Volume 12 Number 12 1984 Nucleic Acids Research

Evolution of muldcellular as deduced from 5S rRNA sequences: a possible early emergence of the

Takeshi Ohama, Tsutomu Kumazaki, Hiroshi Hon and Syozo Osawa

Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464, Japan

Received 9 April 1984; Revised and Accepted 30 May 1984

ABSTRACT The nucleotide sequences of 5S rRNA from a mesozoan Dicyema rmisakiense and three metazoan species, i.e., an acorn- kowaZlevskii, a moss- Bugula neritina, and an octopus Octopus vulgaris have been determined. A phylogenic tree of multicellular animals has been constructed from 73 5S rRNA sequences available at present including those from the above four sequences. The tree suggests that the mesozoan is the most ancient multicellular animal identified so far, its emergence time being almost the same as that of flagellated or ciliated protozoans. The branching points of and are a little later than that of the mesozoan but are clearly earlier than other metazoan groups including and jellyfishes. Many metazoan groups seem to have diverged within a relatively short period.

INTRODUCTION The 5S rRNA is one of the molecules suited for studying the evolutionary process of widely separated organisms because of its universal occurrence with a low nucleotide substitution rate (1). To date, 69 5S rRNA sequences from 54 multicellular animals from almost all the major phyla have been determined (see ref. of (2)). However, the sequences from the Mesozoa, the and the Hemichordata have been wanting. We have therefore determined the 5S rRNA sequences from a mesozoan Dicyema misakiense ( Mesozoa), a moss-animal BuguZa neritina (Phylum Lophophorata), an octopus Octopus vulgaris (Phylum ) and an acorn-worm Saccoglossus kowalevskii (Phylum Hemichordata). Using these sequences together with all multicellular aminal 5S rRNAs now available, we have constructed a phylogenic tree.

MATERIALS AND METHODS BuguZa neritina and Octopus vulgaris were collected near the Sugashima Marine Biological Station, Nagoya University. Saccoglossus kowaZevskii was collected at the Woods Hole Marine Biological Laboratory, USA. For the isolation of Dicyema misakiense, about 120 gm of renal appendages were obtained from eight individuals of the octopus, Octopus vuZgaris, minced with scissors in two volumes of cold sea-water, shaken gently, and centrifuged for 5 min at 100 x g to sediment the bulk of the debris, leaving the mesozoans in

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Dicyema G--UPLCA GGCCMCCACCCUGUCAGCCCCACGGPWAG Bugula GUCAACGCC N3JUCUCGGJCCAJCUAG Octopus CCC(JGU@IGA CUCCCGJ(VAG Saccoglossus GCCWGGCCACG IGCCCCG4AG

CUGCG(JCGGCG3AGUACWGCAUGGGACCCGCCGrGGAiICfa(, GCnrAGGCUU

Fig. 1. Nucleotide sequences of 5S rRNA from Dicyema misakiense (Mesozoa), Bugula neritina (moss-animal, Lophophorata), Octopus vulgaris (Mollusca) and Saccoglossus kowalevskii (acorn-worm, Hemichordata). the supernatant. The mesozoans were then sedimented by centrifugation for 10 min at 500 x g. The RNA was directly isolated by the phenol method (3) from the Octopus livers or whole organisms of the rest of the three species. The nucleotide sequence was determined by the chemical method of Peattie (4) and the enzy- matic method of Donis-Keller (5) using 3'- or 5'-labelled RNA. Certain parts of the sequence were confirmed by electrophoresis on a hot plate (6). A phylogenic tree was constructed by the average-linkage method (= WPGMA, see refs. 7, 8) and the UPGMA matrix method (8) using Knuc values that were calculated by the following equation (9, 7, 10) : Knuc = - (1/2) log [(1 - 2P - Q)(1 - 2Q)012], where Knuc is the evolutionary distance between two sequences compared, P and Q are the fractions of nucleotide sites showing transition- and transversion type differences, respectively. One gap vs. one nucleotide was counted as equal to one transversion-type substitution.

RESULTS AND DISCUSSION SEQUENCE The 5S rRNA preparations from both Bugula and Octopus gave a clear single band on the gel and had 120 nucleotides, while those of Dicyema and Saccoglossus showed two bands. The Dicyema 5S rRNAs from the upper and lower bands consisted of 117 and 116 nucleotides, respectively, revealing no difference in sequence except that the upper RNA had an additional U at its 3'-terminus. The two Saccoglossus 5S rRNAs were both 120 nucleotides long, U at the 111th residue from the 5'-terminus of the upper RNA being replaced by C in the lower RNA. These sequences are shown in Fig. 1. SIMILARITY PERCENTS DISPLAY Fig. 2 shows a graphic display of simi- larity percents between the 5S rRNAs from human, an acorn-worm, a moss- animal, an octopus and a mesozoan Dicyema taken in ordinate and those from other animal species taken in abscissa. The 5S rRNAs from the former four species show rather high similarities (80-85%) to those from animals in most of the representative phyla, with exceptions of nematodes (symbols Jl-J3 in Fig. 2), fresh-water planarians (L2, L3) and a mesozoan (01). This suggests that the phyla to which these four species belong are related with most

