J Mol Evol (2004) 58:424–441
DOI: 10.1007/s00239-003-2564-9
Dating the Monocot–Dicot Divergence and the Origin of Core Eudicots
Using Whole Chloroplast Genomes
Shu-Miaw Chaw,1 Chien-Chang Chang,1 Hsin-Liang Chen,1 Wen-Hsiung Li2
1
Institute of Botany, Academia Sinica, 128 Sec. 2, Academy Road, Taipei 115, Taiwan
2
Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA
Received: 31 July 2003 / Accepted: 23 October 2003
Abstract. We estimated the dates of the monocot–
dicot split and the origin of core eudicots using a
large chloroplast (cp) genomic dataset. Sixty-one
protein-coding genes common to the 12 completely
sequenced cp genomes of land plants were concate-
nated and analyzed. Three reliable split events were
used as calibration points and for cross references.
Both the method based on the assumption of a con-
stant rate and the Li–Tanimura unequal-rate method
were used to estimate divergence times. The phylo-
genetic analyses indicated that nonsynonymous sub-
stitution rates of cp genomes are unequal among
tracheophyte lineages. For this reason, the constant-
rate method gave overestimates of the monocot–dicot
divergence and the age of core eudicots, especially
when fast-evolving monocots were included in the
analysis. In contrast, the Li–Tanimura method gave
estimates consistent with the known evolutionary
sequence of seed plant lineages and with known fossil
records. Combining estimates calibrated by two
known fossil nodes and the Li–Tanimura method, we
propose that monocots branched off from dicots 140–
150 Myr ago (late Jurassic–early Cretaceous), at least
50 Myr younger than previous estimates based on the
molecular clock hypothesis, and that the core eudi-
cots diverged 100–115 Myr ago (Albian–Aptian of
the Cretaceous). These estimates indicate that both
Correspondence to: Shu-Miaw Chaw; email: smchaw@sinica.
edu.tw
the monocot–dicot divergence and the core eudicot’s
age are older than their respective fossil records.
Key words: Chloroplast genome — Divergence of
monocot and dicot — Angiosperm phylogeny —
Age of core eudicots — Molecular clock — Un-
equal rate
Introduction
Fossil evidence suggests that flowering plants (an-
giosperms) first appeared
140 million years (Myr)
ago in the early Cretaceous (Willis and McElwain
2002). They soon diversified and expanded globally in
the mid-Cretaceous (90–100 Myr ago) (Nicholas et al.
1983). Although the angiosperm phylogeny has now
been largely established (Mathews and Donoghue
1999; PS Soltis et al. 1999; Qiu et al. 1999; Parkinson
et al. 1999; DE Soltis et al. 2000; Chase et al. 2000),
the question of why the oldest unequivocal fossil for
angiosperms is nearly 300 and 170 Myr later than the
first vascular plants (ca. 440 Myr ago [Taylor and
Taylor 1993]) and their extant sister group, the
gymnosperms (late Carboniferous, ca. 310 Myr ago
[Doyle 1998]), respectively, remains an ‘‘abominable
mystery’’ (Darwin et al. 1903). A number of hy-
potheses have been proposed to explain the late ar-
rival of angiosperms in the fossil record. These
include (1) the escape of fossilization in the initial
stage of angiosperm evolution (Thomas and Spicer
1987), (2) bias in the fossil record (i.e., angiosperms
 425

Table 1. Comparison of previous estimates of divergence between monocots and dicots

Reference point
Reference Gene used (timea of divergence; Myr) Estimated timea (Myr)

Ramshaw et al. (1972) Cytochrome cb Mammals–birds (280) 220–240

Martin et al. (1989) nrc GapC, CHS Animal–yeast (1000) 319 ± 35
Drosophila–vertebrates (600)
Mammals–chicken (270)
Humane–rat (85)
Wolfe et al. (1989) 12 cpc genes Bryophyte–angioaperm (350–450) 170–230
Maize–wheat (50–70) 150–260
nr 26S, 18S rRNA Plant–animal (1000) 200–250, 200–210
Brandl et al. (1992) cp tRNA Maize–wheat (50–70)
Tracheophyte–bryophyte (350–450) 230–350
Plant–animal (1000)
Martin et al. (1993) nr GapC, cp rbcL Bryophyte–spermatophyte (450) 300
Conifer–angiosperm (330)
Laroche et al. (1995) 12 mtc genes Maize–wheat (50–70) 170–238
Vicieae–Phaseoleae (45–65) 157–226
Goremykin et al. (1997) 58 cp genesb Bryophyte–spennatophyte (450) 160 ± 16
Sanderson (1997) rbcL Marchantia (450) [160–215]d
Yang et al. (1999) mt 1st intron of nad Maize–wheat (50–70) 170–235
Sanderson & Doyle (2001) rbcL, 18S rRNA Land plant (450) 140–190
Wikström et al. (2001) cp rbcL, atpB Fagales–Cucurbitales (84) [158–179]
nr 18S rDNA [131–147]e
a
The unit of time is millon years ago (or before present).
b
The translated amino acid sequence was used.
c
cp, chloroplast; mt, mitochondrial; nr, nuclear.
d
The age of extant angioaperms.
e
The origin date of eudicots.

