J Mol Evol (1996) 42:552-559 JO U R N A L O F MOLECULAR EVOLUTION © Springer-Verlag New York Inc. 1996 18S rRNA Suggests That Entoprocta Are Protostomes, Unrelated to Ectoprocta L.Y. Mackey,1 B. Winnepenninckx,2 R. De Wachter,2 T. Backeljau,3 P. Emschermann,4 J.R. Garey1 1 Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282, USA 2 Departement Biochemie, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium 3 Koninklijk Belgisch Instituut voor Natuurwetenschappen, Vautierstraat 29, B-1040 Brussel, Belgium 4 Institut für Biologie III, Albert-Ludwig-Universität-Freiburg, Schanzlestrasse 1, D-79104 Freiburg, Germany Received: 27 May 1995 / Accepted: 8 January 1996 Abstract. The Ento- and Ectoprocta are sometimes Introduction placed together in the Bryozoa, which have variously been regarded as proto- or deuterostomes. However, En­ The phylum Entoprocta (Kamptozoa) comprises about toprocta have also been allied to the pseudocoelomates, 150 species of small, sessile, solitary or colonial species while Ectoprocta are often united with the Brachiopoda (Emschermann 1985). In their life cycle and larval form and Phoronida in the (super)phylum Lophophorata. they show some similarities to the Ectoprocta, which Hence, the phylogenetic relationships of these taxa are prompted Nielsen (1977) to place both taxa together in still much debated. We determined complete 18S rRNA the Bryozoa (moss animals). According to Jägersten sequences of two entoprocts, an ectoproct, an inarticulate (1972) the Entoprocta can be derived from mollusc-like brachiopod, a phoronid, two annelids, and a platyhel- ancestors, i.e., coelomate protostomes. Nielsen (1994), minth. Phylogenetic analyses of these data show that (1) on the other hand, sees a definite affinity between the entoprocts and lophophorates have spiralian, protosto- Entoprocta/Ectoprocta and the Platyhelminthes (flat mous affinities, (2) Ento- and Ectoprocta are not sister worms) and Nemertini (ribbon worms). Other authors taxa, (3) phoronids and brachiopods form a monophy- (Clark 1921; Cori 1936) have denied any close relation­ letic clade, and (4) neither Ectoprocta or Annelida appear ship between Ecto- and Entoprocta and consider the lat­ to be monophyletic. Both deuterostomous and pseudo- ter as neotenic annelid trochophorae (Emschermann coelomate features may have arisen at least two times in 1982, 1985). All these postulated relationships have been evolutionary history. These results advocate a Spiralia- questioned because the Entoprocta possess only a small Radialia-based classification rather than one based on mesenchymous body cavity, which points to a pseudo- the Protostomia-Deuterostomia concept. coelomate or even acoelomate origin (e.g., Hyman 1951; Brusca and Brusca 1990). Moreover, the phylum Ecto­ Key words: Ectoprocta — Entoprocta — Phoronida procta (moss animals sensu stricto) is also often united — Brachiopoda — Lophophorata — 18S rRNA — Mo­ with Brachiopoda (lamp shells) and Phoronida (horse­ lecular phylogeny — Oligochaeta — Hirudinida — shoe worms) in the (super)phylum Lophophorata or Ten­ Polychaeta taculata (e.g., Valentine 1973; Zimmer 1973; Gutmann 1978; Emig 1984). These three groups are mainly joined on the basis of their overall tripartite body plan and the presence of a lophophore, which is a horseshoe-shaped, Abbreviations: NJ: neighbor-joining; MP: maximum parsimony ciliated, tentacular feeding organ containing coelomic Correspondence J.R.to: Garey extensions. Yet, the phylogenetic relationships of the lo- 553 phophorates remain controversial since they show an position 111, was determined on a restriction fragment cloned into the amalgam of deuterostome and protostome features. On plasmid pBluescript SK+. The major part was determined on a PCR amplification product obtained by means of primers binding to con­ the basis of their oligomerous body plan, the three lo- served areas of the 18S and 28S rRNA gene (Winnepenninckx et al. phophorate groups have traditionally been allied to the 1994; Van der Auwera et al. 1994). The 18S rRNA gene ofLingula deuterostomes (e.g., Zimmer 1973; Emig 1984). Nielsen lingua and Haemopis sanguisuga was amplified in two overlapping ( 1977) however, concluded that ectoprocts have evolved parts using primers complementary to the 5' and 3' of the 18S rRNA from an entoproct-like ancestor and that both groups gene (Winnepenninckx et al. 1994) and primers complementary to a conserved part of the 18S rRNA gene and the 5' end of the 28S rRNA have protostome affinities. The Phoronida and Bra­ gene (Winnepenninckx et al. 1994; Van der Auwera et al. 1994). All chiopoda seem to him more related to Deuterostomia PCR amplification products were ligated into T-tailed PSK+ vector (Nielsen 1977, 1994). According to