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Molecular Phylogenetic Analysis Among Bryophytes and Tracheophytes Based on Combined Data of Plastid Coded Genes and the 18S rRNA Gene Tomoaki Nishiyama and Masahiro Kato Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan The basal relationship of bryophytes and tracheophytes is problematic in land plant phylogeny. In addition to cladistic analyses of morphological data, molecular phylogenetic analyses of the nuclear small-subunit ribosomal RNA gene and the plastid gene rbcL have been performed, but no confident conclusions have been reached. Using the maximum-likelihood (ML) method, we analyzed 4,563 bp of aligned sequences from plastid protein- coding genes and 1,680 bp from the nuclear 18S rRNA gene. In the ML tree of deduced amino acid sequences of the plastid genes, hornworts were basal among the land plants, while mosses and liverworts each formed a clade and were sister to each other. Total-evidence evaluation of rRNA data and plastid protein-coding genes by TOTALML had an almost identical result. Introduction Attempts to elucidate relationships within and between members of land plant lineages have met with conflicting results. The bryophytes, which are characterized by their unbranched and dependent sporophytes, unornamented conducting cell walls, and lack of true lignin, are placed as the most primitive of extant land plants (Bremer et al. 1987). The bryophytes consist of three main groups. Musci (mosses) has the largest number of species, Hepaticae (liverworts or hepatics) is morphologically the most diverse, and Anthocerotae (hornworts) is a relatively small and homogeneous group. The relationships of these bryophyte groups are uncertain. Cladistic analyses of phenotypic characters placed the mosses as a sister group to tracheophytes, hornworts as a sister group to the mosses plus the tracheophytes, and liverworts as a sister group to all other land plants (Mishler and Churchil 1984; Bremer et al. 1987; Mishler et al. 1994). Distribution of three mitochondrial introns also implied that liverworts are basal to all other land plants (Qiu et al. 1998). Cladistic analyses based on male gametogenesis showed that bryophytes were monophyletic and hornworts were basal in the bryophyte clade (Garbary, Renzaglia, and Duckett 1993; Mishler et al. 1994). The phylogenetic relationship of the three bryophyte classes has been inferred based on the plastid gene rbcL (1,350 bp), the nuclear small-subunit rRNA gene (ca. 1,800 bp), the mitochondrial gene cox3 (381 bp), and the mitochondrial 19S rRNA gene (1,716 bp). rbcL analyses did not show enough resolution of the relationship among the bryophytes and other land plants (Manhart 1994; Lewis, Mishler, and Vilgalys 1997). Chapman and Buchheim’s (1991, 1992) cladograms based on partial sequences of 18S and 26S rRNA showed that the liverworts were divided into thallose (Marchantiidae) and leafy liverworts (Jungermaniidae), and that the thallose liverworts were sister to the tracheophytes, but support for the most branches among classes or subclasses was low. Capesius (1995) and Bopp and Capesius (1995, 1996) analyzed 1,705 nt sequences of the 18S rRNA gene and concluded that Hepaticae is polyphyletic, indicating that Jungermaniidae and Marchantiidae do not form a single clade. The analyses by Bopp and Capesius did not contain any tracheophyte species. The analysis of the nuclear-encoded rRNA gene by Hedderson, Chapman, and Rootes (1996) showed that each of the mosses and liverworts was monophyletic and formed a clade sister to the tracheophytes, and that hornworts were basal in land plants. The analyses of the mitochondrial 19S rRNA gene suggested that either liverworts or hornworts are the basal land plant clade but failed to determine which are basal (Duff and Nickrent 1999). An analysis based on cox3 cDNA showed that hornworts were the most basal group among land plants, but the relationships of the species of mosses and liverworts were unclear (Malek et al. 1996). Low resolution of trees and the contradictory results of the previous studies seem to have been caused by insufficient information from short sequences. In this study, we analyzed plastid protein-coding genes to obtain sufficient information on the basal phylogenetic relationship among land plants. The complete plastid genome sequences of several land plants and green algae are available, and it will be possible to determine tens of kilobases of aligned sequences whose positional homology is certain. Another merit of using plastid genes for deep phylogeny is that plastid genes have a slower substitution rate than nuclear genes (Wolfe, Li, and Sharp 1987, 1989), which is supposed to be caused by the reduced mutation rate (Clegg and Zurawski 1992). Abbreviation: CTAB, hexadecyltrimethylammonium bromide. Key words: bryophytes, phylogeny, land plants, plastid, tracheophytes, paralinear distance. Materials and Methods Regions Used for Analysis Address for correspondence and reprints: Tomoaki Nishiyama, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan. E-mail: [email protected]. In addition to rbcL, which is one of the most widely used genes in plant systematics, psaA, psaB, psbD, and rpoC2 were selected from the genes present in the large single-copy region of all known plant plastid genomes. Selection of these regions was due to their dis- Mol. Biol. Evol. 16(8):1027–1036. 