evolved much earlier but went undetected), and (3)
the suggestion that the evolution of angiosperms was
triggered by a particular set of environmental con-
ditions, and/or biotic interactions (such as co-evolu-
tion with faunal groups) (Willis and McElwain 2002).
Is the origin of angiosperms actually much older
than the known fossil record? Since Ramshaw et al.’s
(1972) first application of molecular data to address
this question, three decades have passed. In the in-
terim, molecular phylogenetic studies and critical
fossils of derived angiosperms from older geological
deposits (Magallón et al. 1999; Wikström et al. 2001)
have opened up an opportunity to readdress the age
and evolution of angiosperms. Although all previous
estimates of the monocot–dicot divergence (Table 1)
predate angiosperms’ fossil records, they are highly
variable, ranging from 140–190 Myr (Goremykin
et al. 1997; Sanderson 1997; Wikström et al. 2001;
Sanderson and Doyle 2001) to
200 Myr (Ramshaw
1972; Wolfe et al. 1989; Laroche et al. 1995; Yang
et al. 1999) or even 300–320 Myr (Martin et al. 1989,
1993; Brandl et al. 1992).
Traditionally, the angiosperms were subdivided
into two classes, Liliopsida (the monocots) and
Magnoliopsida (the dicots) (Cronquist 1988). How-
ever, this subdivision was first refuted by rbcL and
18S rRNA gene phylogenies (Chase et al. 1993; Chaw
et al. 1997) and later by analyses of multiple genes
from the three plant genomes (Mathews and Do-
noghue 1999; Parkinson et al. 1999; Qiu et al. 1999;
PS Soltis et al. 1999; DE Soltis et al. 2000; Chaw et al.
2000). These phylogenetic analyses have led to the
conclusion that the dicots were split into the basal
dicots (or the magnoliids) and the eudicots and that
the monocot lineage was derived from one of the
basal magnoliids (Fig. 1A). Parallel to the molecular
data has been the accumulation of pollen fossils of
eudicots, which began in the late Barremian (of
Cretaceous, ca. 120 Myr ago) and spread globally in
the Albian (ca. 110 Myr ago) (Doyle 1992; Hughes
1994). In addition, many new megafossils of basal
eudicots have appeared, such as Tetracentraceae
from the Barremian (110–118 Myr ago) (Magallón
et al. 1999), as well as core eudicots, such as a pos-
sible Rhamnaceae/Rosaceae (rosids) from the early
Cenomanian (94–97 Myr ago [Basinger and Dilcher
1984]). It has also been suggested that the date of
diversification of core eudicots was underestimated.
Wikström et al. (2001) have examined this issue
(Table 1) with nuclear 18S rDNA and two cp (rbcL
and atpB) genes. We now provide additional evidence
by analyzing whole chloroplast (cp) genomic DNA
sequences.
Cp DNA sequences are useful for studying the
plant phylogeny at deep levels of evolution because of
their lower rates of silent nucleotide substitution
426
Fig. 1. Rooted phylogenetic tree for the 12 sampled species. A
Phylogeny of angiosperms based on Qiu et al. (1999) and P. S.
Soltis et al.’s (1999) phylogenetic trees. Solid lines lead to taxa
sampled in this study. B Rooted NJ tree using the Pamilo–Bianchi–
Li distances based on the Ka values concatenated from 61 cp
protein-coding genes. The branches leading to nodes C2 and A are
not drawn to scale. Lengths are indicated. The calibration points
(nodes C1, C2, C3) were used to estimate the divergence between
monocots and dicots (node A) and the origin of core eudicots (node
(Palmer 1985a, b; Wolfe et al. 1989; Clegg et al.
1994). Moreover, concatenating sequences from
many genes may overcome the problem of multiple
substitutions that cause the loss of phylogenetic in-
formation between cp lineages (Lockhart et al. 1999)
and can reduce ‘‘sampling errors due to substitutional
noise and the finite number of characters within a
gene’’ (Sanderson and Doyle 2001). In this study we
analyzed 39,507 sites of cp DNA genomic sequences
from 61 protein-coding genes common to the 12
complete cp genomes of land plants (Table 2). Our
dataset is larger than those used in previous studies,
including that of Goremykin et al. (1997; see also
Table 1), who analyzed 40 proteins of cp genomes
from fewer taxa (five land plants, including only one
dicot and two monocots).
Molecular dating often assumes rate constancy,
but this is frequently violated (PS Soltis et al. 2002
and references herein). For example, substitution
rates of cp genes vary greatly among and within
tracheophyte (or vascular plant) lineages (Bousquet
et al. 1992; Gaut et al. 1992, 1993; Clegg et al. 1994;
Sanderson and Doyle 2001; PS Soltis et al. 2002),
between protein-coding loci (Muse and Gaut 1997;
Matsuoka et al. 2002), and between nonsynonymous
and synonymous sites (Gaut et al. 1997; Matsuoka et
al. 2002). Sanderson and Doyle (2001) believed that
much of the conflict in estimating divergence times
B). Gene loss (open bar), loss but with known transfer to nucleus
(hatched bar), retention (gray bar), and likely gain with no simi-
larity to prokaryotic genes (filled bar) are plotted on the branches
leading to each lineage. The upper numbers at each node denote the
bootstrap percentages (where applicable, values of the interior
branch test indicated after the slash). Total gene number in the cp
genome is given after each species, in parentheses. Branch lengths
and the scale bar are Ka values per 100 sites.
was due to rate variation across lineages. In order to
mitigate this problem we used mean branch lengths of
the sampled monocots and dicots.
The focus of this study is to estimate the dates of
the monocot–dicot split and the origin of core eudi-
cots using a large cp genomic dataset. The date of the
monocot–dicot divergence can be calculated by ex-
trapolation from the reliable dates of other speciation
events by means of phylogeny based on DNA se-
quence distances (Wolfe et al. 1989). Three diver-
gence events with well-supported fossil dates were
used as calibration points and cross references. Both
the method based on the assumption of a constant
rate and Li and Tanimura’s (1987) unequal-rate
method (hereafter the Li–Tanimura method) were
used to estimate divergence times, and the estimates
were compared with known fossil dates. Although
several other methods without the rate constancy
assumption, such as the nonparametric rate
smoothing method (NPRS), have been proposed
(Sanderson 1997 and references cited herein), we
chose the Li–Tanimura method for its simplicity. The
method uses lineages in which the molecular clock
holds better than the others to estimate the diver-
gence time at a particular node. We also discuss
possible reasons for discrepancies among estimates of
divergence dates obtained in this study and previous
studies.
 427

Table 2. Scientific names, classification, and NCBI accessions of species in the dataset

Classificationa Scientific name NCBI accession No. (version date)b/Reference

Bryophyte
Marchantiaceae Marchantia polymorpha NC_001319 (Aug 2002)/Ohyama et al. (1986)
Petridophyte
Psilotaceae Psilotum nudum AP004638 (Nov 2002)/Wakasugi et al. (2000)
Gymnoaperm
Pinaceae Pinus thunbergii NC_001631 (Sep 2002)/Wakasugi et al. (1994)
Angiosperms
Monocots
Poaceae
Andropogoneae Zea mays NC_001666 (Sep 2002)/Maier et al. (1995)
Oryzeae Oryza sativa NC_001320 (Sep 2002)/Hiratsuka et al. (1989)
Triticeae Triticum aestivum NC_002762 (Sep 2002)/Ikeo and Ogihara (2000)
Dicots
Eudicots
Caryophyllidae
Chenopodiaceae Spinacia oleracea NC_002202 (Aug 2002)/Schmitz-Linneweber et al. (2001)
Asteridae
Solanaceae Nicotiana tabacum NC_001879 (Sep 2002)/Shinozaki et al. (1986)
Rosidae
Brassicaceae Arabidopsis thaliana NC_000932 (Sep 2002)/Sato et al. (1999)
Onagraceae Oenothera elata subsp. hookeri NC_002693 (Sep 2002)/Hupfer et al. (2000)
Fabaceae
Papillionoideae
Loteae Lotus japonicus NC_002694 (Sep 2002)/Kato et al. (2000)
Trifolieae Medicago truncatula AC093544c(Nov 2001)/Lin et al. (2001)
a
Ranks of species follow the NCBI’s Taxonomy Guide.
b
Data modified from http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html (vers. 20 Dec 2002).
c
No gene annotation in this accession.

