Download Molecular Phylogenetic Analysis Among Bryophytes and

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. This study was partly supported by
a Grant-in-Aid (08404055) for Scientific Research from
the Ministry of Education, Science, Culture and Sports
of Japan.
LITERATURE CITED
ADACHI, J., and M. HASEGAWA. 1996. MOLPHY version 2.3:
programs for molecular phylogenetics based on maximum
likelihood. Comput. Sci. Monogr. 28:1–150.
ASHTON, N. W., and D. J. COVE. 1977. The isolation and preliminary characterization of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens. Mol.
Gen. Genet. 154:87–95.
BOPP, M., and I. CAPESIUS. 1995. New aspects of the systematics of bryophytes. Naturwissenschaften 82:193–194.
. 1996. New aspects of bryophyte taxonomy provided
by a molecular approach. Bot. Acta 109:368–372.
BREMER, K., C. J. HUMPHRIES, B. D. MISHLER, and S. P. CHURCHILL. 1987. On cladistic relationships in green plants. Taxon 36:339–349.
CAPESIUS, I. 1995. A molecular phylogeny of bryophytes based
on the nuclear encoded 18S rRNA genes. J. Plant Physiol.
146:59–63.
CHAPMAN, R. L., and M. A. BUCHHEIM. 1991. Ribosomal RNA
gene sequences: analysis and significance in the phylogeny
and taxonomy of green algae. Crit. Rev. Plant Sci. 10:343–
368.
. 1992. Green algae and the evolution of land plants:
inferences from nuclear-encoded rRNA gene sequences.
BioSystems 28:127–137.
CHAUDHURI, S., H. CARRER, and P. MALIGA. 1995. Site-specific
factor involved in the editing of the psbL mRNA in tobacco
plastids. EMBO J. 14:2951–2957.
CLEGG, M. T. 1993. Chloroplast gene sequences and the study
of plant evolution. Proc. Natl. Acad. Sci. USA 90:363–367.
CLEGG, M. T., and G. ZURAWSKI. 1992. Chloroplast DNA and
the study of plant phylogeny: present status and future prospects. Pp. 1–13 in P. S. SOLTIS, D. E. SOLTIS, and J. J.
DOYLE, eds. Molecular systematics of plants. Chapman and
Hall, New York.
CUMMINGS, M. P., S. P. OTTO, and J. WAKELEY. 1995. Sampling properties of DNA sequence data in phylogenetic
analysis. Mol. Biol. Evol. 12:814–822.
DUFF, R. J., and D. L. NICKRENT. 1999. Phylogenetic relationships of landplants using mitochondrial small-subunit
rDNA sequences. Am. J. Bot. 86:372–386.
FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39:783–791.
. 1995. PHYLIP (phylogeny inference package). Version 3.572c. Distributed by the author, Department of Genetics, University of Washington, Seattle.
GARBARY, D. J., K. S. RENZAGLIA, and J. G. DUCKETT. 1993.
The phylogeny of land plants: a cladistic analysis based on
male gametogenesis. Plant Syst. Evol. 188:237–269.
GRAHAM, L. E. 1993. Origin of land plants. Wiley, New York.
GRANTHAM, R., C. GAUTIER, and M. GOUY. 1980. Codon frequencies in 119 individual genes confirm consistent choices
of degenerate bases according to genome type. Nucleic Acids Res. 8:1893–1912.
GU, X., and W.-H. LI. 1996. Bias-corrected paralinear and
LogDet distances and tests of molecular clocks and phylogenies under nonstationary nucleotide frequencies. Mol.
Biol. Evol. 13:1375–1383.
HASEBE, M., and K. IWATSUKI. 1990. Chloroplast DNA from
Adiantum capillus-veneris L., a fern species (Adiantaceae);
Land Plant Phylogeny
clone bank, physical map and unusual gene localization in
comparison with angiosperm chloroplast DNA. Curr. Genet.
17:359–364.
HASEGAWA, M., and H. KISHINO. 1994. Accuracies of the simple methods for estimating the bootstrap probability of a
maximum-likelihood tree. Mol. Biol. Evol. 11:142–145.
HASEGAWA, M., T. YANO, and H. KISHINO. 1984. A new molecular clock of mitochondrial DNA and the evolution of
hominoids. Proc. Jpn. Acad. B60:95–98.
