RESEARCH ARTICLES
Chloroplast Phylogeny Indicates that Bryophytes Are Monophyletic
Tomoaki Nishiyama,* Paul G. Wolf, Masanori Kugita,à1 Robert B. Sinclair,§1
Mamoru Sugita,k1 Chika Sugiura,k1 Tatsuya Wakasugi,{1 Kyoji Yamada,{1
Koichi Yoshinaga,à1 Kazuo Yamaguchi,# Kunihiko Ueda,** and Mitsuyasu Hasebe* *National Institute for Basic Biology, Okazaki, Japan; Department of Biology, Utah State University;
àFaculty of Science, Shizuoka University, Shizuoka, Japan; §The Jackson Laboratory, Bar Harbour, Maine;
kCenter 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
Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical
support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome
sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that
extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that
extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not
strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte
monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other
data sets. We propose bryophyte monophyly as the currently best hypothesis.
Introduction
More than 300,000 green plant species now cover the
Earth’s surface, and these plants are descended from a
common ancestor that lived during the Ordovician, more
than 470 MYA (Kenrick and Crane 1997a, 1997b; Kenrick
2000). Recent morphological and molecular phylogenetic
studies have revealed that the closest relative of land plants
is a group of freshwater green algae (reviewed in Graham
and Wilcox [2000] and Chapman and Waters [2002]).
However, the earliest branches of the land plant phylogeny
remain unclear.
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
1
These authors contributed equally to this work.
Key words: land plants, bryophytes, codon usage, nucleotide
composition, LogDet.
E-mail: [email protected].
Mol. Biol. Evol. 21(10):1813–1819. 2004
doi:10.1093/molbev/msh203
Advance Access publication July 7, 2004
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.
Molecular Biology and Evolution vol. 21 no. 10 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
1814 Nishiyama et al.
Table 1
Accession Numbers and Codon Usage for Leucine in Chloroplast Genomes
Organism
Accession No.
UUA
UUG
CUU
CUC
CUA
CUG
%Ca
Calycanthus floridus var. glaucus
Atropa beladona
Lotus cornicularis var. japonicus
Nicotiana tabacum
Spinacia oleracea
Triticum aestivum
Zea mays
Oryza sativa
Arabidopsis thaliana
Pinus thunbergii
Pinus koraiensis
Adiantum capillus-veneris
Psilotum nudum
Marchantia polymorpha
Anthoceros angustus (cDNA)
Anthoceros angustus (genomic)
Physcomitrella patens
Chaetosphaeridium globosum
Chlorella vulgaris
Nephroselmis olivacea
Mesostigma viride
NC_004993.1
NC_004561.1
NC_002694.1
NC_001879.1
NC_002202.1
NC_002762.1
NC_001666.2
NC_001320.1
NC_000932.1
NC_001631.1
NC_004677.1
NC_004766.1
NC_003386.1
NC_001319.1
NC_004543.1
NC_004543.1
NC_005087.1
NC_004115.1
NC_001865.1
NC_000927.1
NC_002186.1
308
335
342
332
353
349
350
348
373
275
276
233
336
636
426
380
625
613
536
237
602
194
188
194
188
171
179
179
178
169
191
192
233
181
54
151
139
51
82
29
197
61
176
180
187
183
192
190
190
188
193
176
181
128
186
205
174
169
201
165
285
282
153
60
56
46
56
40
50
49
52
43
62
61
86
37
5
38
38
4
4
39
98
6
128
124
115
123
136
127
125
121
118
152
148
146
148
51
129
125
69
66
55
87
81
59
61
51
63
49
47
53
56
53
75
70
74
50
8
36
35
16
9
25
57
14
46%
45%
43%
45%
44%
44%
44%
44%
43%
50%
50%
48%
45%
28%
40%
41%
30%
26%
42%
55%
28%
a
Proportion of C in the first codon position of leucine.
