* National Institute for Basic Biology, Okazaki, Japan; Department of Biology, Utah State University;
Faculty of Science, Shizuoka University, Shizuoka, Japan;
The Jackson Laboratory, Bar Harbour, Maine; || Center for Gene Research, Nagoya University, Nagoya, Japan; ¶ Department of Biology, Faculty of Science, Toyama University, Toyama, Japan; # Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan; ** Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan; and
Department of Molecular Biomechanics, Sokendai, Okazaki, Japan
Correspondence: E-mail: mhasebe{at}nibb.ac.jp.
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Abstract |
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Key Words: land plants bryophytes codon usage nucleotide composition LogDet
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Introduction |
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The oldest fossil records of land plants are spore tetrads from the mid-Ordovician (Gray 1993) that are thought to be sporopollenin-bearing meiospores and are suggested to be similar to spores of liverworts because of lamellae in the spore walls. Fossil plant fragments containing similar spores, recently found in Ordovician deposits from Oman, appear to be from embryophytes (Wellman, Osterloff, and Mohiuddin 2003). However, the affinity of these spores to extant liverworts is uncertain because no associated megafossils have been found, and similar tetrad spores are also produced by mosses (Gray 1993). A cladistic analysis of several key morphological characters supports the basal branching of liverworts (Mishler and Churchill 1984), whereas other cladistic analyses of ultrastructural, biochemical, and developmental characters (Garbary and Renzaglia 1998; Renzaglia et al. 2000) support a basal position of hornworts rather than liverworts. Data from antheridial development and the complex process of spermatogenesis (Garbary, Renzaglia, and Duckett 1993; Renzaglia et al. 2000; Renzaglia and Garbary 2001) support a monophyly of extant bryophytes (mosses, liverworts, and hornworts). Results using molecular data are also controversial. Analyses using DNA sequences of multiple genes suggest a basal position for hornworts (Nishiyama and Kato 1999), although the branching patterns are only weakly supported. Absence of group II introns in three mitochondrial genes in both liverworts and green algae supports the liverwort basal hypothesis (Qiu et al. 1998), although repeated intron losses have been observed in land plant lineages (Qiu et al. 1998) and the possibility of parallel evolution cannot be excluded. The consensus of these conflicting results yields an unresolved polytomy at the base of land plants. Determining these relationships would not only resolve a critical area of the land plant phylogeny, but it would also be important for understanding the characteristics of the earliest land plants and their subsequent evolution.
Combined analyses of DNA sequences from multiple loci have proven useful in inferring deep phylogenetic relationships (Qiu et al. 1999; Pryer et al. 2001), as predicted by simulation studies (Cummings, Otto, and Wakeley 1995). With the accumulation of genome information, the entire chloroplast genome sequences of 15 seed plants, one liverwort (Marchantia polymorpha), four green algae, three red algae, and one cryptophyte have been reported in the GenBank RefSeq division. Because these published data do not cover all major lineages of land plants, we determined the entire chloroplast genome sequences of two pteridophytes, Psilotum nudum (accession number: NC_003386) and Adiantum capillus-veneris (accession number: AY178864; Wolf et al. 2003), a hornwort Anthoceros angustus (accession number: AB086179; Kugita et al. 2003), and a moss, Physcomitrella patens (accession number: AP005672; Sugiura et al. 2003). We analyzed 51 genes that are found in every chloroplast genome of the sampled land plants and chlorophytes (26,937 bp / 8,979 amino acid sites in total), to infer the phylogeny of land plants and to examine the utility of the amino acid versus nucleotide sequence analyses.
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Materials and Methods |
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Results |
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Analysis of Deduced Amino Acid Sequence Data
A local rearrangement search starting with the NJ tree (Saitou and Nei 1987) under the JTT-F model (Jones, Taylor, and Thornton 1992; Adachi and Hasegawa 1996) found an ML tree (ln likelihood = 89478.09; fig. 1). The three bryophytes, Marchantia polymorpha, Physcomitrella patens, and Anthoceros angustus, formed a monophyletic group with high local bootstrap probability (99%). Previously well-recognized monophyletic groups, the eudicots, monocots, angiosperms, seed plants, and land plants (reviewed in Judd et al. 2002, pp. 153183), each formed a monophyletic group with high local bootstrap probability. Vascular plants formed a monophyletic group, though the bootstrap value was low (55%). This contrasts previous studies with high bootstrap support for vascular plants (Hedderson, Chapman, and Cox 1998; Nickrent et al. 2000). To test whether other possible trees have a significantly less likelihood than the ML tree, the bootstrap test and AU test (Shimodaira 2002) were used for all 105 possible topologies among seed plants, monilophytes, hornworts, liverworts, mosses, and outgroups. We constrained the topology within seed plants and within outgroup taxa. In the bootstrap test, topology 1 (fig. 2), which had the same topology as the ML tree, had 52.8% bootstrap support. Topology 2 (fig 2), which had the second highest likelihood (ln = 89480.91), occupied 44.3% of the bootstrap replicates. Bryophytes were also monophyletic in topology 2, but the bryophyte clade was nested within vascular plants, and monilophytes were basal among land plants. Other topologies were significantly less likely. The AU test showed that all topologies except topology 2 were significantly less likely than topology 1 at the 5% level (table 2; table S1 in the online Supplementary Material).