1999 q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 1027 1028 Nishiyama and Kato Table 1 Sources of Plastid DNA Sequences Sourcea Species Name Abbreviation Green algae sensu lato Chlorella vulgaris Beijerinck . . . . . . . . . . . . . . . . Coleochaete nitellarum Jost. . . . . . . . . . . . . . . . . . Cvu Cni Wakasugi et al. (1997), AB001684 Unialgal culture, UTEX LB1261; this study, AB013658– AB013663 Hornworts Anthoceros punctatus L. . . . . . . . . . . . . . . . . . . . . Apu Callus, Ono, Murasaki, and Takamiya (1988); this study, AB013664–AB013669 Liverworts Haplomitrium mnioides (Lindb.) Schust. . . . . . . . Hmn Field collected, H. Akiyama 13579, HYO; this study, AB013675– AB013679 Ohyama et al. (1986), X04465 Marchantia polymorpha L. . . . . . . . . . . . . . . . . . . Mosses Physcomitrella patens (Hedw.) B.S.G. . . . . . . . . Mpo Ppa Sphagnum fallax Klinggr. . . . . . . . . . . . . . . . . . . . Tracheophytes Adiantum capillus-veneris L. . . . . . . . . . . . . . . . . Sfa Aca Pinus thunbergii Parl. . . . . . . . . . . . . . . . . . . . . . . Nicotiana tabacum L. . . . . . . . . . . . . . . . . . . . . . . Oryza sativa L. . . . . . . . . . . . . . . . . . . . . . . . . . . . Zea mays L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pth Nta Osa Zma Aseptic culture, Ashton and Cove (1977); this study, AB013652– AB013657 Aseptic culture, H. Rudolph; this study, AB013670–AB013674 Hasebe and Iwatsuki (1990), D14880; this study, AB013680– AB013683 Wakasugi et al. (1994), X15901 Shinozaki et al. (1986), Shimada et al. (1990), Z00044 Hiratsuka et al. 1989, X15901 Maier et al. (1995), X86563 a For the sequences taken from the database, the reference literature and the accession number are shown. For sequences newly determined in this study, the source of plant material and the accession numbers are shown. persal over the whole large single-copy region. As implied by a study of vertebrate mitochondrial genomes, sampling several short stretches distributed from different genomic locations should be better for phylogenetic reconstruction than sampling several short stretches from contiguous sites (Cummings, Otto, and Wakely 1995). Selection of Operational Taxonomic Unit Since it is generally accepted from both morphological and molecular data that the charophycean algae are the closest to land plants, a certain species from the group was supposed to be an appropriate outgroup. Because of the availability of unialgal culture (Starr and Zeikus 1993), Coleochaete nitellarum (UTEX LB1261) was analyzed. The green alga Chlorella vulgaris, whose complete plastid genome sequence is available (Wakasugi et al. 1997), was also used as an outgroup. Five bryophyte species were selected, representing the three classes. From the Anthocerotae, a callus culture of Anthoceros punctatus established by Ono, Murasaki, and Takamiya (1988) was used. From the Musci, which are systematically very divergent, two species from two of the three subclasses recognized by Vitt (1984), Physcomitrella patens from the Bryidae and Sphagnum fallax from the Sphagnidae, were used. From the Hepaticae, with two distinct lineages, Marchantiidae and Jungermaniidae (Schuster 1984), Haplomitrium mnioides (Jungermaniidae) was used, as was the published sequence of Marchantia polymorpha (Marchantiidae). There are available sequences from the seed plants Pinus thunbergii (gymnosperm), Nicotiana tabacum (angiosperm, dicot), Oryza sativa (monocot), and Zea mays (monocot). However, there is no plastid sequence of fern species sufficient for this analysis. Hence, the fern species Adiantum capillus-veneris was analyzed along with the above four seed plant species. The accession numbers of the plastid DNA sequences used in this study are shown in table 1. The following sequences for nuclear-encoded 18S rRNA were from the DNA database: Chlorella vulgaris (X13688), Coleochaete scutata (X68825), Anthoceros agrestis (X80984), Marchantia polymorpha (X75521), Haplomitrium hookeri (U18504), Sphagnum cuspidatum (X80213), Physcomitrella patens (X80986), Adiantum raddianum (U18621), Pinus luchuensis (D38246), Lycopersicon esculentum (X51576), Oryza sativa (X00755), and Zea mays (K02202). Plant Material and DNA Extraction DNA was extracted from samples that were frozen and ground in liquid nitrogen with extraction buffer containing CTAB (Murray and Thompson 1980) precipitated either by a low-ionic-strength CTAB solution or 2propanol. Adiantum capillus-veneris plastid