Materials and Methods
Coding sequences were extracted from the annotated
sequences using bioruby (http://bioruby.org/). Chloroplast
genome sequences were determined for a moss (Physcomitrella patens), a hornwort (Anthoceros angustus), and
two pteridophytes (Adiantum capillus-veneris and Psilotum nudum). In addition to these four taxa, 16 green plant
chloroplast DNA genome sequences in the DNA database
(table 1) were used for phylogenetic analyses. The
nucleotide sequences of 51 genes that are present in all
the chloroplast genomes of green plants were individually
aligned using the program fftnsi in MAFFT (Katoh et al.
2002) version 3.85. The 51 genes are atpA, atpB, atpE,
atpF, atpH, atpI, petA, petB, petD, petG, psaA, psaB,
psaC, psaI, psaJ, psbA, psbB, psbC, psbD, psbE, psbF,
psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ,
rbcL, rpl2, rpl14, rpl16, rpl20, rpl36, rpoB, rpoC1, rpoC2,
rps2, rps3, rps4, rps7, rps8, rps11, rps12, rps14, rps18,
rps19, ycf3, and ycf4. Unambiguously aligned regions
were selected for further analyses and concatenated. The
overlapping region of psbD and psbC was excluded. The
data matrix is available online as Supplementary Material.
The selected regions included 26,937 nucleotide sites.
Because RNA editing is abundant in Anthoceros angustus
(Kugita et al. 2003), we used cDNA sequence of A.
angustus. The amino acid matrix comprised 20 taxa and
8,979 amino acid sites. Pairwise maximum-likelihood
(ML) distances were calculated using ProtML (Adachi and
Hasegawa 1996) and a Neighbor-Joining (NJ) tree was
obtained with Njdist (Adachi and Hasegawa 1996). A local
rearrangement search was performed with the NJ tree as
the starting tree. Local bootstrap probabilities were
calculated with resampling using the estimated log-likelihood method (Adachi and Hasegawa 1996). A user tree
was prepared for each of the 105 possible bifurcating
topologies among six groups of taxa: outgroups (O),
mosses (M), liverworts (L), hornworts (H), monilophytes
(F), and seed plants (S). The monilophytes include ferns,
psilotophytes (whisk ferns), and equisetophytes (horsetails) (Kenrick and Crane 1997b, p. 228; Pryer et al. 2001;
Judd et al. 2002, pp. 162–168), and in our study they are
represented by Adiantum capillus-veneris and Psilotum
nudum. A. capillus-veneris and P. nudum are forced to
form a clade, and the topologies within seed plants and
within the outgroup were constrained as the ML tree found
in the local rearrangement search (fig. 1). The estimated
log-likelihood under the JTT model (Jones, Taylor, and
Thornton 1992), adjusted with the empirical amino acid
frequency (JTT-F model) of each site under the 105
topologies, was calculated using ProtML (Adachi and
Hasegawa 1996). The log-likelihood under the JTT model
(Jones, Taylor, and Thornton 1992) was calculated for
each gene to take account of rate differences among genes.
The likelihood under a discrete gamma model of rate
heterogeneity among sites (Yang 1994) was calculated
with PAML version 3.13d (Yang 1997). The P values for
the bootstrap and approximately unbiased (AU) test
(Shimodaira 2002) were calculated using the estimated
log-likelihood with CONSEL (Shimodaira and Hasegawa
2001). Maximum parsimony analyses were performed
with PAUP* version 4.0b10 (Swofford 1998). The most
parsimonious trees were recovered with a heuristic search
strategy employing tree bisection and reconnection (TBR)
with 1,000 random addition sequence replications. Bootstrap tests were performed with 1,000 replications with 10
replications of random addition sequence in each run.
Analyses to overcome compositional heterogeneity with
paralinear/LogDet distances (Lake 1994; Lockhart et al.
1994) followed Nishiyama and Kato (1999).
