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Home , Charophyta, Lycopodium clavatum

Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 820-823, February 1985 Evolution

Evolution of green as deduced from 5S rRNA sequences (5S rRNA sequencing/phylogenetic tree) HIROSHI HORI, BYUNG-LAK LIM, AND SYOZO OSAWA Laboratory of Molecular Genetics, Department of , Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464, Japan Communicated by Motoo Kimura, September 28, 1984

ABSTRACT We have constructed a phylogenic tree for od using arithmetic averages (WPGMA) or the unweighted green plants by comparing 5S rRNA sequences. The tree sug- pair-group method using arithmetic averages (UPGMA) (13). gests that the emergence of most of the uni- and multicellular green such as Chlamydomonas, , Ulva, and RESULTS AND DISCUSSION Chlorella occurred in the early stage of green evolution. Phylogeny of Various Groups of Organisms and Emergence The branching point of is a little earlier than that of of Green Plants. First, using the two hundred forty-nine 5S land plants and much later than that of the above , rRNA sequences available to date, including those described supporting the view that Nitella-like green algae may be the below and shown in Fig. 2, we have constructed a phylogen- direct precursor to land plants. The Bryophyta and the Pteri- ic tree by the WPGMA to see the phylogenic relationships of dophyta separated from each other after emergence of the representative groups of organisms and, especially, to settle Spermatophyta. The result is consistent with the view that the the emergence point of green plants (Fig. 1). The tree shows Bryophyta evolved from by degeneration. In the Pterido- that the eubacteria separated from the metabacteria/eukar- phyta, Psilotum (whisk ) separated first, and a little later yotes branch. In the eubacterial branch, the (club ) separated from the ancestor common (plus plant chloroplasts) emerged first and this was followed to (horsetail) and Dryopteris (fern). This order is in by the diversification of three major bacterial groups: i.e., accordance with the classical view. During the Spermatophyta Gram-negative having the 120-nucleotide type 5S evolution, the (Cycas, Ginkgo, and Metasequoia rRNA, Gram-positive bacteria having the 116-nucleotide have been studied here) and the angiosperms (flowering type 5S rRNA, and the intermediate type of bacteria such as plants) separated, and this was followed by the separation of Micrococcus and Streptomyces (2). After emergence of the Metasequoia and Cycas ()/Ginkgo (maidenhair tree) on eubacteria, the metabacteria (Halobacterium, Thermo- one branch and various flowering plants on the other. plasma, Sulfolobus, methanogens, and such belong to this group) and the separated from each other and In 1979, we published a phylogenic tree of fifty-four 5S from their common ancestor. Thus, as we have pointed out rRNAs (1). Since then, we as well as others have reported 5S (1, 4), the metabacteria are phylogenically closer to the eu- rRNA sequences of nearly 200 species of organisms with fre- karyotes than to the eubacteria (see also ref. 5). In our previ- quent constructions of 5S rRNA phylogenic trees for certain ous paper (1), the branching order among fungi, plants, and groups of organisms, such as eubacteria (2), the metabac- could not be deduced because the number of 5S teria (3-5), fungi (6), the Protozoa (7), and the Meso- and rRNA sequences was insufficient. The 5S rRNA tree shown Metazoa (8). However, the 5S rRNA tree of green plants has here reveals that, in early eukaryotic evolution, the been wanting. We have thus constructed a phylogenic tree of evolved first and were followed by the various fungi (Basid- representative green plants using twenty-eight 5S rRNA se- iomycetes and Ascomycetes). Green plants, brown algae, quences, and we report it here. and the Protozoa/Oomycetes then emerged at nearly the same time but probably in that order. In any case, these four MATERIALS AND METHODS groups seem to have emerged one by one within a relatively Cytoplasmic 5S rRNAs were isolated from 400-800 g of short time. The occurrence of the Mesozoa and Metazoa fol- whole organisms by the phenol method and purified by poly- lowed. Thus, the red algae seem to be the most anciently acrylamide gel electrophoresis as described (2, 7). The se- emerged group among eukaryotes. Green algae clearly be- quences were determined by both chemical and enzymatic long to the green plants branch (see below). The three types methods (2, 9, 10). Certain parts of the sequences were con- of algae (i.e., red algae, brown algae, and green algae) are firmed by electrophoresis on a hot plate at 70°C (11). The only remotely related to one another phylogenically. sequence alignment was done as described (1, 5) with minor Phylogenic Tree of 5S rRNA from Green Plants (Chloro- manual corrections. phyta). We have determined the sequences of cytoplasmic The evolutionary distance, Knuc, and the standard error 5S rRNAs from two green algae (7, 14) and from six land of the Knuc, 0ok, between two sequences being compared plants [four Bryophyta species (15) and two Pteridophyta were calculated by the equations described by Kimura (12): species (16)] during the last 2 years. In addition, 5S rRNA Knuc = -(1/2)loge[(1 - 2P - Q)(1 - 2Q)½], where P and Q sequences from several angiosperms have been reported are the fractions of nucleotide sites showing transition- and from other laboratories (17-21). Here, we report the se- transversion-type differences, respectively. One gap (repre- quences of the 5S rRNAs from two green algae [Spirogyra sented by a broken line in the alignment shown in Fig. 2) sp. and Nitellaflexilis (stonewort)] because the classical bot- versus one nucleotide was counted as equal to one transver- any suggests that Spirogyra is a taxonomically important sion-type substitution. Using the Knuc values, we construct- multicellular fresh-water green algae [a conjugating green al- ed a phylogenic tree by using the weighted pair-group meth- gae (Gamophyta)] and the Nitella-like organism may be the direct precursor to land plants. We have also determined the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: WPGMA and UPGMA, weighted and unweighted, in accordance with 18 U.S.C. §1734 solely to indicate this fact. respectively, pair-group method using arithmetic averages. 820 Evolution: Hori et aL Proc. Natl. Acad. Sci. USA 82 (1985) 821