Gutmann (1978), the (Biorad, USA) and cloned into DH5 E. coli bacteria. To avoid an monophyletic lophophorates have been derived from a enhancement of the error rate by cloning the PCR products prior to protostome annelid-like metamerous ancestor. Some sequencing (e.g., Bevan et al. 1992), plasmids were extracted from ten different clones and pooled. Dideoxynucleotide sequencing of both workers even consider lophophorates as an intermediary DNA strands of the pooled plasmids was performed with USB (Cleve­ group between protostomes and deuterostomes (e.g., land, OH, USA) and Pharmacia (Uppsala, Sweden) kits using 16 oli­ Siewing 1976, 1980; von Salvini-Plawen 1982). An gonucleotide primers (Winnepenninckx et al. 1994). The 18S rRNA analysis of partial 18S rRNA sequences, (Field et al. gene of Phoronis architecta. Pedicellina cernua. Barentsia benedeni, 1988) suggested that the brachiopod Lingula reevi be­ and Nereis limbata was PCR amplified in two overlapping fragments spanning nearly the complete gene, corresponding to nucleotides 130- longs to a cluster of Protostomia. More robust analyses 1965 of the human sequence (EBI [EMBL] accession number were carried out by Halanych et al. (1995) which indi­ M10098). PCR products were cloned into M13 mpl8 nondirectionally cated that the lophophorates are polyphyletic, with Bra­ with an appropriate restriction enzyme. DNA sequencing was carried chiopoda + Phoronida forming a clade separate from out completely in both directions from the M13 templates of a single Ectoprocta. They found that all three lophophorate phyla clone, with the dideoxynucleotide sequencing method using Sequenase (USB, Cleveland, OH, USA), commercial M13 primers, and conserved were protostomes, forming a clade with Annelida and internal primers. Additional sequencing reactions were carried out us­ Mollusca, which they named Lophotrochozoa as a varia­ ing inosine mixes as needed to resolve some sequencing artifacts (Au- tion of the Eutrochozoa taxon (Ghiselin 1988; Eemisse et subel et al. 1995). Since for Phoronis architecta, Pedicellina cernua, al. 1992). Barentsia benedeni, and Nereis limbata, only a single clone was se­ We further assessed the phylogenetic relationships of quenced, their sequence may contain minor amplification errors. Esti­ mations of the error rate of Taq polymerase range from 2 x 1CT4 to less the “ lophophorate” phyla Ectoprocta, Brachiopoda, and than 1 x IO-5 according to Eckert and Kunkel (1991) and 2.75 x lCT3 Phoronida by determining new complete or nearly com­ according to Bej et al. (1991). plete 18S rRNA sequences of representatives of these three taxa as well as those of two annelids and a platy- Alignment and Tree Construction. Sequences were fitted into an helminth. We also present the first 18S rRNA gene se­ alignment of small-subunit rRNA sequences (Van de Peer et al. 1994) quences from two entoprocts in order to explore the evo­ using a computer program developed by De Rijk and De Wachter lutionary relationships of Entoprocta to Ectoprocta and (1993). Subsequent manual adjustments were made on the basis of secondary-structure features (Van de Peer et al. 1994). Previously pub­ other protostome taxa. lished sequences used for the phylogenetic analyses have the following EBI accession numbers: Acanthopleura japonica, X70210; Anemonia sulcata, X53498; Artemia salina, X01723; Branchiostoma floridae, M97571; Eisenia fetida, X79872; Eurypelma californica, X I3457; Materials and Methods Glottidia pyramidata, U12647; Glycera americana, U19519; Gordius aquaticus, X87985;Herdmania momus, X53538; Homo sapiens, Biological Materials and DNA Extraction. The animals examined X03205; Lanice conchilega, X79873; Limicolana kambeul, X66374; in the present study are the ectoproct Alcyonidium gelatinosum (Oos­ Ochetostoma erythrogrammon, X 79875; Opisthorchis viverrini, tende, Belgium), the brachiopod Lingula lingua (Hong Kong), the an­ X55357; Phascolosoma granulatum, X79874;Phoronis vancouveren­ nelids Nereis limbata (Polychaeta) (Panacea, Florida) and Haemopis sis, U l2648; Placopecten magellanicus, X53899; Plumatella repens, sanguisuga (Hirudinida) (Vrouwenpolder, The Netherlands), the platy- U12649; Saccharomyces cerevisiae, V01335; Schistosoma mansoni, helminth Bipalium sp. (Sao Miguel, Azores), the phoronid Phoronis X53498; Tenebrio molitor, X07801; Terebratalia transversa, U12650; architecta (Panacea, Florida), and the entoprocts Pedicellina cernua and Tripedalia cystophora, L10829. and Barentsia benedeni from laboratory cultures (Emschermann 1987). Distances between sequences were calculated using the method of DNA of Alcyonidium gelatinosum, Lingula lingua, HaemopisKimura san­ (1980), modified to take gaps into account (Van de Peer et al. guisuga, and Bipalium