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Analysis of Nucleotide Sequence Data
We found a single most parsimonious tree (14,487 steps) from a TBR search based on nucleotide sequence data omitting third codon positions. The liverwort Marchantia polymorpha was resolved as the earliest diverging lineage in land plants, but bootstrap support for the phylogenetic relationships among bryophytes was low (fig. 3). When all the leucine codons and third codon positions were excluded, Marchantia polymorpha and Physcomitrella patens formed a monophyletic group (fig. 4). Phylogenetic relationships between this moss + liverwort clade and other taxa did not have high bootstrap support.
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Discussion |
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Phylogenetic analyses using DNA sequences suggest that extant bryophytes are paraphyletic (Lewis, Mishler, and Vilgalys 1997; Duff and Nickrent 1999; Nishiyama and Kato 1999; Nickrent et al. 2000). However, only the MP-3Ti analysis by Nickrent et al. (2000) provided evidence of strong statistical support. Moreover, monophyly of bryophytes was not rejected in the Kishino-Hasegawa (1989) test by Nickrent et al. (2000). The sister relationship of mosses and liverworts received relatively strong support in recent analyses using multiple genes (Nishiyama and Kato 1999; Nickrent et al. 2000). Marchantia appears on a basally diverging branch in land plants with high bootstrap probability in a combined analysis of atpB, rbcL, nad5, and the small subunit rRNA gene (Karol et al. 2001). However, that study used DNA sequences of chloroplast genes without attempting to compensate for the nucleotide composition bias, which means that the bootstrap probabilities may not be a good estimator of the reliability of branches and tree topologies (Lockhart et al. 1994; Swofford et al. 2001; Felsenstein 2003, pp. 272274). Therefore, the extant bryophyte monophyletic hypothesis inferred in this study stands as the best one statistically supported by molecular sequence data.
Phylogenetic studies of land plant relationships using morphological characters are also incongruent. Most studies suggest that extant bryophytes are paraphyletic (Mishler and Churchill 1984, 1985; Renzaglia et al. 2000), whereas monophyly of extant bryophytes and a sister relationship of mosses and liverworts were inferred using characters of spermatogenesis (Garbary, Renzaglia, and Duckett 1993; Renzaglia et al. 2000; Renzaglia and Garbary 2001). Analyses of spermatogenesis characters also supported monilophytes involving ferns, psilotophytes (whisk ferns), and equisetophytes (horsetails), which are supported by DNA sequence data (Nickrent et al. 2000; Pryer et al. 2001). If our inference of monophyletic bryophytes reflects the true relationships, this would suggest that spermatogenesis characters have retained more phylogenetic signal than other morphological characters.
While our hypothesis was supported with high statistical confidence with presently available data, the bryophyte monophyly hypothesis should be tested with more data and further analysis. Current analyses include only one sample per group from mosses, liverworts, and hornworts and lack a major lineage of land plants, lycophytes. Some major lineages of bryophytes and lycophytes should be included based on previous phylogenetic analyses (Lewis, Mishler, and Vilgalys 1997; Garbary and Renzaglia 1998; Hedderson, Chapman, and Cox 1998; Nickrent et al. 2000; Renzaglia et al. 2000). Sphagnum, Takakia, Andreaea, and Andraeobryum from mosses and Treubia, Haplomitrium, Blasia, one simple thalloid, and one leafy liverwort from liverworts are candidate representatives. In addition to one homosporous and one heterosporous lycophyte, equisetophytes should be targeted for vascular plants. Furthermore, data from Charales may be a better outgroup to land plants than Coleochaetales (Turmel et al. 2002). Mitochondrial and nuclear genome data are also candidate sources of phylogenetic information that could provide an independent test of results based on chloroplast genome data.
The fossil record of plants older than rhyniophytes includes cuticles (Edwards, Duckett, and Richardson 1995) and spore tetrads (Gray 1993). These fossils have been considered to be related to extant liverworts (Edwards, Duckett, and Richardson 1995; Taylor 1995). Our results are not in line with this interpretation because we infer that liverworts do not descend from a basal node in bryophytes (fig. 2). Our results, however, are consistent with a "bryophyte-like ancestor" hypothesis (Graham and Gray 2001), whereby the above fossils represent a common ancestor of the three extant groups of bryophytes.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Peter Lockhart, Associate Editor
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References |
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