73 (28) Mesozoa 1 (1) Protozoa 21 (8) Oomycetes Brown algae 2 (2) .Green plants 28 (15) Basidiomycetes 26 Ascomycetes 11 Hyphochyt r idomycetes 2 Red algae 3 (2)

Metabacteria 8 (= Archoebacteria)

*Plant chloroPlasts 13 Cyanobacteria 3 Micrococcus, etc. 5 (3) .Gram (-) bacteria 25 Gram (+) bacteria 28 (3) 1/2 Knuc , I I I | 0.6 0.4 0.2 C 2249 (62)

FIG. 1. Simplified phylogenic tree constructed from two hundred forty-nine 5S rRNA sequences. 1/2 Knuc, relative evolutionary distance; I---o---1, ok (range of standard error of 1/2 Knuc; ref. 12). Major taxon names are followed by numbers of sequences used. Numbers in parentheses are numbers of sequences determined in our laboratory. Most of the sequences used here, except for those reported in this paper, are from ref. 24. The tree determined by the UPGMA generally agrees with that determined by the WPGMA presented here except that, in the UPGMA tree, the Basidiomycetes is closer to the brown algae than to the Ascomycetes. This is probably due to the limited number of brown algae sequences available. Thus, the positions of the Basidiomycetes and brown algae are tentative. The Knuc values of chloroplast 5S rRNAs are about 0.62 times those of the corresponding host cytoplasmic 5S rRNAs (unpublished work). Thus, the branches of the chloroplasts may be 1.5 times as long as that of the cytoplasm. sequences of the 5S rRNAs of five land plants: Lycopodium such as Nitella. We have already pointed out from the 5S clavatum [club moss or ground pine, a fairly primitive Pteri- rRNA sequence comparisons that unicellular as well as mul- dophyta species positioned between Psilotum and Equise- ticellular green algae share a common ancestor with vascular tum (horsetail)], Psilotum nudum (whisk fern, regarded as plants (7, 14). The 5S rRNA comparison between Nitella and the most primitive living ), Cycas revoluta (cy- land plants suggests that the Charophyta emerged just before cad), Ginkgo biloba (maidenhair tree), and Metasequoia the Spermatophyta and the Pteridophyta/Bryophyta sepa- glyptostroboides (the latter three are gymnosperms). Using rated. Thus, our result is consistent with the view that the these 7 sequences together with 23 sequences reported in ancestor of the present-day Nitella would be the precursor to previous papers (7, 14-23) (Fig. 2), we have constructed a land plants. phylogenic tree of green plants (Fig. 3). The 5S rRNA tree shows further that, among land plants, The tree shows that all green plants (; A1-G6) the Spermatophyta diverged from the Pteridophyta and the including vascular plants (Pteridophyta and Spermatophyta), Bryophyta first, and the Pteridophyta and the Bryophyta the Bryophyta (E1-E4), and green algae (G1-G6) belong to separated later (Fig. 3). The Spermatophyta and the Pterido- the branch derived from point A in Fig. 1. On this branch, phyta are sometimes considered vascular plants. The major- emergence of Chlamydomonas occurred at a very early ity opinion is that these vascular plants evolved from Bryo- time. VarioUs green algae and stonewort/land plants then phyta-like organisms lacking a vascular system. As previ- separated from each other. Thus, it is not improbable that ously pointed out (16), the 5S rRNA tree does not agree with green plants originated from some type of green flagellated this view and is consistent with the opinion that the Bryo- organism such as Chlamydomonas (see ref. 7). Among the phyta evolved from ferns by degeneration (25). green algae, Ulva separated from Spirogyra/Chlorella-Scen- Within the Bryophyta, the 5S rRNA tree shows that the edesmus first and diversification of the latter followed. It is separated from both the liverworts and the interesting that a multicellular fresh-water green algae (Spi- first and this was followed by diversification of the rogyra) is more closely related to a unicellular fresh-water latter (see ref. 15). This picture is in agreement with the clas- green algae (Chlorella-Scenedesmus) than to a multicellular sical view that the hornworts are evolutionally distinct from marine green algae (Ulva). Chlorella and Scenedesmus are the liverworts and the mosses (26). closely related to each other, as already pointed out (7). Pre- Among the Pteridophyta, the species reported in this pa- viously, we estimated that Chlamydomonas, Chlorella- per may be arranged, on the basis of anatomical evidence, in Scenedesmus, and plants separated at about the same time; the order of Psilotum (whisk fern), Lycopodium (club moss), in this paper, we report that Chlamydomonas separated Equisetum (horsetail), and Dryopteris (fern) from primitive first. The difference is due to the limited number of 5S rRNA to advanced (26). The 5S rRNA tree shows that Psilotum, sequences used in the previous study. Even at present, the known as the oldest and simplest vascular plant, separated number of sequences of green algae species available is not first, that a little later Lycopodium separated from the ances- sufficient for construction of an extensive phylogenic tree of tor common to Equisetum and Dryopteris, and that those this group. separated more recently. Thus, the branching order of the It has generally been accepted that land plants and green Pteridophyta members deduced from the 5S rRNA se- algae have a common ancestor and, among green algae, the quences agrees perfectly with the classical view. direct precursor to land plants would be some Charophyta There are two major hypotheses for the evolutionary proc- 822 Evolution: Hori et aL Proc. Natl. Acad Sci. USA 82 (1985)