Data and Methods
Database Search for Cp Genome Sequences
Individual genes of the 12 published cp genome sequences (Table
2) were downloaded from GenBank, National Center for Bio-
technology Information (NCBI). Nomenclatures of the cp pro-
tein-coding genes complied by Hallick and Bairoch (1994), Stoebe
et al. (1998), Martin et al. (2002), and Swiss-Prot Protein
Knowledgebase (2003) were used as guides. When synonyms were
encountered, their sequence homologies with the typified names
were carefully verified. Two homology criteria were considered:
(1) the alignable length between two proteins is larger than 80%
of the longer sequence, and (2) the sequence identity in the
aligned region is at least 40% if L > 150, or at least 0.06 +
4.8L)0.032 (1 þ exp()L/1000)) (Rost 1999; Gu et al. 2002). Note that we
raise the identity to 40% instead of 30% because the taxa we
sampled are comparatively recent and cp genes are highly con-
served (Wolfe et al. 1989).
Since the sequence of Medicago was not annotated, its protein-
coding genes were annotated using the Nucleotide query–Protein
database (BLASTX) algorithm at NCBI with each known gene
from Lotus as query. If a particular gene was missing from Lotus,
that gene from the rest of the 10 taxa was used instead. Open
reading frames annotated by us were also verified using the BLAST
2 sequences algorithm and the Nucleotide query–Translated db
algorithm in NCBI against the corresponding gene and the whole
genome of Arabidopsis, respectively. A query sequence with more
than 40% identity to the specific known genes was then considered
as a putative homologous gene. A remnant of the accD gene in the
rice was reported previously (Hiratsuka et al. 1989) but could not
be detected by Katayama and Ogihara (1996) or Ogihara et al.
(2002) using Southern hybridization. We were not able to locate it
from the rice cp genome either. We used the reviews of Millen et al.
(2001) and Martin et al. (2002) cp genes as guides to confirm our
BLAST searches, especially for those genes lost or with unknown
functions.
After careful comparison and annotation, a total of 98 protein-
coding genes was found in the cp genomes of the 12 sampled
species (Table 2). The lengths as well as the presence or absence of
those genes in each taxon are presented in Appendix 1. An open
reading frame homologous to a known gene was given the same
name to facilitate comparison and alignment. For some unanno-
tated genes filtered by using BLASTX search, their positions in the
corresponding genomes were indicated. We excluded pseudogenes
and genes duplicated in the inverted repeat regions. The cp encoded
RNA genes were previously shown to be problematic in early cp
phylogeny (Martin et al. 1998; Lockhart et al. 1999) and in the
present study as well (data not shown). Therefore, RNA genes were
excluded from analysis.
Alignment of All Cp Genes and Phylogenetic Analyses
Amino acid sequences of each gene from the 12 taxa were first
aligned one by one using GeneDoc (Nicholas and Nicholas 1997)
with minor adjustments. The alignment was then used as a guide
for aligning the corresponding nucleotide sequences. Unknown
sites, start and stop codons, and regions difficult to align were
removed from each gene alignment. All aligned individual gene
sequences were then assembled using the Text Editor in MEGA 2.1
(Kumar et al. 2001). Gaps were completely deleted from the as-
sembled alignment concatenated from the 61 cp protein-coding
genes common to the 12 sampled taxa (see also Results). The
working data file (in MEGA format) is shown in Appendix 2,
available in the Supplementary Material Section at the JME Web
site.
428
Nucleotide sequence divergence between a pair of taxa (or
groups) was calculated in terms of the numbers of substitutions per
synonymous site (Ks) or per nonsynonymous site (Ka), using the
Pamilo–Bianchi–Li method implemented in MEGA 2.1. Diver-
gence value between two groups is presented as average distance ±
standard error, obtained from the option Compute Between
Groups Means in MEGA 2.1. Average distance between two
groups is the arithmetic mean of all pairwise distances between taxa
in the intergroup comparisons. To date the divergence between the
monocot and the dicot lineages, Saito and Nei’s (1987) neighbor-
joining (NJ) method and the Ka values (not Ks, because substitu-
tions at the third codon positions are saturated across sampled land
plant lineages; see Results) were used to reconstruct the phylo-
genetic trees, rooted at the top of the Pinus lineage. Because the six
sampled dicots (Table 2) represent the two large clades (the rosids
and the asterids) of core eudicots and one of the remaining four
small core eudicot clades, they can be used to infer the age or
diversification date of core eudicots. The NJ trees reconstructed by
Ka values and Ks values were rooted at the monocot lineage (see
Results). Relative support for each node was evaluated using the
bootstrap test and the interior branch test implemented in MEGA
2.1 with 2000 replicates. The latter test is constructed based on the
interior branch length and its standard error. If this value is higher
than 95% for a given branch, then the inferred length for that
branch is considered significantly higher than 0 (Kumar et al.
2001). To compare the evolutionary rates of sampled fern, pine,
monocot, and dicot lineages, Tajima’s relative rate test (1993) im-
plemented in MEGA 2.1 was applied. Because the method does not
distinguish between Ka, and Ks, the first and second codon posi-
tions of the combined 61 cp protein-coding genes were used
instead.
Calibration Points
To date the divergence between the monocots and the dicots, three
split events (see Figs. 1 and 4) with reliable fossil dates were used as
reference nodes: (C1) the Psilotum (fern)–seed plant split (400–420
Myr old [Pryer et al. 2001]); (C2) the Pinus (conifer)–angiosperm
split (280–310 Myr old); and (C3) the maize–wheat split (50–60
Myr old). Since uncertainties about the age of the reference node
were a probable reason behind the discrepancies among previous
estimates of angiosperm origin (Bremer 2000; Sanderson and Doyle
2001), we have carefully examined the dates of our calibration
nodes.
Fossil Dates
Psilotum has been repeatedly suggested as a member of ferns by
molecular data (e.g., Nickrent et al. 2000; Pryer et al. 2001), but the
architecture of its sperm cell suggests that Psilotum is an early
divergent fern (Renzaglia et al. 2001) with relatively remote affin-
ities to Ophioglossaceae (a basal fern family) and Equisetaceae
(sphenopsids). Kenrick and Crane (1997) considered that the basal
dichotomy of Euphyllophytina occurred in the early–mid Devo-
nian (ca. 400–420 Myr ago) and resulted in two clades: one con-
taining the extinct Psilophyton and the other ferns, horsetails, and
seed plants. We took this splitting date as the lower bound for the
divergence between Psilotum and seed plants.
Pinus is a genus of Pinaceae, which contains over 230 species
and is the largest and most basal family of conifers (Hart 1987;
Price et al. 1993; Chaw et al. 1997). Delevoryas and Hope (1973)
and Miller (1977, 1988) proposed that the Triassic (206–248 Myr
ago) period may represent a time when modern conifers were
evolving. Cladistic and stratigraphic analyses of living seed plants
(Doyle and Donoghue 1987; Crane 1988; Doyle 1998) suggested
that diversification of modern seed plants occurred from the lower
Pennsylvanian to the upper Triassic (215–310 Myr ago). The
earliest fossil evidence of trees bearing the typical conifer’s bisac-
cate pollen that germinates distally dates from the late Carbonif-
erous to early Permian (ca. 250–290 Myr ago) and conifer relatives
are known from ca. 310 Myr ago (Rai et al. 2003). Gymnosperms
and angiosperms are the two major taxa of seed plants, distinct
since the end of the Carboniferous,
300 Mya (Bow et al. 2003).
From the above considerations, we took 280–310 Myr as an upper
bound for the split between the conifer and the angiosperm line-
ages.
Fossil leaves of rice (belonging to the grass family Poaceae)
have been described from the upper Eocene, about 40 Myr ago
(Stebbins 1981), and the earliest unequivocal evidence of grass
fossils (including spikelets and inflorescence with pollen) were
found in Paleocene–Eocene deposits, about 50–60 Myr ago (Crepet
and Feldman 1991). Initial radiation of the grass family was sug-
gested to be 65 Myr ago (Stebbins 1987; Thomasson 1987). Bremer
(2000) regarded the 50–70 Myr ago estimate of a maize–wheat
divergence used by Wolfe et al. (1989) as rather uncertain. More-
over, phylogenetic analyses of the cp rpl16 intron sequences (Zhang
2000), eight character sets (GPWG 2001), cp genome structure
(Ogihara et al. 2002), and cp genomic comparison (Matsuoka et al.
2002) indicated that in the grass family, Oryzoideae (rice) and
Pooideae (wheat) diverged after the subfamily Panicoideae (maize),
which was preceded by four other subfamilies (Zhang 2000).
Therefore, we took 50–60 Myr as a reasonable estimate of the
maize–wheat split.
Results
Cp Genome Data
The concatenated lengths of all known cp functional
protein-coding genes (Appendix 1) in the 12 sampled
species (Table 2) range from 58,095 bp in the Triticum
to 71,509 bp in the Marchantia; the average is 63,661
± 4,764 bp. Sixty-one cp protein-coding genes, which
encode two envelope membrane proteins (cemA,
ycf9), 1 maturase (matK), 1 protease (clpP), 34 pho-
tosynthetic light reactions (atpA, atpB, atpE, atpF,
atpH, atpI, petA, petB, petD, petG, petL, petN, psaA,
psaB, psaC, psaI, psaJ, psbA, psbB, psbC, psbD, psbE,
psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN,
psbT), 18 ribosomal proteins (rpl2, rpl14, rpl16, rpl20,
rpl32, rpl33, rpl36, rps2, rps3, rps4, rps7, rps8, rps11,
rps12, rps14, rps15, rps18, rps19), 4 RNA polym-
erases (rpoA, rpoB, rpoC1, rpoC2), and 1 cytochrome
c biogenesis protein (ccsA), are in common to all 12
taxa. After elimination of unknown sites, regions
difficult to align, start and stop codons, and all gaps,
39,507 sites were used for comparison and tree
reconstruction.
As shown in Table 3 (the first row), the 12 cp ge-
nomic sequences are AT-rich. This bias is particularly
strong at the third codon positions, primarily because
of the high T nucleotide contents. These data are
consistent with the high AT content found earlier in
the plastid genome (Whitfeld and Bottemley 1983).
Across the 11 tracheophytes nucleotide base compo-
sitions are homogeneous at the first and second co-
 429

Table 3. Nucleotide base composition (%) of the concatenated 61 cp protein-coding genes in Marchantia and 11 sampled tracheophytes

