Dating the Monocot–Dicot Divergence and the Origin of Core Eudicots Using Whole Chloroplast Genomes

8/8/2019 Dating the MonocotDicot Divergence and the Origin of Core Eudicots Using Whole Chloroplast Genomes

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Dating the MonocotDicot 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 wereused as calibration points and for cross references.

Both the method based on the assumption of a con-

stant rate and the LiTanimura 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 monocotdicot

divergence and the age of core eudicots, especially

when fast-evolving monocots were included in the

analysis. In contrast, the LiTanimura method gaveestimates 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 LiTanimura method, we

propose that monocots branched off from dicots 140

150 Myr ago (late Jurassicearly Cretaceous), at least

50 Myr younger than previous estimates based on the

molecular clock hypothesis, and that the core eudi-

cots diverged 100115 Myr ago (AlbianAptian of

the Cretaceous). These estimates indicate that both

the monocotdicot divergence and the core eudicots

age are older than their respective fossil records.

Key words: Chloroplast genome Divergence ofmonocot 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 (90100 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

J Mol Evol (2004) 58:424441DOI: 10.1007/s00239-003-2564-9

Correspondence to: Shu-Miaw Chaw; email: smchaw@sinica.

edu.tw


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volved much earlier but went undetected), and (3)

he suggestion that the evolution of angiosperms wasriggered by a particular set of environmental con-

itions, and/or biotic interactions (such as co-evolu-

ion with faunal groups) (Willis and McElwain 2002).

Is the origin of angiosperms actually much older

han the known fossil record? Since Ramshaw et al.s

1972) first application of molecular data to address

his question, three decades have passed. In the in-

erim, molecular phylogenetic studies and critical

ossils of derived angiosperms from older geological

eposits (Magallo n et al. 1999; Wikstro m et al. 2001)

ave opened up an opportunity to readdress the age

nd evolution of angiosperms. Although all previous

stimates of the monocotdicot divergence (Table 1)

redate angiosperms fossil records, they are highly

ariable, ranging from 140190 Myr (Goremykin

t al. 1997; Sanderson 1997; Wikstro m et al. 2001;

anderson and Doyle 2001) to 200 Myr (Ramshaw

972; Wolfe et al. 1989; Laroche et al. 1995; Yang

t al. 1999) or even 300320 Myr (Martin et al. 1989,

993; Brandl et al. 1992).

Traditionally, the angiosperms were subdividednto two classes, Liliopsida (the monocots) and

Magnoliopsida (the dicots) (Cronquist 1988). How-

ver, this subdivision was first refuted by rbcL and

8S rRNA gene phylogenies (Chase et al. 1993; Chaw

t 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 (110118 Myr ago) (Magallo n

et al. 1999), as well as core eudicots, such as a pos-

sible Rhamnaceae/Rosaceae (rosids) from the early

Cenomanian (9497 Myr ago [Basinger and Dilcher

1984]). It has also been suggested that the date of

diversification of core eudicots was underestimated.

Wikstro m et al. (2001) have examined this issue

(Table 1) with nuclear 18S rDNA and two cp (rbcL

and atpB) genes. We now provide additional evidenceby 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

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

Reference Gene used

Reference point

(timea of divergence; Myr) Estimated timea (Myr)

Ramshaw et al. (1972) Cytochrome cb Mammalsbirds (280) 220240

Martin et al. (1989) nrc GapC, CHS Animalyeast (1000) 319 35

Drosophilavertebrates (600)

Mammalschicken (270)

Humanerat (85)

Wolfe et al. (1989) 12 cpc genes Bryophyteangioaperm (350450) 170230Maizewheat (5070) 150260

nr 26S, 18S rRNA Plantanimal (1000) 200250, 200210

randl et al. (1992) cp tRNA Maizewheat (5070)

Tracheophytebryophyte (350450) 230350

Plantanimal (1000)

Martin et al. (1993) nr GapC, cp rbcL Bryophytespermatophyte (450) 300

Coniferangiosperm (330)

aroche et al. (1995) 12 mtc genes Maizewheat (5070) 170238

VicieaePhaseoleae (4565) 157226

Goremykin et al. (1997) 58 cp genesb Bryophytespennatophyte (450) 160 16

anderson (1997) rbcL Marchantia (450) [160215]d

Yang et al. (1999) mt 1st intron of nad Maizewheat (5070) 170235

anderson & Doyle (2001) rbcL, 18S rRNA Land plant (450) 140190Wikstro m et al. (2001) cp rbcL, atpB FagalesCucurbitales (84) [158179]

nr 18S rDNA [131147]e

The unit of time is millon years ago (or before present).