1 2 3 4 5 6 7 8 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1 -GGGUGCGAUC AUACCAGCAC UAGAGCACCG GAUCCCAUCA GAACUCCGAA GUUAAGCGUG CUUGGGCGAG AGCAGUACUA 2 -GGAUGCGAUC AUACCAGCAC UAAAGCACCG GAUCCCAUCA GAACUCCGAA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 3 -GGAUGCGAUC AUACCAGCAC UAAAGCACCG GAUCCCAUCA GAACUCCGAA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 4 -GGUUGCGAUC AUACCAGCAC UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 5 -GGAUGCGAUC AUACCAGCAC UAACGCACCG GAUCCCAUCA GAACUCCGAA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 6 -GGGUGCGAUC AUACCAGCAC UAAUGCACCG GAUCCCAUCA GAACUCCGAA GUUAAACGUG CUUGGGCGAG AGUAGUACUA 7 -AGGUGCGAUC AUACCAGCAC UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 8 -AGGUGCGAUC AUACCAGCAC UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 9 -AGGUGCGAUC AUACCAGCAC UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGCG CUCGGGCGAG AGUAGUACUA 10 -GGGUGCGAUC AUACCAGCAC UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGUG CUUGGGCGAG AGUAGUACUA 11 -GGGUGCGAUC AUACCAGCGU UAGUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGCG CUUGGGCCGG AGUAGUACUG 12 -GGGUGCGAUC AUACCAGCGU UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCACG CUUGGGCUGG AGUAGUACUA 13 -GGGUGCGAUC AUACCAGCGU UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGCG CUUGGGUUGG AGUAGUACUA 14 GUGGUGCGGUC AUACCAGCGC UAAUACACCG GAUCCCAUCA GAACUCCGAA GUUAAGCGCG CUUGGGCCAG AACAGUACUG 15 GUGGUGCGGUC AUACCAGCGC UAAUGCACCG GAUCCCAUCA GAACUCCGCA GUUAAGCGCG CUUGGGCCAG AACAGUACUG 16 GUGGUGCGGUC AUACCAGCAC UACUAGACCG GAUCCCAUCA GAACUCCGAA GUUAAGCGUG CUUGGGCCUG AAUAGUACUG 17 GUGGUGCGGUC AUACCACCGU UAAUGCACCG GAUCCCGUCG GAACUCCGUA GUUAAGCGCG CUUGGGCCGG AAUAGUACUG 18 --GGJGCGGUC AUACCAGGGC UACUACACCG GAUCCCAUCA GAACUCCGUA GUUAAGCGCC CUUGGGCCGG AUCAGUACUG 19 -GGAUGCGGUC AUACCAAGGC UACUACACCA GAUCCCAUCA GAACUCUGAA GUUAAGCGCC UUUGGGCCGG AAUAGUACUG 20 -GGAUGCGGUC AUACCAAGGC UACUACACCA GAUCCCAUCA GAACUCUGCA GUUAAGCGCC UUUGGGCCGG AAUAGUACUG 21 -GGAUGCGGUC AUACCAGGGC UACUACACCA GAUCCCAUCA GAACUCUGCA GUUAAGCGCC CUUGGGCCGG AAUAGUACUG 22 AUGGUACGGUC AUACCACGGC UAAUGCGCCC GAUCCCAUCC GAACUCGGAA GCCAA.GCGCC GUUGGGCCGG AAUAGUACUG 23 AUGCUACGGUC AUACCACCAC GAAAGCACCC GAUCCCAUCA GAACUCGGAA GUUAGACGUG GUUGGGCCAG AUUAGUACUG 24 AUGCUACGUUC AUACCACCAC GAAAGCACCC GAUCCCAUCA GAACUCGGAA GUUAAACGUG GUUGGGCUCG ACUAGUACUG 25 AUGCUACGUUC AUACCACCAC GAAAGCACCC GAUCCCAUCA GAACUCGGAA GUUAAACGUG GUUGGGCUCG ACUAGUACUG 26 AUGCUACGUUC AUACCACCAC GAAAGCACCC GAUCCCAUCA