HÉBANT, C. 1977. The conducting tissues of bryophytes. J.
Cramer, Vaduz, Switzerland.
HEDDERSON, T. A., R. L. CHAPMAN, and W. L. ROOTES. 1996.
Phylogenetic relationships of bryophytes inferred from nuclear-encoded rRNA gene sequences. Plant Syst. Evol. 200:
213–224.
HILLIS, D. M., J. P. HUELSENBECK, and C. W. CUNNINGHAM.
1994. Application and accuracy of molecular phylogenies.
Science 264:671–667.
HIRATSUKA, J., H. SHIMADA, R. WHITTIER et al. (16 co-authors). 1989. The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid
DNA inversion during the evolution of the cereals. Mol.
Gen. Genet. 217:185–194.
INOUE, H., H. NOJIMA, and H. OKAYAMA. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene
96:23–28.
JONES, D. T., W. R. TAYLOR, and J. M. THORNTON. 1992. The
rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8:275–282.
KIMURA, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies
of nucleotide sequences. J. Mol. Evol. 16:111–120.
KISHINO, H., and M. HASEGAWA. 1990. Converting distance to
time: application to human evolution. Methods Enzymol.
183:550–570.
KISHINO, H., T. MIYATA, and M. HASEGAWA. 1990. Maximum
likelihood inference of protein phylogeny and the origin of
chloroplasts. J. Mol. Evol. 30:151–160.
LAKE, J. A., 1994. Reconstructing evolutionary trees from
DNA and protein sequences: paralinear distances. Proc.
Natl. Acad. Sci. USA 91:1455–1459.
LEWIS, L. A., B. D. MISHLER, and R. VILGALYS. 1997. Phylogenetic relationships of the liverworts (Hepaticae), a basal
embryophyte lineage, inferred from nucleotide sequence
data of the chloroplast gene rbcL. Mol. Phylogenet. Evol.
7:377–393.
LOCKHART, P. J., M. A. STEEL, M. D. HENDY, and D. PENNY.
1994. Recovering evolutionary trees under a more realistic
model of sequence evolution. Mol. Biol. Evol. 11:605–612.
MAIER, R. M., K. NECKERMANN, G. L. IGLOI, and H. KÖSSEL.
1995. Complete sequence of the maize chloroplast genome:
gene content, hotspots of divergence and fine tuning of genetic information by transcript editing. J. Mol. Biol. 251:
614–628.
MALEK, O., K. LÄTTIG, R. HIESEL, A. BRENNICKE, and V.
KNOOP. 1996. RNA editing in bryophytes and a molecular
phylogeny of land plants. EMBO J. 15:1403–1411.
MANHART, J. R. 1994. Phylogenetic analysis of green plant
rbcL sequences. Mol. Phylogenet. Evol. 3:114–127.
MISHLER, B. D., and S. P. CHURCHILL. 1984. A cladistic approach to the phylogeny of the ‘‘bryophytes.’’ Brittonia 36:
406–424.
MISHLER, B. D., L. A. LEWIS, M. A. BUCHHEIM, K. S. RENZAGLIA, D. J. GARBARY, C. F. DELWICHE, F. W. ZECHMAN,
T. S. KANTZ, and R. L. CHAPMAN. 1994. Phylogenetic re-
1035
lationships of the ‘‘green algae’’ and ‘‘bryophytes.’’ Ann.
Mo. Bot. Gard. 81:451–483.
MIYATA, T., and H. HAYASHIDA. 1981. Extraordinarily high
evolutionary rate of pseudogenes: evidence for the presence
of selective pressure against changes between synonymous
codons. Proc. Natl. Acad. Sci. USA 78:5739–5743.
MURRAY, M. G., and W. F. THOMPSON. 1980. Rapid isolation
of high molecular weight plant DNA. Nucleic Acids Res.
8:4321–4325.
OHYAMA, K. 1996. Chloroplast and mitochondrial genomes
from a liverwort, Marchantia polymorpha—gene organization and molecular evolution. Biosci. Biotech. Biochem.
60:16–24.
OHYAMA, K., H. FUKUZAWA, T. KOHCHI et al. (13 co-authors).
1986. Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322:572–574.
OLMSTEAD, R. G., and J. D. PALMER. 1994. Chloroplast DNA
systematics: a review of methods and data analysis. Am. J.