DNA (separated by CsCl density gradient centrifugation) and the clone bank of A. capillus-veneris plastid DNA (Hasebe and Iwatsuki 1990) were provided by Dr. M. Hasebe. PCR Amplification and Sequencing A 50-ml PCR mix contained 5 ml of 10 3 PCR buffer (0.1 M Tris-HCl [pH 9], 0.5 M KCl, 25 mM MgCl2, 1% Triton X-100, or 0.1 M Tris-HCl [pH 8.3], 0.5 M KCl, 15 mM MgCl2), 0.2 mM dATP, dCTP, dGTP, and dTTP, 0.5 mM each of two primers, 1.3 U recombinant Taq DNA polymerase, and 1–5 ml of DNA solution. The sequences of primers used in this study will be provided by the author (T.N.) on request. The PCR mix was incubated at 948C for 2 min, and 25 or 30 amplification cycles were carried out in a thermal cycler (Nippon Genetics QTP-1, Astec PC-700, or Taitec TR- Land Plant Phylogeny 100). Each cycle included a 1-min period for denaturation at 948C, a 1-min period for annealing, and a 2-min period for extension at 728C. The annealing temperature was 458C in most cases. Amplified fragments were electrophoresed in 1% or 1.2% Agarose (Seakem GTG, FMC biotech), TAE buffer. The fragments were recovered from the gel using the GeneCleanII kit (Bio101). Purified fragments were directly sequenced using fluorescein-labeled primer having the same nucleotide sequence as the one used for the PCR amplification with the AutoCycle sequencing kit (Pharmacia) or the Thermosequenase fluorescent labeled primer sequencing kit (Amasham) according to the manufacturer’s instructions. The product of sequence reaction was electrophoresed in an A.L.F. autosequencer (Pharmacia), and the data obtained were processed by ALF Manager, version 2.5. Plasmid Manipulation Competent cell preparation and transformation of Escherichia coli DH5a were performed according to Inoue, Nojima, and Okayama (1990). Plasmid DNA from E. coli culture was extracted by alkali lysis, followed by precipitation with polyethylene glycol (Sambrook, Fritch, and Maniatis 1989). After digestion by restriction enzyme, vectors were dephosphorilated using calf intestine phosphatase (Boeringer Manheim) according to the manufacturer’s instructions. Ligation was performed with the Takara ligation kit, version 1. Plasmids were sequenced using the AutoRead sequencing kit (Pharmacia). For the A. capillus-veneris rpoC1/rpoC2 gene, some subclones of pACP3 (Hasebe and Iwatsuki 1990) were sequenced. pACP3 was digested with XhoI and electrophoresed in 1% Agarose, and six fragments were recovered using GeneCleanII. Every fragment except for the largest was subcloned into pUC18 at the SalI site (pACP3-2 to pACP3-6). Sequencing each subclone, the relative position to the gene was determined and pACP3-3 was further subcloned. pACP3-3 was digested with HindIII and electrophoresed in agarose gel. The second and third fragments were recovered using GeneCleanII, and the end was filled with T4 DNA polymerase and ligated in pUC18 at the SmaI site. Most of the sequence used in the phylogenetic analysis for the rpoC2 gene was also confirmed by PCR direct sequencing of plastid DNA. Alignment and Phylogenetic Analysis The nucleotide sequences from each gene were aligned separately using CLUSTAL W, version 1.6 (Thompson, Higgins, and Gibson 1994). Each sequence for the rpoC1/rpoC2 region was separated into two sequences for rpoC11spacer and rpoC2 at the site that was homologous to the start codon of M. polymorpha. Default parameters were used for rbcL, psaA, psaB, and psbD, but an increased gap penalty of 30 for both pairwise alignment and multiple alignment was used for rpoC2. Unambiguous parts of the alignment were cut to fit in the codon position and concatenated to a single data matrix. Nucleotide sequences were translated to 1029 Table 2 GC Content of Each Codon Position from Plastid Genes CODON POSITION SPECIESa First Second Third Cvu . . . . . . . . Cni . . . . . . . . Ppa . . . . . . . . Sfa . . . . . . . . . Apu . . . . . . . . Hmn . . . . . . . Mpo . . . . . . . Aca . . . . . . . . Pth . . . . . . . . . Nta . . . . . . . . Osa . . . . . . . . Zma . . . . . . . . 0.546 0.500 0.512 0.543 0.496 0.548 0.511 0.542 0.538 0.544 0.531 0.529 0.443 0.435 0.430 0.433 0.423 0.445 0.427 0.444 0.437 0.439 0.439 0.437 0.216 0.125 0.157 0.258 0.234 0.381 0.136 0.417 0.308 0.292 0.328 0.322 a Abbreviations for taxon names are shown in table 1. amino acid sequences based on the universal code by using the program NUC2PTN of the MOLPHY, version 2.3b3, package (Adachi and Hasegawa 1996). Analysis of Nucleotide Sequence It has been recognized that synonymous codons are not used equally and the usage pattern differs from organism to organism (Grantham, Gautier, and Gouy 1980). The usage is supposed to be subject to selective pressure (Miyata and Hayashida 1981). Although Clegg (1993) noted little variation in codon usage in rbcL among flowering plants, a base composition difference in the rbcL sequence was recognized by Lewis, Mishler, and Vilgalys (1997). Prior to phylogenetic analysis, nucleotide