Codon positiona A C G T pb

All 33.2/29.2 ± 0.4 (1.4%) 14.4/18.4 ± 0.5 (2.7%) 17.8/21.5 ± 0.4 (1.9%) 34.6/31.0 ± 0.5 (1.6%) 0.000
1st 30.3/28.2 ± 0.4 (1.4%) 16.9/19.6 ± 0.3 (1.5%) 29.1/30.2 ± 0.3 (1.0%) 23.7/22.0 ± 0.3 (1.4%) 0.793
2nd 29.1/27.2 ± 0.3 (1.1%) 20.8/21.7 ± 0.2 (0.9%) 17.4/19.1 ± 0.3 (1.6%) 32.6/32.0 ± 0.3 (0.9%) 0.981
3rd 40.1/32.3 ± 0.8 (2.5%) 5.3/13.8 ± 1.0 (7.2%) 7.0/15.0 ± 0.8 (5.3%) 47.5/39.0 ± 1.1 (2.8%) 0.000
a
The start and stop codons were not included in analysis. Data on Marchantia are before the slash and the average of 11 sampled
tracheopytes is after the slash and presented as mean ± SE (coefficient of variation).
b
Probabihty (p) was based on v2 tests for homogeneity across the 11 sampled tracheophytes using PAUP 4.0b1 (Swofford 1998).

Fig. 2. Uncorrected pairwise sequence

divergence (P distance) plotted against
corrected distances (Kimura two-pa-
rameter) for transitions (Ts) and trans-
versions (Tv) at first and second codon
positions (A) and third codon position
(B). Each plot presents 66 data points.

don positions (v2 test, p = 0.793 and 0.981) but not
so at the third codon positions (p < 0.000). The G
content is particularly high at the first codon posi-
tions in all taxa and Marchantia much prefers the use
of synonymous codons ending with A or T.
The mean Ka/Ks ratio for all species pairs is 0.19.
The mean Ka/Ks ratio difference between the mono-
cot (0.156) and the dicot (0.158) lineages is small.
These data are suggestive of stringent selective con-
straints on amino acid substitutions and correlate
well with the observed higher GC contents at the first
and second positions (Table 3).
The Inferred Phylogenetic Trees
Figure 1A was simplified from the topology of the
maximum parsimony (MP) trees reconstructed with
multigenes by Qiu et al. (1999) and by S. P. Soltis
et al. (1999). Figure 1B is a NJ tree reconstructed with
Ka values using Marchantia as the outgroup. The
topology of this tree strongly indicates that, to the
exclusion of the fern (Psilotum) lineage, the seed
plants form a monophyletic clade, within which the
conifer (Pinus) lineage and the angiosperms comprise
two separate subgroups. The sampled angiosperms
are subdivided into two well-supported lineages, the
monocots and the eudicots. Both bootstrap and in-
terior branch tests for the above-mentioned major
tracheophyte lineages are 100%, and the latter test
yielded a higher percentage support for the rice +
wheat clade. The phylogenetic relationships of the
monocot lineage and the six core eudicots generally
agree well with those in recent multigene trees (Fig.
1A [Qiu et al. 1999; PS Soltis et al. 1999; DE Soltis et
al. 2000]) except that in our NJ tree the Caryophyll-
ales (represented by Spinacia) and asterid (repre-
sented by Nicotiana) are well resolved as sister clades.
This relationship was previously revealed in the trees
made by Wolfe et al. (1989) and Goremykin et al.
(2003).
The NJ tree reconstructed from the Ks values ap-
pears to be unreliable because it placed Arabidopsis as
basal to the remaining dicots (data not shown),
contrary to the most recent multigene phylogenies of
angiosperms (Mathews and Donoghue 1999; Qiu et
al. 1999; PS Soltis et al. 1999; DE Soltis et al. 2000;
Chase et al. 2000). These data caused us to question if
the third codon position, where most synonymous
substitution occurs, is saturated with substitutions.
To assess levels of sequence saturation with the
concatenated cp genes, pairwise uncorrected numbers
of transitions and transversions (uncorrected P) were
plotted against corrected (Kimura’s two-parameter)
sequence distance (Fig. 2). Sixty-six paired points [12
· (12 ) 1)/2] are presented in Fig. 2. The curves of
both uncorrected transitions and transversions
430
Fig. 3. Relative branch lengths (based on Ka values) of monocots
and dicots, using Marchantia (A), Psilotum (B), and Pinus (C) as
outgroup, respectively. Gray branches are with Aratbidopsis, Spi-
nacia, and Nicotiana excluded (for their slower rates). Branch
lengths are Ka values per 100 sites. D Rooted NJ tree of the core
eudicot lineages based on Ks values, using the three grasses as
outgroup. Italicized numbers denote bootstrap percentages (boot-
strap test before the slash, interior branch test after the slash).
Branch lengths are Ks values per 100 sites.
against sequence divergence at the first two codon
positions were nearly linear (Fig. 2A). In contrast, the
curves at the third codon position revealed a signifi-
cant trend toward asymptotic saturation (Fig. 2B),
indicating that substitutions at the third codon posi-
tion are saturated and not suitable for inferring
phylogenetic relationships among the sampled taxa
or for dating purposes. For this reason, we used only
the NJ tree based on the Ka values.
Because Fig. 1B did not resolve the relationships
among sampled eudicots, we reconstructed a phylo-
genetic tree of eudicots using the three monocots as
the outgroup. The NJ tree based on the Ks values
yielded a reasonable topology (i.e., in agreement with
the phylogenetic relationships of the orders of flow-
ering plants compiled by APG [1998]) for the sampled
six eudicots (Fig. 3A), whereas the NJ tree based on
Ka values did not (data not shown). Based on the
limited number of eudicots, Fig. 3A suggests that the
six core eudicots first split (at node B) into two well-
supported monophyletic clades, the rosids (repre-
sented by Oenothera, Arabidopsis, Lotus, and Medi-
cago) and the asterids + Caryophyllales (represented
by Nicotiana and Spinacia, separately).
Both NJ trees reconstructed from Ka (Fig. 1B) and
Ks (Fig. 3A) values suggested a close relationship
between the Ehrhartoideae (rice) and the Pooideae
(wheat) with the maize as an outgroup, but the
bootstrap values for the rice–wheat clade are low to
moderate. Recently, using the NJ method with the
variable sites of 98 genes (including not only all
protein-coding but also RNA genes) common to the
cp genomes of these three cereals and rooting at the
Nicotiana lineage, Matsuoka et al. (2002) also placed
maize as sister to the rice–wheat clade.
Phylogenetic Distribution of Cp Genes During
Tracheophyte Evolution
We examined a total of 98 protein-coding genes
(Appendix 1) that are present among the 12 studied
taxa. Figure 1B also presents the protein-coding gene
numbers held in each sampled species and specific
gene loss, transfer, and retention in the 11 lineages of
tracheophytes. Although Martin et al. (2002) have
done a similar evolutionary analysis for deeper
groups. Fig. 1B is focused on the tracheophyte line-
ages using Marchantia as the outgroup with addi-
tional 5 core eudicots and 1 fern.
Compared with the cp genome of bryophyte
(Marchantia), those of tracheophytes have lost three
genes: two transporters (cysA and cysT) and one with
unknown function (cys66) (Fig. 1B). During trache-
ophyte evolution, the fern (Psilotum) and angiosperm
lineages have parallel losses of three chlorophyll
biosynthesis genes, chlB, chlB, and chlN. The seed
plant lineage has commonly transferred the rpl21
(Martin et al. 2002 and references cited therein) to its
nucleus. In the spinach and Arabidopsis lineages that
gene, however, has been unusually replaced by
a nuclear RPL21c gene of mitochondrial origin
(Martin et al. 1990; Gallois et al. 2001).
Within seed plants, the conifer (Pinus) lineage has
uniquely lost all 11 NADH dehydrogenase subunit
genes (ndhA–K; 4 are completely missing and 7 are
pseudogenes [Wakasugi et al. 1994]) but has gained a
new gene of unknown function, ycf68 (Martin et al.
2002). The angiosperm lineage has further lost two
genes, one, psaM, involved in the photosynthetic light
reaction and the other, ycf12, of uncertain function.
Within angiosperms the three grasses have lost
three genes: one metabolism related, accD (Hiratsuka
et al. 1989; Maier et al. 1995; Ogihara et al. 2002), and
two genes of unknown function, ycf1 and ycf2. How-