GAACUCGGAA GUUAAACGUG GUUGGGCUCG AUUAGUACUG 27 GUGAUACGGUC AUACCACCAG GAAAACAGGC GAUCCCAUCA GAACUCGCAA CUUAAGCCUG GUUGGGCAGG AUUAGUACUG 28 AUGGU-CGUUC AUACUAGCAC UACUGCACCC UAACCCGUCA GAUCUAGGAA GUUAAGCGUG CUCAGGCGAG GCCAGUAGUA 1 1 9 0 1 1234567890 1234567890 1234567890 123456789 GGAUGGGUGA CCUCCUGGGA AGUCCUCGUG UUGCACCCU- Al Lemna minor (ref. 21) 1 GGAUGGGUGA CCUCCUGGGA AGUCCUCGUG UUGCAUUCCC A2 Triticum aestivum (ref. 17) 2 GGAUGGGUGA CCUCCUGGGA AGUCCUCGUG UUGCAUUCCC A3 SecaLe cereaLe (ref. 21) 3 GGAUGGGUGA CCCCCUGGGA AGUCCUCGUG UUGCAACCCC B1 Helianthus annuus (ref. 21) 4 GGAUGGGUGA CCCCCUGGGA AGUCCUCGUG UUGCAUCCCU B2 Lycopersicum esculentum (ref. 21) 5 AGAUGGGUGA CCUCUUGGGA AGUCCUCGUG UUGCACCCCC B3 Linum usitatissimum (ref. 19) 6 GGAUGGGUGA CCUCCUGGGA AGUCCUCGUG UUGCACUUU- B4 Phaseolus vulgaris (ref. 21) 7 GGAUGGGUGA CCUCCUGGGA AGUCCUUGUG UUGCACCUC- B5 Visia faba (ref. 21) 8 GGAUGGGUGA CCUCCUGGGA AGUCCUCGUG UUGCACCUCC B6 Lupius Luteus (ref. 20) 9 GGAUGGGUGA CCUCCUGGGA AGUCCUCGUG UUGCACCCCU B7 Spinacia oLeracea (ref. 18) i0 GGAUGGGUGA CCUCCCGGGA AGUCCCGGUA UUGCACCCUU C1* Metasequoia gLyptostroboides (this paper) 11 GGAUGGGUGA CCUCCUGGGA AGUCCCAGUG UUGCACCCUC C2* Ginkgo biloba (this paper) 12 GGAUGGGUGA CCUCCUGGGA AGUCCUAAUA UUGCACCCUU C3* Cycas revoluta (this paper) 13 GGAUGGGUGA CCUCCCGGGA AGUCCUGGUG CUGCACCCUU D1* Dryopteris acuminata (ref. 16) 14 GGAUGGGUGA CCUCCCGGGA AGUCCUGGUG CCGCACCCC- D2* Equisetum arvense (ref. 16) 15 GGAUGGGUGA CCUCCCGGGA AGUCCGGGUG CCGCACCCUC D3* Lycopodium clavatum (this paper) 16 GGAUGGGUGA CCUCCCGGGA AGUCCCGGUd CCGCGCCCAC D4* PsiLotum nudum (this paper) 17 GGAUGGGUGA CCUCCCGGGA AGUCCCGGUG CUGCACCCU- E1* Anthoceros punctatus (ref. 15) 18 GGAUGGGUGA CCUCCCGGGA AGUCCCGGUG CUGCAUCCA- E2* Plagiomnium trichomanes (ref. 15) 19 GGAUGGGUGA CCUCCCGGGA AGUCCCGGUG CUGCAUCCA- E3* LophocoLea heterophylta (ref. 15) 20 GGAUGGGUGA CCUCCCGGGA AGUCCCGGUG CUGCAUCCA- E4* Marchantia polymorpha (ref. 15) 21 GGAUGGGUGA CCUCCUGGGA AGUCCCGGUG CUGUACCUAU Fl* Nitelta fLexilis (this paper) 22 GGUUGAGGGA UCACUUGGGA ACCCCUGGUG CUGUAGUGU- G1* Spirogyra sp. (this paper) 23 GGUUGAGGGA UUACCUGGGA ACCCCGAGUG ACGUAGUGU- G2 ChLoreLLa pyrenoidosa (ref. 22) 24 GGUUGAGGGA UUACCUGGGA ACCCCGAGUG ACGUAGUGU- G3 Senedesmus quadricauda (ref. 22) 25 GGUUGAGGGA UUACCUGGGA ACCCCGAGUG ACGUAGUGU- G4 Senedesmus obLiquus (ref. 23) 26 GGCUGAGUGA UCUCCUGGGA AUCCCCUGUG CUGUAUCGC- G5* Ulva pertusa (ref. 14) 27 CGGUGGGUGA CCACGUGCGA AGCCCUCGUG ACG-AUCGU- G6* Chiamydomonas sp. (ref. 7) 28