composition of each sequence was examined for first, second, and third codon positions. As shown in table 2, the GC contents of first and second codon positions fell within a very small range, from 0.496 (A. punctatus) to 0.548 (H. mnioides) and from 0.422 (A. punctatus) to 0.444 (H. mnioides), respectively. However, the GC contents of third codon positions varied from 0.125 (C. nitellarum) to 0.417 (A. capillus-veneris). This difference in the GC contents of the third codon positions clearly indicates that the evolutionary process under which these sequences have evolved cannot be stationary. In other words, different lineages must have had different evolutionary trends. As most of the currently available methods of phylogenetic inference are based on the stationary Markov process, their results may be misleading under nonstationary conditions. The only methods applicable to nonstationary cases are the paralinear distance (Lake 1994) and the LogDet distance (Steel 1994), which are very similar to each other. Recently, a method to correct the bias of those distances for short sequences has been developed by Gu and Li (1996). A program to calculate the bias-corrected paralinear distance was developed, and phylogenetic analysis based on the bias-corrected paralinear distance was performed. The calculation followed Gu and Li (1996), but because the determinant of divergence matrix F may be negative, the logarithm of absolute value of the determinant was used for the estimation, as was also done 1030 Nishiyama and Kato for the original LogDet distance by Steel (1994). To utilize the power of the paralinear or LogDet distance, it is important to use only those sites that evolve at the same rate. If constant sites are included, the phylogeny based on the LogDet distance may fall into the same topology as that inferred using Jukes-Cantor distance, but by using only parsimony sites or third codon positions, correct topologies were obtained in some data sets (Lockhart et al. 1994; Penny et al. 1994). Therefore, the bias-corrected paralinear distance using third codon positions and fourfold-degenerate sites was calculated. As the paralinear distance is additive, the neighbor-joining method (Saitou and Nei 1987) was used to construct phylogenetic trees. This procedure was done by NEIGHBOR in PHYLIP, version 3.572c (Felsenstein 1995). The bootstrap test (Felsenstein 1985) was performed by analyzing 1,000 data sets resampled by SEQBOOT with random seed 5. Occurrence of each subgroup was counted using CONSENSE. Analysis of Deduced Amino Acid Sequence Another way to avoid the defect by the change of codon usage or GC content is to translate nucleotide sequences to amino acid sequences. Maximum-likelihood (ML) analysis was performed by PROTML in the MOLPHY, version 2.3b3, package (Adachi and Hasegawa 1996). The internal stop codons found in A. punctatus and A. capillus-veneris were treated as unknown. Using constraint phylogeny within seed plants (Pth, (Nta, (Osa, Zma))), 135,000 trees were examined for their approximate likelihoods under the JTT-F model (Jones, Taylor, and Thornton 1992) with the ‘‘-e’’ option, and the best 3,000 trees were extracted. The likelihood of each of those 3,000 trees was evaluated. Local bootstrap probability was estimated using the resampling-of-estimated-log-likelihood (RELL) method (Kishino, Miyata, and Hasegawa 1990; Hasegawa and Kishino 1994) with the best tree found in the search described above. To test other hypotheses, we performed a bootstrap test for all of the 15 possible topologies with fixed subtrees for mosses, liverworts, tracheophytes, and algae. To test the contribution of the individual region, the likelihood of each region was estimated for the 15 topologies under the JTT-F model. To test the dependency on the substitution model, the 15 topologies were examined for their likelihoods under other substitution models implemented in PROTML. Reevaluation of 18S Data Set The 18S rRNA sequences were aligned by CLUSTAL W, version 1.6, with default parameters. All positions with any gaps were removed, and the data were analyzed by NUCML under the Kimura (1980) two-parameter model and the HKY85 model (Hasegawa, Yano, and Kishino 1984) with the restriction in the subtree of angiosperms. Total ML Analysis Using Plastid Genes and 18S rRNA Sequences Although amino acid sequences of plastid genes and nucleotide sequences of nuclear 18S rRNA genes FIG. 1.