Results
Heterogenetity in base composition at all codon positions is recognized as a problem for phylogenetic analyses
of chloroplast genes (Steel, Lockhart, and Penny, 1993;
Chloroplast Phylogeny Indicates Monophyletic Bryophytes 1815
Nicotiana tabacum
Atropa belladonna
Spinacia oleracea
Triticum aestivum
Oryza sativa
Land plants
Zea mays
Vascular plants
Calycanthus floridus var. glaucus
Seed plants
Angiosperms
Lotus corniculatus var. japonicus
Arabidopsis thaliana
Pinus koraiensis
Adiantum capillus-veneris
Psilotum nudum
Marchantia polymorpha
Liverworts
Physcomitrella patens
Mosses
Anthoceros angustus (cDNA)
Hornworts
Bryophytes
Monilophytes
Pinus thunbergii
Outgroups
Chaetosphaeridium globosum
Nephroselmis olivacea
Chlorella vulgaris
Mesostigma viride
0.1 substitutions/site
FIG. 1.—Phylogenetic relationship of representative lineages in land plants inferred from a maximum-likelihood analysis using 8,979 amino acid
residues for 51 genes from 20 chloroplast genomes. Local bootstrap probabilities are shown on the branches. The horizontal branch lengths are
proportional to the estimated number of amino acid substitutions per site. This tree is rooted using all the taxa other than embryophytic land plants.
Higher taxonomic categories and common names are indicated.
Nishiyama and Kato 1999). We observed such compositional differences across entire chloroplast genomes; GC
composition ranged from 12.9% in the third codon of
Marchantia polymorpha to 38.3% in Adiantum capillusveneris. We also found differences in codon usage for the
first codon positions of leucine (table 1). Fifty-five percent
of the leucine codons start with U in Nephroselmis
olivacea compared to 26% in Chaetosphaeridium globosum. Most conventional methods of phylogenetic analysis
are statistically consistent (i.e., reach the true topology
with infinite data) only when the sequences evolved under
a stationary Markov process (Gu and Li 1996). The large
differences in the base composition indicate that the
evolutionary process is not stationary. Therefore, conventional methods may give positively misleading results with
high bootstrap probabilities (Lockhart et al. 1994). To
overcome this problem, we applied three approaches: (1)
we eliminated codon usage differences by analyzing amino
acids sequences; (2) we removed the sites exhibiting
compositional heterogeneity, i.e., all the leucine codons
and third codon positions; and (3) we used LogDet/
paralinear distances (Lake 1994; Lockhart et al. 1994;
Steel 1994; Gu and Li 1996), which are consistent under
nonstationary processes.
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 ¼ 289478.09; 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. 153–183), 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% boot-
1
S
F
H
L
M
O
2
S
H
L
M
F
O
3
S
H
L
M
F
O
FIG. 2.—The best three alternative topologies found in the analyses
of 105 trees. S, seed plants; F, monilophytes (Adiantum capillus-veneris
plus Psilotum nudum); H, a hornwort (Anthoceros angustus); L,
a liverwort (Marchantia polymorpha); M, a moss (Physcomitrella
patens); O, outgroup taxa.
1816 Nishiyama et al.
Table 2
Statistical Test for the 105 Topologies Under Various
Models of Rate Heterogeneity
JTT-F
a
b
Independent Gene
b
Topology
AU
1
2
3
0.707 0.528
0.532 0.443
0.019 0.005
BP
Spinacia oleracea
Lotus corniculatus var. japonicus
88 Arabidopsis thaliana
100
Atropa belladonna
99
100 Nicotiana tabacum
100
Calycanthus floridus var. glaucus
Zea mays
100
Triticum aestivum
100
67 Oryza sativa
78
100 Pinus koraiensis
Pinus thunbergii
100
65
Psilotum nudum
Adiantum capillus-veneris
56
Anthoceros angustus
Physcomitrella patens
Marchantia polymorpha
Chaetosphaeridium globosum
Nephroselmis olivacea
Chlorella vulgaris
Mesostigma viride
64
Gamma 8 Category
AU
BP
AU
BP
0.981
0.151
0.018
0.847
0.107
0.006
0.843
0.396
0.005
0.645
0.321
0.003
a
Topologies are shown in figure 2.
