Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 820-823, February 1985 Evolution Evolution of green plants 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 Biology, 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 algae such as Chlamydomonas, Spirogyra, Ulva, and RESULTS AND DISCUSSION Chlorella occurred in the early stage of green plant evolution. Phylogeny of Various Groups of Organisms and Emergence The branching point of Nitella 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 green algae, 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 ferns by degeneration. In the Pterido- that the eubacteria separated from the metabacteria/eukar- phyta, Psilotum (whisk fern) separated first, and a little later yotes branch. In the eubacterial branch, the Cyanobacteria Lycopodium (club moss) separated from the ancestor common (plus plant chloroplasts) emerged first and this was followed to Equisetum (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 bacteria having the 120-nucleotide type 5S evolution, the gymnosperms (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 (cycad)/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 eukaryotes 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- animals 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 red algae 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 vascular plant), 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 (Chlorophyta; 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.