—The tree obtained using the bias- corrected paralinear distance of third codon positions. The number on each branch indicates the bootstrap probability (%) by 1,000 resamplings. The horizontal length of each branch is proportional to the estimated distance. The number in parentheses after each species name indicates that species’ GC content (%) of the third codon positions. cannot be assumed to evolve under the same evolutionary model, both sequences should have evolved under the same topology. In cases in which the rRNA sequence from the same species as the plastid genes was not available, a closely related species was assumed to form the same topology. For each of the 15 topologies, the log-likelihood of each character for the ML estimate of branch lengths was determined with the ‘‘-l’’ option. The data were summarized by TOTALML, and bootstrap probability of each tree being the ML tree was estimated by the RELL method. Results Internal Stop Codons The sequence from A. punctatus contained 24 internal stop codons. It has been reported that the Megaceros enigmaticus rbcL gene contains two internal stop codons (Manhart 1994). In the Anthoceros formosae rbcL gene, 20 positions, including T of two internal stop codons, are shown to be edited (Yoshinaga et al. 1996). The stop codons found in A. punctatus may be translated after RNA editing. The genomic sequence of the A. capillus-veneris rpoC2 gene contained one internal stop codon. The sequence around the stop codon was determined by sequencing a plasmid clone and confirmed by PCR direct sequencing. The most probable explanation is that the fern has RNA editing, as in the case of the internal stop codons in the A. formosae rbcL gene. No internal stop codon was found in any other species used in this study. Plastid Gene Trees The tree obtained using the bias-corrected paralinear distance is shown in figure 1. In this tree, based on third codon positions, the bootstrap probabilities for branches of nonseed plants were quite low. When fourfold-degenerate sites were used instead of third codon positions, the bootstrap probability became lower (data not shown). In the ML tree based on the JTT-F model (fig. 2), each of the mosses, liverworts, and tracheophytes Land Plant Phylogeny 1031 Table 3 The Likelihood Values for Alternative Topologies Under the JTT-F Model, Obtained from the Deduced Amino Acid Sequences of Plastid Genes Treea FIG. 2.—The ML tree based on deduced amino acid sequences of plastid genes. The horizontal length of each branch is proportional to the number of amino acid substitutions estimated by the ML method based on the JTT-F model. The number on each branch is the local bootstrap probability (%) estimated by the RELL method. formed a clade. Mosses and liverworts formed a clade. The moss-plus-liverwort clade was sister to the tracheophyte clade, and the hornworts were basal. Local bootstrap probabilities of those branches supporting the mosses, liverworts, tracheophytes, and land plants were quite high (97%–99%). The likelihood value and relative bootstrap value (estimated by the RELL method) of all possible 15 topologies are shown in table 3. The relative bootstrap probability of the ML tree was 58%. In the second-greatest-likelihood tree, A. punctatus was sister to tracheophytes, and the moss-plus-liverwort clade was basal in land plants. In the three trees with the highest likelihood, the mosses and liverworts were sisters to each other. Table 4 shows the comparisons of the likelihoods for the 15 topologies estimated from regions belonging to each gene. Of the five regions, only psaB showed the best likelihood to topology 1, which is supported as the ML tree by the whole data set (fig. 2). Analysis of combined data excluding psaB favored the topology of the whole data set (table 5), although the difference between topology 1 and topology 2 was small (0.5 6 3.7). Under substitution models other than the JTT-F model, the same topology (topology 1) had the maximum likelihood. From the point of Akaike Information Criterion (AIC) (Kishino and Hasegawa 1990), the JTTF model fits the data better than any other model. Analysis of 18S rRNA A total of 1,680 nucletotide sites from the alignment without indels were used for phylogenetic analysis. The ML tree under the Kimura two-parameter model is shown in figure 3, which has a better AIC value than the HKY85 model. The result under the HKY85 model had the same topology, with little difference in branch lengths and local bootstrap probabilities (data not shown). The topology of the tree by 18S rRNA gene data (fig. 3) generally resembled the ML tree of plastid genes (fig. 2), although the relationship of Sphagnum and Physcomitrella was different between the two trees. The difference between the log-likelihood of the ML tree of 18S rRNA gene data and that of the alternative tree with the same topology as the ML tree of plastid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... DlnLb SEc TBLd RELL-BPe 210,972.8 23.8 25.3 210.0 211.3 212.5 213.9 214.5 214.8 217.3 217.3 217.6 217.9 218.0 219.7 ML 4.9 4.3 9.8 11.7 12.8 9.9 12.7 8.6 11.4 10.6 11.6 11.2 8.8 11.5 ME 0.2 0.3 0.2 0.4 0.4 0.3 0.5 0.4 0.4 0.5 0.6 0.5 0.5 0.6 0.5771 0.1451 0.0168 0.0761 0.0730 0.0632 0.0133 0.0243 0.0004 0.0031 0.0017 0.0003 0.0047 0.0001 0.0008 a The tree topologies are defined as follows: 1—(Cvu, Cni, (Apu, (((Ppa, Sfa), (Mpo, Hmn)), (Aca, (Pth, (Nta, (Osa, Zma))))))); 