AU and BP are P values for the approximately unbiased test and bootstrap
probability for the bootstrap test, respectively. Topologies 1 and 2 constitute the
95% confidence set, as they are the two highest-likelihood topologies and the sum
of their bootstrap probabilities exceeds 95%. A topology with a P value less than
0.05 was rejected as the maximum-likelihood tree at a significance level of 5%.
b
100
100
100
500 changes
strap support. Topology 2 (fig 2), which had the second
highest likelihood (ln ¼ –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).
To consider rate heterogeneity among genes and sites,
we performed separate gene analyses, in which each gene is
assumed to have evolved independently (‘‘independent
gene’’ in table 2). This was performed under the JTT model
using ProtML. We also performed a discrete gamma
approximation with eight categories (‘‘gamma 8 category’’
in table 2) using PAML (Yang 1997). The log-likelihood of
every tree was calculated. These analyses also supported
topology 1 as the ML tree (table 2). We also obtained the
same topology when we used the genomic sequence
of Anthoceros angustus to deduce the amino acid sequence
(data not shown), indicating that RNA editing does not cause
serious problems for these phylogenetic analyses. We also
tested 20 data sets, each one omitting one taxon from
the matrix. Although the monophyly of vascular plants
changed when an outgroup taxon or Adiantum capillusveneris was removed, the bryophyte monophyly was robust
against taxon removal (data not shown).
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 1 liverwort
clade and other taxa did not have high bootstrap support.
Because LogDet/paralinear distances assume a uniform rate among sites (Lake 1994; Lockhart et al. 1994;
Steel 1994; Gu and Li 1996), we used only the fourfold
FIG. 3.—The most parsimonious tree using nucleotides of the first
and second codon positions. 4,228 of 17,958 sites were parsimonyinformative. Tree length ¼ 14,487. Consistency index excluding
uninformative characters ¼ 0.52. Retention index ¼ 0.62. Bootstrap
probabilities are indicated on branches. Branch lengths are proportional to
the number of substitutions on the branches by ACCTRAN optimization.
degenerate sites, of which there were 2,695 across taxa. An
NJ tree was calculated using paralinear/LogDet distances
(fig. 5). Again, the bootstrap values for the basal branches
were low.
Discussion
The monophyly of extant bryophytes was inferred by
our analyses using translated amino acid sequence data of
currently available chloroplast genomes. Analyses using
nucleotide sequences did not resolve phylogenetic relationships of the three bryophytes with statistical confidence. Amino acid and nucleotide data each have their
own merits and drawbacks. Amino acid sequences have
frequently been used to infer deep phylogeny because they
avoid problems with saturation of silent substitution and
differential GC content (Russo, Takezaki, and Nei 1996;
Nishiyama and Kato 1999; Simmons, Ochoterena, and
Freudenstein 2002 and references therein). But, Simmons,
Ochoterena, and Freudenstein (2002) criticized the use of
amino acid translation, showing that nucleotide data
outperform deduced amino acid data in a phylogenetic
analysis of angiosperms, which diverged during the last
200 Myr (Judd et al. 2002, pp. 175–176). However,
analyses using nucleotide sequences may not be robust
when nucleotide compositions are apparently different
among taxa, because phylogenetic analyses usually assume a stationary Markov process (Lockhart et al. 1994).
In an attempt to remove characters with different nucleotide compositions, we excluded third codon positions (fig.
3) or the nucleotide sites coding leucine and third codon
positions (fig. 4). We also compensated for differences in
nucleotide composition by LogDet transformation (fig. 5).