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Fig. 3. Phylogenic tree of 5S rRNAs using 73 sequences from 54 multicellular animal and 11 protozoan sequences. Representative 50 species are displayed in the figure. The sequences from two or more species are identical or almost the same (e.g., human, rat, and bovine), only one of these species is shown. Also, usually only one out of two or more sequences in one species, if they exist, is shown. I---o--i : range of standard error of Knuc, ak (9). The following abbreviations are used for the species-name. [A] Vertebrata, Al : Homo sapiens (human), A2 : Cricetus cricetus (hamster), A3 : Iguana igu- ana (iguana), A4 : GaZZus gaZZus (hen), A5 : Xenopus Zaevis (toad), A6 : Not- ophthaZmus viridescens (newt). [B] Protochordata, Bl : HaZocynthia roretzi ascidian)**. [C] Echinodermata, Cl : Hemicentrotus puicherrimus (sea-urchin)* , C2 : Lytechinus variegatus (sea-urchin), C3 : Asterias vuZgaris ()*

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C4 : Asterina pectinifera (starfish), C5 : Stichopus oshimae (sea-cucumber) *. [D] Hemichordata, Dl : Saccoglossus kowalevskii (acorn-worm)*. [E] Lopho- phorata, El : LinguZa anatina (lamp-shell), E2 : BuguZa neritina (moss-animal )*. [F] Sipunculoidea, Fl : Phascolopsis gouZdii (sipunculid-worm). [G] Arth- ropoda, Gl : Bombyx mori (silkworm), G2 : PhiZosamia cynthia (moth), G3 : DrosophiZa meZanogaster (fly), G4 : Acyrthosiphon magnoZiae (aphid), G5 : Ar- temia saZina (brine shrimp). [H] Annelida, Hl : Urechis unicinctus (spoon- worm)*, H2 : Perinereis brevicirris (sea-worm)*, H3 : SabeZZastarte japonica (sea-worm)*. [I] Mollusca, Il : Octopus vuZgaris (octopus)*, I2 : IZZex ille- cebrosus (squid), I3 : MytiZus eduZis (mussel), I4 : Arion rufus (snail), I5 : Helix pomatia (snail). [J] Aschelminthes, Jl : Caenorhabditis eZegans (ne- matode)**, J2 Caenorhabditis briggsae (), J3 : Rhabditis tokai (ne- matode)*, J4 Brachionus pZicatiZis ()*. [K] Nemertinea, Kl : Lineus geniculatus (ribbon-worm)*, K2 : Emplectonema graciZe (ribbon-worm)*. [L] P1- atyhelminthes, Ll : PZanocera reticuZata (marine )*, L2 & L3 : Duge- sia japonica Furuyu & Sanage (fresh-water planarians)*. [Ml , Ml : Spirocodon saltatrix (jellyfish)*, M2 : Chrysaora quinquecirrha (jellyfish) *, M3 : Nemopsis dofleini (jellyfish)*, M4 : AureZia aurita (jellyfish)**, M5 Anthopleura japonica (sea-anemone)*. [N] Porifera, Nl : Haliclona ocuZata (), N2 : Hymeniacidon sanguinea (sponge), N3 : HaZichondria panicea (sponge). [0] Mesozoa, 01 : Dicyema misakiense*. [P] , P1 : Crithidia fasciculata (trypanosome), P2 : Tetrahymena thermophila (ciliated protozoan) **, P3 : Euglena gracilis**. Symbols * and ** : the sequence determined in our laboratory and in our as well as other laboratories, respectively. metazoan phyla to almost an equal extent. The octopus sequence is close to a squid sequence (I2, ref. 11) as expected (90% identity). The sequence from the moss-animal is clearly similar to that of a lamp-shell LinguZa anatina (El, ref. 12), supporting the classical view that these two animals are related. The acorn-worm 5S rRNA shows higher similarities to those of the species and the ribbon-worm species throughout (H1-H3, Kl-K2), than to those of the species (Al-A6) and the ascidian species (Bl). This might mean that, in contrast to the anatomical evidence, the acorn-worm (the Hemichordata) is not particularly connected either to the or the ascidian (the Protochordata), but rather to the annelid species. The mesozoan 5S rRNA (01, Dicyema) is the least similar not only to the sequences here reported but also to all of the metazoans and the protozoans so far sequenced (62-70%, see Fig. 2). Also notable is that the 5S rRNAs from the fresh-water planarians (L2, L3) and the nematodes (Jl-J3) are rela- tively less similar to the sequences of other multicellular animal groups (65 -70%) as previously emphasized (13, 14). These situations are shown by asteriscs with an arrow in Fig. 2. PHYLOGENIC TREE More detailed analyses have been made by constructing a phylogenic tree to deduce the sequence of appearance of the multicellular animals during . The Knuc values of every possible pair in the sequences of 5S rRNAs from 73 sequences from 54 multicellular animals and 11