Bot. 81:1205–1224.
ONO, K., Y. MURASAKI, and M. TAKAMIYA. 1988. Induction
and morphogenesis of cultured cells of bryophytes. J. Hattori Bot. Lab. 65:391–401.
QIU, Y., Y. CHO, J. C. COX, and J. D. PALMER. 1998. The gain
of three mitochondrial introns identifies liverworts as the
earliest land plants. Nature 394:671–674.
PENNY, D., P. J. LOCKHART, M. A. STEEL, and M. D. HENDY.
1994. The role of models in reconstructing evolutionary
trees. Pp. 211–230 in R. W. SCOTLAND, D. J. SIEBERT, and
D. M. WILLIAMS, eds. Models in phylogeny reconstruction.
No. 52 in Systematics Association Special Volume. Oxford,
New York.
SAITOU, N., and M. NEI. 1987. The neighbor-joining method:
a new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4:406–425.
SAMBROOK, J., E. F. FRITSCH, and T. MANIATIS. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.
SCHUSTER, R. M. 1984. Evolution, phylogeny and classification of the Hepaticae. Pp. 892–1092 in R. M. SCHUSTER,
ed. New manual of bryology. Vol. 2. The Hattori Botanical
Laboratory, Nichinan, Japan.
SHIMADA, H., M. FUKUTA, M. ISHIKAWA, and M. SUGIURA.
1990. Rice chloroplast RNA polymerase genes: the absence
of an intron in rpoC1 and the presence of an extra sequence
in rpoC2. Mol. Gen. Genet. 221:395–402.
SHINOZAKI, K., M. OHME, M. TANAKA et al. (23 co-authors).
1986. The complete nucleotide sequence of the tobacco
chloroplast genome: its gene order and expression. EMBO
J. 5:2043–2049.
STARR, R. C., and J. A. ZEIKUS. 1993. UTEX—the culture
collection of algae at the University of Texas at Austin 1993
list of cultures. J. Phycol. 29(Suppl.):1–106.
STEEL, M. 1994. Recovering a tree from the leaf colourations
it generates under a Markov model. Appl. Math. Lett. 7:
19–23.
THOMPSON, J. D., D. G. HIGGINS, and T. J. GIBSON. 1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.
VITT, D. H. 1984. Classification of the Bryopsida. Pp. 696–
759 in R. M. SCHUSTER, ed. New manual of bryology. Vol.
2. The Hattori Botanical Laboratory, Nichinan, Japan.
WAKASUGI, T., T. HIROSE, M. HIROHATA, T. TSUDZUKI, and H.
KÖSSEL. 1996. Creation of a novel protein-coding region at
the RNA level in black pine chloroplasts: the pattern of
1036
Nishiyama and Kato
RNA editing in the gymnosperm chloroplast is different
from that in angiosperms. Proc. Natl. Acad. Sci. USA 93:
8766–8770.
WAKASUGI, T., T. NAGAI, M. KAPOOR et al. (15 co-authors).
1997. Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: the existence
of genes possibly involved in chloroplast division. Proc.
Natl. Acad. Sci. USA 94:5967–5972.
WAKASUGI, T., J. TSUDZUKI, S. ITO, K. NAKASHIMA, T. TSUDZUKI, and M. SUGIURA. 1994. Loss of all ndh genes as determined by sequencing the entire chloroplast genome of
the black pine Pinus thunbergii. Proc. Natl. Acad. Sci. USA
91:9794–9798.
WOLFE, K. H., W.-H. LI, and P. M. SHARP. 1987. Rates of
nucleotide substitution vary greatly among plant mitochon-
drial, chloroplast, and nuclear DNAs. Proc. Natl. Acad. Sci.
USA 84:9054–9058.
WOLFE, K. H., P. M. SHARP, and W.-H. LI. 1989. Rates of
synonymous substitution in plant nuclear genes. J. Mol.
Evol. 29:208–211.
YOSHINAGA, K., H. IINUMA, T. MASUZAWA, and K. UEDA.
1996. Extensive RNA editing of U to C in addition to C to
U substitution in the rbcL transcripts of hornwort chloroplasts and the origin of RNA editing in green plants. Nucleic Acids Res. 24:1008–1014.
MASAMI HASEGAWA, reviewing editor
Accepted April 22, 1999