicyemids Dicyema acuticephalum and Dicyema orientale were collected from the urine of Octopus vulgaris and Sepioteuthis lessoniana, respectively (Furuya et al., 1992a). Specimens of Convoluta naikaiensis (Acoela) and Planocera multitentaculata (Polycladida) were collected on the shore near the Ushimado Marine Laboratory. Specimens of Dugesia japonica (Tricladida) were obtained from the brook near the Ushimado Marine Laboratory. All were frozen quickly and kept at -- 80 [degrees] C until use.

In addition to the sequences of the above five species, we used the sequences of 23 eukaryotes -- including animals, protists, plants, and fungi -- for which almost complete 18S RDNA sequences were available in databases. The species used and their accession numbers are as follows: Paramecium tetraurelia (Ciliophora), X03772; Oxytricha nova (Ciliophora), X03948; Crypthecodinium cohnii (Dinozoa), M64245; Theileria annulata (Apicomplexa), M64243; Sarcocystis muris (Apicomplexa), M64244; Hartmanella vermiformis (Rhizopoda), M95168; Saccharomyces cerevisiae (Fungi), J01353; Filobasidiella neoformans (Fungi), X60183; Arabidopsis thaliana (Plantae), X16077;…