Table 4. Estimates of the monocot–dicot divergence and the age of core eudicots based on the constant rate method
Outgroup Calibration event (fossil dates; Myr)
Marchantia C1: Fern–seed plant divergence (400–420)
Psilotum C2: Conifer–angiospenn divergence (280–310)
Pinus C3: Maize–wheat divergence (50–60)
Pinus C3: Maize–wheat divergence (50–60)
Monocots C3: Maize–wheat divergence (50–60)
a
K denotes the number of substitutions per 100 synonymous (Ks) or nonsynonymous (Ka) sites between pair of taxa or groups.
b
Rate (r) is defined as the number of substitutions per site per year, r = K/(2T) (Li and Grauer 1991).
ever, we found that the grass lineage has also recruited
nine novel genes; one of them, ycf68, is shared with the
pine lineage, and the remaining eight, ycf69–76, are
unique. Functions of these genes are not known yet
and they have no detectable homology to prokaryotic
genes (Martin et al. 2002). Except for spinach, all
sampled eudicots have lost the translational initiation
factor 1 (infA). According to an extensive survey of
more than 300 diverse angiosperms by Millen et al.
(2001), the infA gene of the cp genome has repeatedly
become defunct in about 24 separate angiosperm lin-
eages, including almost all rosid species.
Nucleotide Substitution Rates
Before applying molecular calibration, we assessed
the assumption of rate constancy. Fig. 1B shows that
the branches from the calibration point C1 leading to
the Psilotum (fern) lineage and the Pinus lineage are
not equal in length. The NJ trees in Figs. 3B, C, and
D, using Marchantia, Pinus, and Psilotum as the
outgroup, respectively, also indicate that the Ka rates
in the monocot and the dicot lineages are unequal.
The monocot lineage has evolved faster than the di-
cots, by 39.6, 37.3, and 32.3%, respectively, for the
three outgroups. In Fig. 1B the branches from node
A leading to Arabidopsis, Spinacia, and Nicotiana are
strikingly shorter than those leading to the other
three dicots and the monocots. Tajima’s relative rate
test using rice, Marchantia, Psilotum, and Pinus as
outgroups, respectively, confirmed this observation
(all p’s < 0.001). However, exclusion of the above
three slower dicot lineages (gray lines in Figs. 3B–D)
led to even higher estimates of divergence dates (data
not shown). We therefore used the entire dataset.
By applying the equation, r = K/(2T), where K is
the distance and T is the divergence time between the
two taxa compared, nonsynonymous rates were cal-
ibrated and are shown in Table 4. Based on the three
divergence events, C1, C2, and C3, and the dataset
with all six dicots, the Ka rates are 0.215–0.225 · 10)9,
0.232–0.257 · 10)9, and 0.164–0.197 · 10)9 substi-
431
Ka Rateb (·10)9) Ka Time (Myr)
Monocot–dicot divergence
Ka: 18.03 ± 0.22 0.215–0.225 Ka: 9.30 ± 0.24 206 ± 5–217 ± 6
Ka: 14.40 ± 0.29 0.232–0.257 Ka: 9.28 ± 0.34 180 ± 7–200 ± 7
Ka: 1.97 ± 0.23 0.164–0.197 Ka: 9.35 ± 0.29 237 ± 5–285 ± 9
Origin of core eudicots
Ka: 1.97 ± 0.23 0.164–0.197 Ka: 6.08 ± 0.26 154 ± 7–185 ± 8
Ks: 12.10 ± 0.11 1.008–1.210 Ks: 36.06 ± 0.51 149 ± 2–181 ± 3
tutions per nonsynonymous site per year, respec-
tively. Clearly, these three calibrated Ka rates are
unequal, differing from one another by from 8%
[(0.232 ) 0.215)/0.215] to nearly 42% [(0.232 ) 0.164)/
0.164], and the conifer–angiosperm’s Ka rate is the
highest.
Dates of the Monocot–Dicot Divergence and the
Origin of Core Eudicots
Molecular Clock or Rate Constancy Method. The
date of the monocot–dicot divergence was estimated
by applying the equation T = K/(2r). As indicated in
Table 4, based on the entire dataset and the calibra-
tion points C1, C2, and C3, three time estimates for
the monocot–dicot divergence, 206 ± 5–217 ± 6, 180
± 7 –200 ± 7, and 237 ± 5–285 ± 9 Myr, were
obtained. These estimates suggest that the monocot–
dicot divergence took place
220 ± 40 Myr ago.
Using either the Ka or the Ks rates of the maize–
wheat divergence and the mean Ka values (see node B
in Fig. 1B and Fig. 4) of all six eudicots or the Ks
values between the rosid clade and the asterid +
Caryophyllales clade (see node B in Fig. 3A), the
divergence for core eudicots was estimated to be 154
± 7–185 ± 8 and 149 ± 2–181 ± 3 Myr ago (Table
4), respectively. These two estimates are close to each
other, and their average is
170 Myr ago.
Li–Tanimura Method. Figure 4 was simplified
from the phylogenetic tree Fig. 1B with all branch
lengths indicated. The branch length of core eudicots
was calculated as the mean length of the branch
leading from their emergence point (node B) to the six
core eudicots. We then used the Li–Tanimura meth-
od, which uses lineages in which the molecular clock
holds better than the others, to estimate the diver-
gence time at points A and B. For example, we know
that the branching date for Pinus (node C2) is 280–
310 Myrs ago and want to estimate the branching
dates between the monocot and the dicot lineages.
The distances from node C2 to Pinus, monocots, and
432
Fig. 4. A phylogenetic tree simplified from Fig. 1B. Nodes and
lineages correspond to those in Fig. 1B. C1 was used to estimate the
divergence dates of C2, the monocot–dicot divergence (node A),
and the origin of core eudicots (node B) (refer to Table 5 and the
text for detail). The number on each branch is the Ka value per 100
sites.
dicots are 6.05, 9.02 (=3.91 + 4.22 + 0.89), and 7.66
(=3.91 + 0.90 + 2.85), respectively. Since the
monocot lineage has a longer branch length than do
the Pinus and dicot lineages, it is not used. Based on
the branch length of the dicot lineage, the monocot–
dicot divergence (node A in Fig. 1B and Fig. 4) was
estimated to be 137–152 Myr ago, which is derived
from (280 or 310) · (0.90 + 2.85)/7.66; the origin of
core eudicots (node B in Fig. 1B and Fig. 4) was
estimated as 104–115 Myr ago, which is calculated
from (280 or 310) · 2.85/7.66. As shown in Fig. 4 the
distance from node C1 to dicots is 10.4 (=2.74 +
3.91 + 3.75), and we assume that the molecular clock
along the dicot lineage is approximately constant.
Similarly, using the branching date of Psilotum, 400–
420 Myr ago (node C1 in Fig. 1 and Fig. 4), the
monocot–dicot divergence (node A in Fig. 1 and Fig.
4) was estimated to be (400 or 420) · (0.90 + 2.85)/
10.4 = 144–151 Myr ago, and the origin of core
eudicots was estimated to be (400 or 420) · 2.85/10.4
= 110–115 Myr ago. Table 5 shows that these dates
are highly close to those estimated from C2. Com-
bining estimates calibrated from both C1 and C2, we
estimated that monocots and dicots diverged at 140–
150 Myr ago and the core eudicot lineages originated
100–115 Myr ago.
Discussion
Rate Variation Among Tracheophyte Lineages
Our phylogenetic analyses (Figs. 1B, 3, and 4) in-
dicate that nonsynonymous substitution rates of cp
Table 5. Ages of nodes (Myr) inferred from the phylogenetic tree
in Fig. 3 using the Li–Tanimura method (1987)
Node C1 (400–420 Myr) C2 (280–310 Myr)
C1 —a 380–421
C2 295–309 —
A l44–151 137–152
B 110–115 104–115
a
Nonapplicable.
genomes are unequal among the six eudicot line-
ages, between the two angiosperm lineages (i.e.,
monocots and dicots), and among the tracheophyte
lineages (i.e., all sampled seed plants and a fern,
Psilotum). These observations were confirmed by
Tajima’s relative rate test using Marchantia as the
outgroup and the first two coding positions (data
not shown).
Using 40 cp proteins, Goremykin et al. (1997)
found that the average substitution rates (equiva-
lent to the Ka rate) along the branches from the
common node (equivalent to node C1 in our
Fig. 1B) of seed plants to Nicotiana and to Pinus
were quite similar. However, our cp genomic data
(Fig. 1B) suggest that the former branch is signifi-
cantly longer than the latter when Psilotum is
used as the outgroup (Tajima’s relative rate test:
p < 0.001).
As revealed in Fig. 1B and Figs. 3B–D, the Ka
rate in the grass lineage has evolved much faster
than in the dicots. In addition, Fig. 1B (Ka rate)
and Fig. 3A (Ks rate) also indicate that among the
six annual dicots sampled, the Nicotiana and Spi-
nacia evolved more slowly than the rest. Extensive
rate variation among annual plants has also been
observed in other cp genes at nonsynonyrnous sites
(Wolfe et al. 1987, 1989). Generally, there is a
resonance of Bousquet et al.’s observation (1992)
on the rbcL gene of seed plant lineages. They
found that the annual form evolved more rapidly
on average than the perennial form (represented in
our study by Psilotum and Pinus) and that the
grass family has the fastest evolution rate. Com-
paring the rbcL and ndhF loci in the grass family,
Gaut et al. (1997) found that at Ka sites rate var-
iation was not correlated between those two plastid
loci. Most recently, examining the whole cp ge-
nomes (106 genes) of maize, rice, and wheat,
Matsuoka et al. (2002) also found variation in Ka
rates. The Ka rate variation seems to correlate well
with the evolutionary divergence pattern depicted
in the cp genomic NJ tree (Fig. 1B), which shows
that the angiosperm lineage has evolved faster than
the gymnosperm lineage and that the latter in turn
has evolved more rapidly than the fern lineage.
Since the number of completely sequenced cp ge-