5105 Nucleic Acids Research protozoan sequences were calculated according to the equation shown in Materials and Methods. Using these values, we constructed a phylogenic tree by means of the UPGMA method or the WPGMA method with the standard errors (7). Since the trees from these two methods were fundamentally the same, only the latter is shown in Fig. 3. The mesozoan (symbol 01 in Fig. 3), the ciliated protozoans (P2) and the flagellated protozoans (P1, P3) have separated from one another at about the same time in an early stage of evolution. The separation took place at about the 1/2 Knuc value of 0.21 to 0.22 ± 0.04. This corresponds to about 0.90 to 0.94 ± 0.17 x 109 (billion) years ago, if the yeast-animal divergence time is taken to be 1.2 billion years ago (15). The Knuc values of these three groups of organisms are so close to each other that their branching order is difficult to decide. After the emergence of the protozoans and the mesozoan, the fresh-water planarians (L2, L3) and then the nematodes (Jl-J3) separated from the ances- tors for majority of the metazoan phyla. Their branching points are located at the 1/2 Knuc values of 0.18 ± 0.04 and 0.17 ± 0.04, respectively. The most plausible phylogenic explanation for these pictures would be that the fresh-water planarians and the nematodes are of relatively ancient origins in the animal evolution. Contrary to the fresh-water planarians (Order Tricladida), the 5S rRNA from a marine planarian (Order Polycladida; Ll in Fig. 3) revealed rather high similarities to 5S rRNAs from majority of the phyla (14), suggesting that the origin of the Polycladida is more recent than the Tricladida, and these two groups of animals are phylogenically much more remote than generally accepted. With exceptions of the three groups, i.e., the mesozoan, the fresh-water planarians and the nematodes, the emergence points of most of the metazoan phyla are situated within a range of 0.13 to 0.05 1/2 Knuc values, forming a cluster. Thus, many metazoan groups seem to have diverged one by one within a relatively short period. The acorn-worm (Dl), the moss-animal (E2) and the octopus (Il) studied here are also located in this cluster. The 5S rRNAs from jellyfishes (16), a sea anemone (3) and sponges (17) (symbols Ml-M4, M5, N1-N4 in Fig. 3) show relatively high similarities to those from majority of the 5S rRNAs (83% on average). Sponges and jellyfishes have been believed in traditional as primitive multicellular animals (18). However, their branching points deduced from the 5S rRNA are located in the cluster of majority . Thus the emer- gence time of these animals may not be so old as generally believed; their morphological simplicity could be a result of degeneration. The branching

5106 Nucleic Acids Research points of several other animals are also against expectation. For example, a brine shrimp (19, 20) does not behave with insects; an ascidian (21) occupies a peculiar position in the tree; two (11, 22) are situated sepa- rately, etc. Some of such "anomalous" branchings may be the results of involvements of rather large standard errors, sequence heterogeneity (7, 23) and/or sequencing errors. Thus, the branching order in the cluster discussed above should be taken as tentative. More definitive tree may be constructed by comparing a sufficiently large number of 5S rRNA sequences from each phylum so as to cancel the anomalous influences of the errors. MESOZOAN AND EARLY METAZOAN EVOLUTION Because of the morphological and functional simplicity, mesozoan animals were originally supposed to be most primitive multicellular animals (24). However, the recent majority opinion has been that mesozoans evolved from planarians by degeneration (see ref. 18). However, the phylogenic tree of 5S rRNA deduced from the Knuc values (Fig. 3) suggests strongly that the mesozoan is the most ancient multicellu- lar animals so far known, and is not such an organism that has been derived from the planarians by degeneration. The relationship of the mesozoans to the ciliated protozoans may be especially close because both have many cilia on the cell surface, and both have nuclei with DNA of exceedingly low G+C con- tents (22 to 35% in the ciliated protozoans and 23% in the mesozoan (24)). Hadzi (25) proposed that the most ancient metazoan is a planarian-like animal which has been originated from some ciliated protozoans. The planarian would then evolve to nematode-like organisms and serve as the common ancestor to various metazoan groups including jellyfishes. This view is consistent with the 5S rRNA data, since the planarians and nematodes are situated bet- ween the protozoans and majority of the metazoan phyla (Fig. 4), although there is no definitive evidence as to whether the mesozoans are more related to the ciliated protozoans than to the flagellated protozoans, or vice versa. HAdzi did not mention the role of the mesozoans in metazoan evolution, but the present study points to a possibility that some mesozoan-like organism is the ancestor common to all metazoans including planarians and nematodes. Another possibility would be that the mesozoans were specialized in the protozoan branch and do not serve as a direct ancestor to the metazoans. At present, we do not have any conclusive evidence to decide which alternative is correct; the Knuc values of the mesozoan and the protozoan groups are so close that their exact sequence of emergence is difficult to estimate.

ACKNOWLEDGEMENTS We thank Drs. Masami Sato, Eizo Nakano, Motoki Hoshi and R. A. Zimmermann for valuable advices and collecting materials. This work

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was supported by grants 58880025, 58113004 and 58540404 (Special Project Research) from the Ministry of Education of Japan.

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