FIG. 2. Sequence alignment of twenty-eight 5S rRNAs from green plants. The symbols Al, A2, etc., preceding the species names corre- spond to those preceding the common names in Fig. 3. Al-A3, ; Bl-B7, dicotyledons; Cl-C3, gymnosperms; Dl-D4, Pterido- phyta; El-E4, Bryophyta; F1, Charophyta; G1, Gamophyta; G2-G6, green algae. *, sequences determined in our laboratory.

ess within the Spermatophyta. The first hypothesis is that, include flowering plants. Thus, the most important differ- after separation from the Pteridophyta, the ancestor of the ence between these two hypotheses lies in the evolutionary Spermatophyta evolved into two groups, one group contain- position of the . The former assumes that the cycads ing the Ginkgopsida (maidenhair tree), the Coniferopsida are more closely related to the flowering plants than to the (coniferous trees), and the Gnetopsida (e.g., joint fir) and maidenhair tree and coniferous trees while, in the latter, cy- another group containing the Cycadopsida (cycads) and the cads, maidenhair tree, and coniferous trees are more closely angiosperms (flowering plants). The latter two share the related to one another than to flowering plants. The 5S common ancestor called the pteridosperms (seed-ferns; 27). rRNA phylogenic tree clearly shows that Metasequoia (a co- The second hypothesis supposes that the gymnosperms and niferous tree), Cycas (a cycad), and Ginkgo (maidenhair the angiosperms separated sometime during Spermatophyta tree) are closely related. The separation of these three.spe- evolution (26). Here, the gymnosperms include the cycads, cies occurred after their separation from the ancestor of the maidenhair tree, and coniferous trees and the angiosperms flowering plants, supporting the second hypothesis de- Evolution: Hori et al. Proc. NatL. Acad Sci. USA 82 (1985) 823