Neither the removal of characters nor the LogDet transformation resolved the relationships of extant bryophytes
with high statistical support, whereas resolution with high
statistical support was achieved with amino acid translation. These results might be explained if a uniform rate of
change does not describe evolution of the fourfold degen-
Chloroplast Phylogeny Indicates Monophyletic Bryophytes 1817
Atropa belladonna
Nicotiana tabacum
100
Lotus corniculatus var. japonicus
68
Arabidopsis thaliana
82
Spinacia oleracea
100
Calycanthus floridus var. glaucus
Zea mays
100
100
Triticum aestivum
76 Oryza sativa
Pinus koraiensis
Pinus thunbergii
50
Anthoceros angustus
Marchantia polymorpha
77
Physcomitrella patens
Psilotum nudum
100
Adiantum capillus-veneris
Chaetosphaeridium globosum
Nephroselmis olivacea
Chlorella vulgaris
Mesostigma viride
100
100
100
100
100 changes
FIG. 4.—The most parsimonious tree excluding the nucleotide sites coding leucine and third codon positions. 2,731 of 14,860 sites were
parsimony-informative. Tree length ¼ 8,998. Consistency index excluding uninformative characters ¼ 0.63. Retention index ¼ 0.65. Bootstrap
probabilities are indicated on branches. Branch lengths are proportional to the number of substitutions on the branches by ACCTRAN optimization.
erate sites. These results may also suggest that nucleotide
composition bias can increase in proportion to divergence
time among taxa. Nucleotide sequences including the third
codon position data are more likely to be useful for
inferring phylogeny among taxa that diverged relatively
recently, such as angiosperms, which diverged within
;200 Myr (Judd et al. 2002, pp. 175–176). If compositional heterogeneity is not extreme and the spatial pattern
of substitution is simple, it would follow that nucleotide
data will outperform deduced amino acid data because the
former has more phylogenetic information than the latter.
However, when sequences become highly diverged, the
spatial substitution pattern may become more complex and
problematic for distance-based correction formulae (Steel,
Huson, and Lockhart 2000). The main lineages of land
plants probably diverged as many as 470 MYA (Kenrick
and Crane 1997a, 1997b; Kenrick 2000).
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. 272–
274). 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
100 Atropa belladonna
Nicotiana tabacum
Spinacia oleracea
Lotus corniculatus var. japonicus
Arabidopsis thaliana
Calycanthus floridus var. glaucus
100
78 Triticum aestivum
100
Oryza sativa
85
Zea mays
17
Pinus
koraiensis
100
Pinus thunbergii
63
Psilotum nudum
35
Adiantum capillus-veneris
77
Anthoceros angustus
Marchantia polymorpha
71
Physcomitrella patens
Chaetosphaeridium globosum
Nephroselmis olivacea
Chlorella vulgaris
Mesostigma viride
45
48
98
92
56
52
0.1
FIG. 5.—Neighbor-Joining tree using LogDet distance based on
2,695 fourfold degenerate sites. The number on each branch indicates
a bootstrap probability (%) estimated by 10,000 replicates. Branch lengths
are proportional to the LogDet distance.
1818 Nishiyama et al.
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.
Supplementary Material
The complete table for statistical tests among 105
topologies is available online as a supplementary table.
The nucleotide data matrix is available online in nexus
format.
Acknowledgments
We thank two anonymous reviewers, Brent Mishler,
Hiroyuki Akiyama, Hironori Deguchi, and members of the
Hasebe laboratory for discussion and comments on the
manuscript. Work on Anthoceros angustus has been published under the name of A. formosae. Thanks also to
Carol Rowe for help sequencing the Adiantum chloroplast
genome. The computations were partly done in the
Computer Lab of the National Institute for Basic Biology.
The research was supported by National Science Foundation grant #DEB-0228432 to P.G.W. and grants by
MEXT and JSPS. Adiantum capillus-veneris, Anthoceros
angustus, Physcomitrella patens, and Psilotum nudum
were sequenced by P.G.W., R.B.S, and M.H.; M.K. and
K.Y.; M.S. and C.S.; and T.Y. and K.Y, respectively.
Correspondence in general should be addressed to M.H.
(e-mail: [email protected]). Correspondence and requests for materials for chloroplast DNAs should be addressed to P.G.W. (Adiantum, e-mail: [email protected].
edu), K.Y. (Anthoceros, e-mail: [email protected].
jp), M.S. (Physcomitrella, e-mail: [email protected].
ac.jp), and K.Y. (Psilotum, e-mail: [email protected].
ac.jp).
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Accepted June 28, 2004