nomes is quickly increasing, this trend may be re-
tested soon.
Significant rate variation in the cp genomes of the
tracheophyte lineages is also consistent with the fin-
ding of P. S. Soltis et al. (2002), who studied one
nuclear and three plastid genes using MP analyses. In
summary, the molecular clock hypothesis does not
hold for the Ka rates among the cp genomes of tra-
cheophyte plants.
Reference Fossil Dates and the Phylogenetic Tree
Obtained
In the Data and Methods section we have carefully
cross-examined the three fossil dates by adopting
updated phylogenies and documented fossil records.
Bremer (2000, pp 4709, 4710) suggested that in
phylogenetic dating rate calibration rather than
unequal substitution rates is the major source of
error and is behind the discrepancies in earlier es-
timates of monocot and flowering plant evolution.
Indeed, in Table 4 the three calibrated rates based
on the molecular clock are discrete, and the ob-
tained dates for monocot–dicot divergence do not
agree with one another. To evaluate if the fossil
dates and the cp Ka rates corresponded well with
each other with respect to the two dating methods,
we also used the divergence rates and the Ka dis-
tances (Table 4) from the fern–seed plant and con-
ifer–seed plant splits to date the other’s divergences.
The rate constancy method led to an estimate of
350–390 Myr ago for the former event and 320–335
Myr ago for the latter. These two estimates differ
widely from the fossil records. In contrast, the di-
vergence times (Table 5) of these two events esti-
mated from the Li–Tanimura method are highly
compatible with the paleobotanical data.
Sanderson and Doyle (2001) proposed that (1)
biases in the data or the statistical estimation
method used, (2) variation in rate across sites which
‘‘causes sequence divergences to be estimated in-
correctly,’’ and (3) incorrect phylogenies are the
underlying sources of error in molecular dating. P.
S. Soltis et al. (2002) added that ‘‘inadequate sam-
pling of taxa...can compound the problem.’’ The
same concern could be raised about the results we
present here. However, the effect of these problems
is likely to have been considerably reduced by the
sampling of 12 evolutionary successive land plants
(Table 2; including all three living subclasses of
eudicots), the use of 61 genes (>39,000 bp long)
with different functions from the complete cp ge-
nomes, and the highly reliable NJ tree (Fig. 1B),
which is consistent with the NJ tree of Goremykin
et al. (1997), inferred from concatenating 14,295
amino acids of cp genomes.
433
Comparison of Estimates from the Molecular Clock
and Li–Tanimura Methods
Tables 4 and 5 show that the dates of the monocot–
dicot split and the origin of core eudicots estimated
by the rate constancy and Li–Tanimura methods
differ greatly, with estimates from the former method
predating the latter by
50 Myr. Estimates calibrated
from nodes C1 and C2 using the molecular clock
method vary more than those obtained from the Li–
Tanimura method.
In the rate constancy method we used the arith-
metic mean of all pairwise Ka distances between
monocots and dicots to estimate the divergence date
(Table 4). As a result, the obtained dates for the
monocot–dicot split (
220 Myr) and for the origin of
the core eudicots (
170 Myr) appear to be severely
overestimated because the three high-rate grasses
were included in the distance calculation. This was
also the case in most previous estimates (Wolfe et al.
1989; Martin et al. 1989, 1993; Brandl et al. 1992;
Laroche et al. 1995; Yang et al. 1999), which not only
used the molecular clock hypothesis but also included
one (maize) or several fast-evolving grass species (or
annual Liliales [such as Ramshaw 1972]).
Together with the preceding age estimates for the
monocot–dicot split and the origin of core eudicots,
we concluded that the Li–Tanimura method can
substantially reduce the effect of rate variation among
lineages and provide an estimate more in line with
known fossil data.
Comparison of Our Estimates with Previous Estimates
Since our estimates based on the rate constancy
method seem unreliable, we shall compare only esti-
mates obtained from the Li–Tanimura method with
those from other methods. Goremykin et al. (1997)
used a very similar framework of the Li–Tanimura
method (1987) and claimed that their approach is
‘‘independent of the rate fluctuation on the grass
(high rate) and Marchantia (low rate) branches.’’
They estimated the divergence time between the Zea–
Oryza lineage and the Nicotiana lineage to be 160
Myr ago, which predates ours by about 10–20 Myr
(Table 5). Based on our cp genome data (Figs. 1B and
3A), the Nicotiana lineage has the slowest Ka and Ks
rates among the sampled dicots but was used as half
of the denominator by Goremykin et al. (1997) in
estimating the monocot–dicot divergence time. Since
our estimates of the dates for the monocot–dicot
lineage and the origin of core eudicots were based on
the mean branch length of six dicots, our data should
be more reliable than data based on single species.
In order to reduce the effects of unequal rates,
Bremer (2000) used the mean branch lengths from a
434
group of terminal taxa to their common node (which
has a known fossil age) for calculating the change
rate (distance/age by Bremer’s definition). Using the
rbcL gene, the MP tree of monocots, and the eight
reference nodes with known fossil dates, Bremer
(2000) estimated the split between Acorus, presuma-
bly the basalmost extant monocot (APG 1998; Chase
et al. 2000), and the remaining monocots at 134 Myr
ago. According to the integrated and widely used
phylogenetic tree for the orders of flowering plants
(APG 1998), the separation of the monocot lineage
from the other magnoliids predated the branching-off
of eudicots. Therefore, Bremer’s estimate is compat-
ible with ours for the monocot–dicot split (140–150
Myr ago) and the core eudicot divergence (100–115
Myr ago).
Using a single gene (rbcL) and the NPRS meth-
od, two genes (plus 18S rRNA) and maximum
likelihood analyses, and the calibration date of
Marchantia (450 Myr ago), Sanderson (1997) and
Sanderson and Doyle (2001) estimated that the age
of crown angiosperms originated 160 and 140–190
Myr ago, respectively. Combining a three-gene da-
taset (rbcL, atpB, and 18S rRNA), the NPRS
method, and the split between Fagales and Cu-
curbitales (84 Myr ago), Wikström et al. (2001)
proposed the origin of the extant angiosperms to be
158–179 Myr old and that of eudicots to be 131–147
Myr old. These estimates are in good agreement
with ours, as the dicots we sampled are all eudicots.
Recent multigene analyses of angiosperm evolution
have revealed that the monocot–dicot divergence
was preceded by five living basal dicot lineages, the
Amborellaceae, the Nymphaeales, and a group in-
cluding Illiciaceae, Trimeniaceae, and Austrobailey-
aceae (i.e., the so-called ANITA group) (Qiu et al.
1999; PS Soltis et al. 1999; DE Soltis et al. 2000; see
Fig. 1A), and an extinct basal angiosperm lineage,
the Archaefructaceae (Sun et al. 2002). Therefore,
previous estimates for angiosperms’ origin based on
the monocot–dicot split have underestimated the age
of angiosperms themselves. The above authors’ es-
timates are consistent with ours if we postulate that
approximately 20 (=160 ) 140) to 40 (=190 ) 150)
Myr separates the angiosperm origin and the split
between the ancestors of the monocot and eudicot
lineages.
Our estimated date for the origin of core eudicot
lineages is 100–115 Myr ago (Table 5). This is earlier
than the many documented fossil-based estimates for
core eudicots, such as a possible Rhamnaceae/Rosa-
ceae (both are rosids, represented here by our sam-
ples: Lotus, Medicago, Arabidopsis, and Oenothera)
from the early Cenomanian (94–97 Myr [Basinger
and Dilcher 1984]), 89 Myr for the Caparales (rep-
resented by our sample: Arabidopsis), 84 Myr for
Myrtales (Magallon et al. 1999) (represented by our
sample: Oenothera), and 83 Myr for the Caryophyll-
ales clade (represented by our sample: Spinacia)
(Magallón et al. 1999). In addition, our estimate for
the age of core eudicots is reasonably shorter than the
fossil age of a basal eudicot, Tetracentraceae, from
the Barremian (110–118 Myr ago [Magallón et al.
1999]). Collectively, our cp genomic data indicate
that the core eudicots’ age is also older than known
fossil records indicate.
Conclusions
We observed significant Ka rate variation in cp ge-
nome data among major tracheophyte lineages.
Therefore, the rate constancy method is not appro-
priate for dating the divergence between monocots
and dicots or the age of eudicots, especially if fast-
evolving monocots are included. Using cp genome
data, we demonstrated that the Li–Tanimura method
gives estimates that better reflect the known evolu-
tionary sequence of tracheophyte lineages and cor-
respond well with the fossil records of calibration
points we used.
Combining our estimates calibrated by two known
fossil nodes and the Li–Tanimura method, we pro-
pose that the monocot lineage branched off from
dicots 140–150 Myr ago, in the late Jurassic to early
Cretaceous, and that the core eudicots radiated 100–
115 Myr ago, between the Albian and the Aptian of
Cretaceous. These estimates are in accordance with
those of Sanderson (1997) and P. S. Soltis et al.
(2002), who analyzed one to three genes and used
MP and ML branch lengths with the NPRS method.
In summary, methods that accommodate unequal
rates give smaller estimates than the rate constancy
method and appear to agree well not only with one
another, but also with the recently documented fossil
evidence.
Our results confirm all previous conclusions that
molecular data indicate a pre-Cretaceous origin for
angiosperms, but our estimates for the monocot–
dicot divergence postdate previous estimates based
on the molecular clock hypothesis by at least 50 Myr
(=200–150 Myr ago).
Acknowledgments. We thank Robert Friedman for critical com-
ments on an early version of the manuscript and Yoshihiro
Matsuoka and Shu-Shin Wu for help with the gene group assign-
ment for the three grasses and other taxa. We also thank the two
reviewers’ critical and valuable comments and suggestions. This
work was supported in part by National Science Council Grant
NSC912311B001103, and Academia Sinica Grant IB91 to S.M.C.,
and NIH Grant GM30998 to W.H.L.
Appendix
Appendix Table A1 continues on next page.
Table A1. Names and sizes of protein-coding genes in the chloroplast genomes of the 12 species (Table 2) analyzed in this study