2—(Cvu, Cni, ((Apu, (Aca, (Pth, (Nta, (Osa, Zma))))), ((Ppa, Sfa), (Mpo, Hmn)))); 3—(Cvu, Cni, ((Apu, ((Ppa, Sfa), (Mpo, Hmn))), (Aca, (Pth, (Nta, (Osa, Zma)))))); 4—(Cvu, Cni, (Apu, (((Ppa, Sfa), (Aca, (Pth, (Nta, (Osa, Zma))))), (Mpo, Hmn)))); 5— (Cvu, Cni, (((Apu, (Aca, (Pth, (Nta, (Osa, Zma))))), (Ppa, Sfa)), (Mpo, Hmn))); 6—(Cvu, Cni, ((Apu, ((Ppa, Sfa), (Aca, (Pth, (Nta, (Osa, Zma)))))), (Mpo, Hmn))); 7—(Cvu, Cni, (((Apu, (Ppa, Sfa)), (Mpo, Hmn)), (Aca, (Pth, (Nta, (Osa, Zma)))))); 8—(Cvu, Cni, (((Apu, (Ppa, Sfa)), (Aca, (Pth, (Nta, (Osa, Zma))))), (Mpo, Hmn))); 9—(Cvu, Cni, (Apu, ((Ppa, Sfa), ((Mpo, Hmn), (Aca, (Pth, (Nta, (Osa, Zma)))))))); 10—(Cvu, Cni, ((Apu, (Ppa, Sfa)), ((((Mpo, Hmn), (Aca, (Pth, (Nta, (Osa, Zma))))))); 11—(Cvu, Cni, (((Apu, (Aca, (Pth, (Nta, (Osa, Zma))))), (Mpo, Hmn)), (Ppa, Sfa))); 12—(Cvu, Cni, ((Apu, (Mpo, Hmn)), ((Ppa, Sfa), (Aca, (Pth, (Nta, (Osa, Zma))))))); 13—(Cvu, Cni, ((Apu, ((Mpo, Hmn), (Aca, (Pth, (Nta, (Osa, Zma)))))), (Ppa, Sfa))); 14—(Cvu, Cni, (((Apu, (Mpo, Hmn)), (Ppa, Sfa)), (Aca, (Pth, (Nta, (Osa, Zma)))))); 15—(Cvu, Cni, (((Apu, (Mpo, Hmn)), (Aca, (Pth, (Nta, (Osa, Zma))))), (Ppa, Sfa))). b The log-likelihood for the maximum-likelihood (ML) tree or the difference of the log-likelihood from that of the ML tree. c Standard error of the difference of the log-likelihood from that of the ML tree. d Difference of total branch length from that of the minimal-evolution (ME) tree. e Relative bootstrap value estimated by the RELL method among all 15 topologies possible under the constraint for local groups. genes (fig. 2), in which Sphagnum and Physcomitrella form a sister group, is not statistically significant (21.0 6 8.1). In the analysis of amino acid sequences, the difference between the log-likelihood of the ML tree (fig. 2) and that of the tree with the topology of Sphagnum being sister to liverworts (fig. 3) is significant (225.8 6 11.5). Total ML Analysis Using Plastid Genes and 18S rRNA Sequences The result of TOTALML analysis was almost identical to the result from plastid genes. The trees with the highest, second-highest, and third-highest likelihoods had the same topologies as and similar relative bootstrap values to those obtained by the analysis of plastid genes. The relative bootstrap probability of the ML tree was 57% for the whole data set, 59% for the plastid gene data, and 20% for 18S rRNA data. 1032 Nishiyama and Kato Table 4 Log-Likelihood Differences of 15 Topologies for Individual Regions of Plastid Genes Based on Their Deduced Amino Acid Sequences RBCL TREEa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... a b c (432 aa) PSAB (402 aa) PSBD RPOC2 (274 aa) PSAA (135 aa) SEc DlnL SE DlnL SE DlnL SE DlnL SE 20.5 0.0 21.8 28.6 27.1 212.0 210.0 213.8 210.3 210.3 26.5 212.0 28.1 29.2 29.3 3.6 Best 2.7 10.1 6.2 10.1 6.9 9.1 9.8 9.8 6.9 10.1 9.7 7.4 9.8 0.0 23.3 22.6 20.1 24.5 22.9 22.3 22.6 22.0 22.0 26.7 25.4 24.9 25.4 27.1 Best 3.8 4.2 4.2 6.2 7.3 7.1 7.7 3.2 6.7 5.3 6.7 5.1 5.9 6.1 20.0 20.0 20.0 20.0 20.0 20.0 0.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Best 0.0 0.0 0.0 20.3 0.0 20.8 22.1 20.4 21.3 22.4 21.3 22.2 23.1 21.3 22.4 21.4 21.9 21.4 1.9 Best 1.5 3.6 3.8 4.2 2.4 4.1 3.4 3.3 3.5 3.8 3.7 2.7 3.7 22.0 22.0 22.0 22.0 22.0 22.1 0.0 20.0 22.0 20.0 22.0 22.0 22.0 22.0 22.0 2.6 2.6 2.6 2.6 1.6 1.6 Best 0.0 2.6 0.0 2.6 2.6 2.6 2.6 2.6 The tree numbers are identical to those in table 1. Difference of the log-likelihood from that of the maxim-likelihood (ML) tree. Standard error of the difference of the log-likelihood from that of the ML tree. Discussion Change of Codon Usage and Editing Although certain sites are supposed to be edited at the RNA level, nucleotide sequences were translated according to the universal code. It is shown in the psbL mRNA of tobacco that a site-specific factor is involved in editing (Chaudhuri, Carrer, and Maliga 1995). This means that a mutation in genomic sequence may survive due to editing, but for this situation to occur, a change in the site-specific factor must be involved. Thus, delTable 5 Analysis Excluding psaB Treea 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (278 aa) DlnLb ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... DlnLb SEc TBLd RELL-BPe 28,172.1 20.5 22.3 28.7 26.5 28.8 211.0 211.3 212.0 214.6 210.2 212.2 211.8 211.8 212.2 ML 3.7 2.6 8.9 10.0 11.0 7.2 10.6 7.8 9.5 9.3 10.0 10.1 7.0 10.3 ME 0.1 0.3 0.1 0.1 0.2 0.2 0.4 0.4 0.5 0.4 0.4 0.4 0.4 0.4 0.4024 0.2797 0.0302 0.0493 0.1109 0.0819 0.0025 0.0050 0.0006 0.0004 0.0119 0.0011 0.0148 0.0010 0.0083 NOTE.