Fern Horsetail1 > C1ubmoss Whisk fern $ X Moss Liverwort A la Liverwort B 0 Stonewort o Spirogyr A Chiore 110a Senedesmus A Senedesmus B Ulva Chlamydomonas 1 /2 Knuc- 0.2 0.1 0 FIG. 3. Phylogenic tree of twenty-eight 5S rRNAs from green plants. The symbols Al, A2, etc., correspond to those in Fig. 2. * and **, sequences determined in our laboratory and in both our and other laboratories, respectively. scribed above. The sequence identity among Metasequoia, 11. Nazar, R. N. & Wildeman, A. G. (1981) Nucleic Acids Res. 9, Cycas, and Ginkgo is 93-96%, while that between Metase- 5345-5358. quoia/Cycas/Ginkgo and flowering plants is 85-92% (88% 12. Kimura, M. (1980) J. Mol. Evol. 16, 111-120. 13. Sneath, P. H. A. & Sokal, R. R. (1973) Numerical on average). Among the gymnosperms, Metasequoia sepa- (Freeman, San Francisco), pp. 227-240. rated from the ancestor of Cycas/Ginkgo first and the latter 14. Lim, B.-L., Hori, H. & Osawa, S. (1983) Nucleic Acids Res. two species separated later. Among the angiosperms, a pre- 11, 1909-1912. cise evolutionary process cannot be deduced because the 15. Katoh, K., Hori, H. & Osawa, S. (1983) Nucleic Acids Res. 11, Knuc values among the species are so close. 5671-5674. 16. Hori, H., Osawa, S., Takaiwa, F. & Sugiura, M. (1984) Nucle- This work was supported by Grants 58540404, 58880025, and ic Acids Res. 12, 1573-1576. 58113004 (Special Project Research) from the Ministry of Education 17. Barber, C. & Nichols, J. L. (1978) Can. J. Biochem. 56, 357- of Japan. 364. 18. Delihas, N., Andersen, J., Sprouse, H. M., Kashdan, M. & 1. Hori, H. & Osawa, S. (1979) Proc. Natl. Acad. Sci. USA 76, Dudock, B. (1981) J. Biol. Chem. 256, 7515-7517. 381-385, and correction (1979) 76, 4157. 19. Goldsbrough, P. B., Ellis, T. H. N. & Lomonossoff, G. P. 2. Dekio, S., Yamasaki, R., Jidoi, J., Hori, H. & Osawa, S. (1982) Nucleic Acids Res. 10, 4501-4514. (1984) J. Bacteriol. 159, 233-237. 20. Rafalski, J. A., Wiewiorowski, M. & Soil, D. (1982) Nucleic 3. Fox, G. E., Luehrsen, K. R. & Woese, C. R. (1982) Zentralbl. Acids Res. 10, 7635-7642. Bakteriol. Hyg. Abt. 1, Orig. C3, 330-345. 21. Vandenberghe, A., Chen, M. W., Dams, E., De Baere, R., De 4. Osawa, S. & Hori, H. (1979) in Ribosomes, eds. Chamblis, G., Roeck, E., Huysmans, E. & De Wachter, R. (1984) FEBS Lett. Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, 171, 17-22. M. (Univ. Park Press, Baltimore), pp. 333-355. 22. Luehrsen, K. R. & Fox, G. E. (1981) Proc. Natl. Acad. Sci. 5. Hori, H., Itoh, T. & Osawa, S. (1982) Zentralbl. Bakteriol. USA 78, 2150-2154. Hyg. Abt. I Orig. C3, 18-30. 23. Green, G. A., McCoy, J. M. & Jones, D. S. (1982) Nucleic Ac- 6. Gottschalk, M. & Blanz, P. A. (1984) Nucleic Acids Res. 12, ids Res. 10, 6389-6392. 3951-3958. 24. Erdmann, V. A., Wolters, J., Huysmans, E., Vandenberghe, 7. Kumazaki, T., Hori, H. & Osawa, S. (1983) J. Mol. Evol. 19, A. & De Wachter, R. (1984) Nucleic Acids Res. 12, r133-r166. 411-419. 25. Schuster, H. (1966) Hepaticae and Anthocerotae of North 8. Ohama, T., Kumazaki, T., Hori, H. & Osawa, S. (1984) Nucle- America (Columbia Univ. Press, New York), Vol. 1, p. 802. ic Acids Res. 12, 5101-5108. 26. Bold, H. C. (1970) The Plant (Prentice-Hall, Engle- 9. Peattie, D. A. (1979) Proc. Natl. Acad. Sci. USA 76, 1760- wood Cliffs, NJ), 3rd Ed. pp. 77-86. 1764. 27. Margulis, L. & Schwartz, K. V. (1982) Five Kingdoms (Free- 10. Donis-Keller, H. (1979) Nucleic Acids Res. 8, 3133-3142. man, San Francisco), pp. 248-271.