Taxon

Genea Marchantia Psilotum Pinus Triticum Oryza Zea Lotus Medicago Arabidopsis Spinacia Nicotiana Oenothera
b c
accD 951 933 966 — — — 1,506 1,512 1,467 1,569 1,539 1,317
(ORF316)
atpA 1,524 1,527 1,485 1,515 1,524 1,524 1,533 1,536 1,524 1,296 1,524 1,518
atpB 1,479 1,479 1,479 1,497 1,497 1,497 1,497 1,497 1,497 1,497 1,497 1,497
atpE 408 402 414 414 414 414 402 402 399 405 402 402
atpF 555 555 555 552 543 552 555 552 555 555 555 555
atpH 246 246 246 246 246 246 246 246 246 246 246 246
atpI 747 747 747 744 744 744 744 738 750 744 744 744
ccsA 963 933 963 969 966 966 972 972 987 972 942 960
(ORF320) (ORF320) (ycf5) (ORF321) (ORF321) (ycf5) (ycf5) (ycf5) (ycf5) (ycf5) (ycf5)
cemA 1,305 1,350 786 693 693 693 690 690 690 702 690 690
(ORF434) (ycf10) (ORF261) (ORF230) (ycf10) (ORF229) (ycf10)
chlB 1,542 — 1,533 — — — — — — — — —
(ORF513)
chlL 870 — 876 — — — — — — — — —
(frxC)
chlN 1,398 — 1,404 — — — — — — — — —
(108667–110064)
chlP 612 597 591 651 651 651 591 588 591 591 591 750
(ORF203) (ORF216)
cysA 1,113 — — — — — — — — — — —
(mbpX)
cysT 867 — — — — — — — — — — —
(ORF288)
infA 237 243 237 342 324 324 — — — 177 Wd —
matK 1,113 1,512 1,548 1,629 1,629 1,635 1,527 1,521 1,581 1,518 1,530 1,539
(ORF370i) (ORF542)
ndhA 1,107 1,116 — 1,089 1,089 1,089 1,092 1,077 1,083 1,098 1,092 1,092
(ndh1)
ndhB 1,504 1,491 Ye 1,533 1,533 1,533 1,164 1,164 1,164 1,533 1,533 1,533
(ndh2)
ndhC 363 363 Y 363 363 363 363 363 363 363 363 363
(ndh3)
ndhD 1,500 1,545 Y 1,503 1,503 1,503 1,494 1,494 1,503 1,383 1,503 1,503
(ndh4)
ndhE 303 321 Y 306 306 306 306 306 306 306 306 306
(ndh4L)
ndhF 2,079 2,223 — 2,220 2,205 2,217 2,244 2,235 2,241 2,229 2,223 2,211
(ndh5)
ndhG 576 567 — 531 531 531 531 531 531 531 531 531
(ORF191)
435