—The combined data set of plastid genes excluding psaB was analyzed under the JTT-F model. The topology of the tree is the same as that in table 1. a The tree numbers are identical to those in table 1. b The log-likelihood of the maximum-likelihood (ML) tree and the difference of the log-likelihood from that of the ML tree. c Standard error of the difference of the log-likelihood from that of the ML tree. d Difference of total branch length from that of the minimal-evolution (ME) tree. e Relative bootstrap value estimated by the RELL method among all 15 topologies possible under the constraint for local groups. eterious substitutions rescued by editing should be rare compared with neutral substitutions. The number of sites that suffer editing is supposed to be rare except in the case of hornworts. Edited sites are completely lacking in M. polymorpha (Ohyama 1996). In maize, 0.13% of amino acids are estimated to be altered by editing (Maier et al. 1995). Twenty-six sites in 14 genes or open reading frames were found in black pine (Wakasugi et al. 1996). In the phylogenetic analysis by Yoshinaga et al. (1996), the genomic and cDNA sequences of A. formosae rbcL clustered to each other. Thus, we assume that phylogenetic analysis can be performed using amino acid sequence deduced from genomic nucleotide sequence without serious error. Phylogenetic Relationship The trees inferred from nucleotide sequences differed from any trees reported so far. In the tree based on third codon positions, the two species with the high- FIG. 3.—The ML tree based on the 18S rRNA gene. The horizontal length of each branch is proportional to the number of nucleotide substitutions estimated by the ML method based on the Kimura two-parameter model. The number on each branch is the local bootstrap probability (%) estimated by the RELL-method. The accession number is shown in parentheses after each taxon name. Land Plant Phylogeny est GC contents, A. capillus-veneris and H. mnioides, formed a sister group, while those species with low GC contents, C. nitellarum, P. patens, and M. polymorpha, came to the basal position (fig. 1). This suggests that the similar GC contents resulted in this topology. Selection of the third codon positions may not be enough to remove all invariant sites and reduce the diagonal element of the divergence matrix, as nondegenerate sites that are subject to functional constraints were included. In the tree based on fourfold-degenerate sites, no branch except for the angiosperm clade had a bootstrap probability greater than 50%. The variance was so large that the phylogeny was unresolved in effect, suggesting that the number of sites, which was only 508 bp after selecting fourfold-degenerate sites, was too small. There should be quite a large sampling error for a small number of sites, although the magnitude is dependent on the method used and the true phylogeny (Hillis, Huelsenbeck, and Cunningham 1994). Although phylogenetic reconstruction based on paralinear distance may be consistent, it was not efficient in this case. With the strict requirement for identical distribution, selecting fourfolddegenerate sites, only one in nine of the sites could be utilized. The paralinear distance has a larger variance than do distances based on stronger assumptions. When each plastid gene was used for phylogenetic inference individually, all the genes except psaB suggested different topologies from the ML tree of the total data. The combined data without psaB supported the same topology as the combined data (table 5). This means that the result is not dependent on the psaB sequences, but is supported by the total sequences. The tree obtained here from the amino acid sequences for the plastid genes indicates that the mosses and liverworts form a monophyletic group which is sister to the tracheophytes. Previous molecular, morphological, or combined data have suggested various relationships for land plants (Mishler and Churchill 1984; Chapman and Buchheim 1991, 1992; Manhart 1994; Hedderson, Chapman, and Rootes 1996; Lewis, Mishler, and Vilgalys 1997; Duff and Nickrent 1999). The relationships inferred from rbcL data are not supported with high probability (Manhart 1994) and should be omitted from discussion. Although morphological data and combined rRNA and morphological data (Mishler and Churchill 1984; Mishler et al. 1994) have suggested a sister relationship of mosses to tracheophytes, the nuclear- encoded rRNA data and the present combined plastid gene data did not support the inference. The characters supporting the moss- plus-tracheophyte clade were ‘‘xylem,’’ ‘‘phloem,’’ ‘‘aerial sporophyte axis,’’ and ‘‘perine layers on spores.’’ Although conducting tissues in mosses (hydroid and leptoid) have been thought to be comparable with xylem and phloem of vascular plants (Hébant 1977), hydroids lack secondary wall thickenings and lignin, which are characteristics of tracheids. Waterconducting tissue and food-conducting tissue present in liverworts were not thought to be comparable with xylem and phloem of vascular plants (Hébant 1977). Our result suggests that morphological characters should be 1033 reevaluated, especially the homology of conducting tissue. The basal placement of hornworts inferred by rRNA (Hedderson, Chapman, and Rootes 1996) and cox3 cDNA nucleotide sequences (Malek et al. 1996) was supported by this analysis, indicating that the relationship may be robust. It has been pointed out that hornworts and Coleochaete have similarly large pyrenoids and the same number (one per cell) of plastids (Graham 1993). Pyrenoids are shared by green algae in general. The monophyletic relationships of mosses (P. patens, S. fallax) and liverworts (H. mnioides, M. polymorpha) are congruent to the cladistic results based on morphological characters (Garbary, Renzaglia, and Duckett 1993; Mishler et al. 1994). The ML analysis of the mitochondrial gene cox3 (Malek et al. 1996), the nuclear rRNA analysis by Hedderson, Chapman, and Rootes (1996), and the mitochondrial rRNA (Duff and Nickrent 1999) also supported the monophyly of mosses. On the other hand, this result conflicts with the results obtained by Bopp and Capesius (1996) and those obtained by Chapman and Buchheim (1992) based on rRNA sequences. In Bopp and Capesius’s tree, Sphagnum and other species of Musci (including P. patens) were paraphyletic. In Chapman and Buchheim’s tree, thallose liverworts solely were sister to tracheophytes, and liverworts were not monophyletic. In the reanalysis of 18S rRNA data with taxa comparable to those used in this study, Musci were paraphyletic, but the relationship is not statistically significant (fig. 3). The inconsistent results obtained from 18S rRNA may be due to the large variance of the estimated likelihood, or, in other words, too little information, which is implied by the small contribution of 18S rRNA to the difference of the sums of the log-likelihoods in this analysis (table 6). Furthermore, 18S rRNA has a limited size of about 1,800 bp, and there is always ambiguity in its alignment when divergent taxa are analyzed. It is important to use the sequence that can be aligned without ambiguity. It should be also noted that rRNA suffers direct constraint of secondary structure, which will violate the basic assumption of independence and may interfere with phylogenetic analysis. In this study, we inferred the basal relationship of land plants using plastid genes and 18S rRNA gene data with high bootstrap support, and previous studies also support the relationship as discussed above. Therefore, it should be concluded that hornworts are basal and that mosses and liverworts each are monophyletic and sister to each other. Since it is possible to determine longer sequences, plastid coded proteins are suitable for further phylogenetic analysis of unresolved problems. As shown in this study, psaA, psaB, psbD, rpoC2, and rbcL are good sources of phylogenetic information at a deep level. Other suitable genes are psbC, psbA, psbB, atpB, and atpA, as suggested by Olmstead and Palmer (1994). There are a number of genes coding ribosomal proteins that are not listed by Olmstead and Palmer (1994) due 1034 Nishiyama and Kato Table 6 Total Likelihood Analysis of the Plastid Genes and the Nuclear 18S rRNA Gene PLASTID (1,521 sites) TREEa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... 18SRRNA (1,665 sites) TOTAL (3,186 sites) Likelihoodb SEc RELL-BPd Likelihood SE RELL-BP Likelihood SE RELL-BP 210,972.8 3.8 5.3 10.0 11.3 12.5 13.9 14.5 14.8 17.3 17.3 17.6 17.9 18.0 19.7 ML 4.9 4.3 9.8 11.6 12.8 9.9 12.7 8.6 11.4 10.6 11.6 11.2 8.8 11.5 0.5902 0.1353 0.0164 0.0753 0.0712 0.0614 0.0152 0.0248 0.0006 0.0029 0.0014 0.0001 0.0041 0.0001 0.0010 26,048.6 2.2 3.0 0.2 4.5 1.0 8.3 3.3 2.7 6.1 6.3 2.7 7.0 6.1 7.8 ML 4.9 4.4 7.5 7.7 9.6 7.4 8.9 6.4 8.2 6.9 9.3 7.9 7.9 8.0 0.2056 0.0873 0.0589 0.1739 0.0110 0.1682 0.0001 0.0842 0.0831 0.0209 0.0006 0.0776 0.0053 0.0212 0.0021 217,021.4 6.0 8.2 10.2 15.7 13.5 22.2 17.8 17.5 23.4 23.6 20.2 25.0 24.1 27.4 ML 6.9 6.2 12.4 14.0 16.0 12.4 15.5 10.7 14.0 12.6 14.8 13.7 11.9 14.0 0.5680 0.1144 0.0218 0.1169 0.0322 0.0991 0.0016 0.0290 0.0050 0.0053 0.0005 0.0023 0.0024 0.0009 0.0006 a The tree numbers are identical to those in table 1. Log-likelihood of the maximum-likelihood (ML) tree or the difference of the log-likelihood from that of the ML tree. c Standard error of the difference of log-likelihood. d Relative bootstrap probability estimated by the RELL method. b to their short lengths (,1,000 bp). However, because it is possible to use combined data from multiple genes, genes of short lengths will be a possible source of phylogenetic information. Acknowledgments We thank Dr. Noriaki Murakami for his useful comments and advice during the experiment, Dr. Mitsuyasu Hasebe for his valuable advice on writing the manuscript and for providing the plastid DNA and clone bank of A. capillus-veneris, Prof. Masahiro Sugiura for providing the corrected sequence of tobacco, Dr. Masayuki Takamiya for providing the A. punctatus cultures, Prof. Hans Rudolph for providing the S. fallax culture, and Mr. Kenjiro Fujiwara for a strain of P. patens. T.N. is a research fellow of the Japan Society for the Promotion of Science. 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