Continued
Table A1. Continued
436

Taxon

Genea Marchantia Psilotum Pinus Triticum Oryza Zea Lotus Medicago Arabidopsis Spinacia Nicotiana Oenothera

ndhH 1,179 1,182 Y 1,182 1,182 1,182 1,182 1,182 1,182 1,182 1,182 1,182
(ORF392) (ORF393)
ndhI 552 498 Y 543 537 543 486 486 519 513 504 498
(frxB) (ORF178)
ndhJ 510 477 — 480 480 480 477 477 477 477 477 477
(ORF169) (ORF480)
ndhK 732 624 Y 738 741 747 693 684 678 846 744 744
(psbG) (psbG) (psbG) (psbG)
petA 963 966 960 963 963 963 963 963 963 963 963 957
petB 648 648 648 648 648 705 648 648 648 648 636 648
petD 483 483 543 483 483 483 483 483 483 483 483 483
petE 114 114 114 114 114 114 114 114 114 114 114 114
(ORF37) (petG) (petG) (petG) (petG) (petG) (petG) (petG) (petG) (petG)
petL 96 96 189 96 96 96 96 96 96 96 96 96
(ORF31) (ORF62b) (ORF31) (ORF31) (ycf7) (ORF31)
petN 90 90 90 90 90 90 90 90 90 90 90 90
(5168–5257) (ORF29) (ycf6) (ORF29) (ORF29) (ycf6) (ycf6) (ycf6) (ycf6) (ycf6) (ycf6)
psaA 2,253 2,253 2,262 2,253 2,253 2,253 2,253 2,277 2,253 2,253 2,253 2,256
psaB 2,205 2,205 2,205 2,205 2,205 2,208 2,205 2,205 2,205 2,205 2,205 2,205
psaC 246 246 246 246 246 246 246 246 246 246 246 246
(frxA)
psaI 111 111 159 111 111 111 105 105 114 102 111 105
(ORF36b) (ORF36)
psaJ 129 129 135 129 135 129 135 135 135 135 135 132
(ORF42b) (ORF44)
psaM 99 99 93 — — — — — — — — —
(ORF32)
psbA 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062
psbB 1,527 1,527 1,527 1,527 1,527 1,527 1,527 1,527 1,527 1,527 1,527 1,527
psbC 1,422 1,386e 1,422 1,422 1,422 1,422 1,422 1,422 1,422 1,422 1,386 1,422
(1422)
psbD 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062 1,062
psbE 252 252 252 252 252 252 252 252 252 252 252 252
psbF 120 120 120 120 120 120 120 120 120 120 120 120
psbH 225 225 228 222 222 222 222 219 222 222 222 222
(ORF74)
psbI 111 111 158 111 111 111 111 111 111 111 111 111
(ORF36a) (8398–8508)
psbJ 123 123 123 123 123 123 123 123 123 123 123 123
(ORF40) (ORF40)
psbK 168 177 180 186 186 186 186 186 180 180 186 180
(ORF55) (ORF98)
psbL 117 117 117 117 117 117 117 117 117 117 117 117
(ORF38)
psbM 105 105 114 105 105 105 105 105 105 105 105 105
(ORF34)
psbN 132 132 132 132 132 132 132 132 132 132 132 132
(ORF43)
psbT 108 99 108 117 108 102 102 108 102 102 105 108
(ORF35) (ORF35) (ORF33)
rbcL 1,428 1,428 1,428 1,434 1,434 1,431 1,428 1,428 1,440 1,428 1,434 1,428
rpl2 612 834 831 822 822 822 825 792 825 819 825 825
(ORF203)
rpl14 369 369 369 372 372 372 369 369 369 366 372 369
rpl16 432 423 405 411 411 411 408 408 408 408 405 408
rpl20 351 345 360 360 360 360 366 360 354 387 387 393
rpl21 351 390 — — — — — — — — — —
rpl22 360 351 429 447 450 447 — — 483 600 468 414
rpl23 276 273 276 282 282 282 282 282 282 W 282 282
(ORF42)
rpl32 210 183 213 192 192 180 153 180 159 174 168 156
(ORF69) (ORF63)
rpl33 198 198 207 201 201 201 201 201 201 201 201 201
rpl36 114 114 114 114 114 114 114 114 114 114 114 114
(secX)
rpoA 1,023 1,023 1,008 1,020 1,014 1,020 1,002 1,002 990 1,008 1,014 1,104
rpoB 3,198 3,201 3,228 3,321 3,228 3,228 3,213 3,213 3,219 3,213 3,213 3,219
rpoC1 2,055 2,025 2,091 2,052 2,049 2,052 2,049 2,061 2,043 2,034 2,067 2,040
rpoC2 4,161 4,227 3,675 4,440 4,542 4,584 3,999 4,145 4,031 4,086 4,179 4,161
rps2 708 720 705 711 711 711 711 711 711 711 711 711
rps3 654 663 654 720 720 675 657 636 657 657 657 657
rps4 609 600 597 606 606 606 606 606 606 606 606 612
rps7 468 468 468 471 471 471 468 474 468 468 468 468
rps8 399 399 399 411 411 411 405 405 405 405 405 417
rps11 393 393 393 432 432 432 417 417 417 417 426 435
rps12 372 372 372 369 375 375 372 372 372 372 372 372
rps14 303 303 300 312 312 312 303 303 303 303 303 303
rps15 267 261 267 273 273 237 273 276 267 273 264 264
rps16 — — — 258 189 258 243 — 240 267 258 267
rps18 228 228 303 513 492 513 315 297 306 306 306 306
rps19 279 279 279 282 282 282 279 279 279 279 279 279
ycf1f 3,207 5,112 5,271 — — — Y — 1,032 5,502 1,053 7,305
(ORF1068) (ORF1756) (ORF350)
ycf2g 6,411 6,942 6,165 — — — 6,897 5,658 6,885 6,396 6,843 6,843
(ORF2136) (ORF2054)
ycf3 513e 516 510 513 510 513 381 381 381 498 507 477
(ORF167) (ORF169) (ORF170) (ORF170)
(378)
ycf4 555 555 555 558 558 558 603 576 555 555 555 558
(ORF184) (ORF184) (ORF185) (ORF185)
437

Continued
Table A1. Continued
438

Taxon

Genea Marchantia Psilotum Pinus Triticum Oryza Zea Lotus Medicago Arabidopsis Spinacia Nicotiana Oenothera

ycf9 189 189 189 189 189 189 189 189 189 189 189 189
(ORF62) (ORF62) (ORF62) (ORF62)
ycf12 102 102 102 — — — — — — — — —
(ORF33) (ORF33)
ycf15 — — — — — 300 204 — 234 192 264 Y
(ORF99) (140818–141021) (ORF77) (90848–91039)
ycf66 408 — — — — — — — — — — —
(ORF135)
ycf68 — — 228 435 402 405 — — — — — —
(ORF75a) (92991–93425) (ORF133) (ORF133)
ycf69 — — — 177 216 177 — — — — 396 —
(124696–124872) (ORF72) (ORF58) (ORF131)
ycf70 — — — 129 270 210 — — — — — —
(14538–14666) (ORF91) (ORF69)
ycf71 — — — 153 249 225 — — — — — —
(80773–80925) (ORF82) (ORF75)
ycf72 — — — 414 414 414 — — — — — —
(81048–81461) (ORF137) (ORF137)
ycf73 — — — 750 750 522 — — — — — —
(83758–84507) (ORF249) (ORF173)
ycf74 — — — 150 330 150 — — — — — —
(94467–94616) (ORF109) (ORF49)
ycf75 — — — — 192 192 — — — — — —
(ORF63) (ORF63)
ycf76 — — — 255 258 258 — — — — — —
(124382–124636) (ORF85) (ORF85)
Total No. genes 87 81 73 84 85 86 77 74 79 79 80 78
Total length 71,509 68,355 60,470 58,095 58,677 58,581 61,908 60,296 63,543 67,839 64,551 70,110
Total No. genes 98 Average length, 63,661 ± 4,764
a
Gene names follow those of Martin et al. (2002) and Swiss-Prot Protein Knowlegebase (2003) and each NCBI accession of a given taxon (refer to Table 2).
b
Gene length (bp) is given after each gene name under each species. Within parentheses are the position ranges (where no annotation was available but a putatively respective gene homologue was
detected using the BLASTX in NCBI), or original gene names, or ORF names in a given taxon, respectively.
c
Absence of the gene in a given chloroplast genome.
d
Pseudogene.
e
The gene length we used was different from the NCBI annotation of a given species due to an earlier stop or longer reading frame detected.
f
Martin et al. (2002) considered that this gene is not related to prokaryotic genes and designated it ycf78.
g
A FtsH-like protein gene designated ycf77 by Martin et al. (2002).

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