The translated amino acid sequence was used.

cp, chloroplast; mt, mitochondrial; nr, nuclear.

The age of extant angioaperms.

The origin date of eudicots.

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(Palmer 1985a, b; Wolfe et al. 1989; Clegg et al.

1994). Moreover, concatenating sequences frommany 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

was due to rate variation across lineages. In order to

mitigate this problem we used mean branch lengths ofthe sampled monocots and dicots.

The focus of this study is to estimate the dates of

the monocotdicot split and the origin of core eudi-

cots using a large cp genomic dataset. The date of the

monocotdicot 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 Tanimuras (1987) unequal-rate

method (hereafter the LiTanimura 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 LiTanimura method for its simplicity. The

method uses lineages in which the molecular clockholds 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.

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 PamiloBianchi

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

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.

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Data and Methods

Database Search for Cp Genome Sequences

ndividual genes of the 12 published cp genome sequences (Table

) were downloaded from GenBank, National Center for Bio-

echnology Information (NCBI). Nomenclatures of the cp pro-

ein-coding genes complied by Hallick and Bairoch (1994), Stoebe

t al. (1998), Martin et al. (2002), and Swiss-Prot Protein

Knowledgebase (2003) were used as guides. When synonyms were

ncountered, their sequence homologies with the typified names

ere carefully verified. Two homology criteria were considered:

) the alignable length between two proteins is larger than 80%

f the longer sequence, and (2) the sequence identity in the

ligned region is at least 40% if L > 150, or at least 0.06 +

8L)0.032 (1 exp()L/1000)) (Rost 1999; Gu et al. 2002). Note that we

aise the identity to 40% instead of 30% because the taxa we

ampled are comparatively recent and cp genes are highly con-

erved (Wolfe et al. 1989).

Since the sequence of Medicago was not annotated, its protein-

oding genes were annotated using the Nucleotide queryProtein

atabase (BLASTX) algorithm at NCBI with each known gene

om Lotus as query. If a particular gene was missing from Lotus,

hat gene from the rest of the 10 taxa was used instead. Open

eading frames annotated by us were also verified using the BLAST

sequences algorithm and the Nucleotide queryTranslated db

lgorithm in NCBI against the corresponding gene and the whole

enome of Arabidopsis, respectively. A query sequence with more

han 40% identity to the specific known genes was then considered

s a putative homologous gene. A remnant of the accD gene in the

ce was reported previously (Hiratsuka et al. 1989) but could not

e 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 ourBLAST 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 thepresent 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.

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

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

ryophyte

Marchantiaceae Marchantia polymorpha NC_001319 (Aug 2002)/Ohyama et al. (1986)

etridophyte

Psilotaceae Psilotum nudum AP004638 (Nov 2002)/Wakasugi et al. (2000)

Gymnoaperm

Pinaceae Pinus thunbergii NC_001631 (Sep 2002)/Wakasugi et al. (1994)

AngiospermsMonocots

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)

Ranks of species follow the NCBIs Taxonomy Guide.

Data modified from http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html (vers. 20 Dec 2002).

No gene annotation in this accession.

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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

PamiloBianchiLi 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 Neis (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 thatbranch is considered significantly higher than 0 (Kumar et al.

2001). To compare the evolutionary rates of sampled fern, pine,

monocot, and dicot lineages, Tajimas 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 asreference nodes: (C1) the Psilotum (fern)seed plant split (400420

Myr old [Pryer et al. 2001]); (C2) the Pinus (conifer)angiosperm

split (280310 Myr old); and (C3) the maizewheat split (5060

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 earlymid Devo-

nian (ca. 400420 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 (206248 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 (215310 Myr ago). The

earliest fossil evidence of trees bearing the typical conifers bisac-

cate pollen that germinates distally dates from the late Carbonif-

erous to early Permian (ca. 250290 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 280310 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 PaleoceneEocene deposits, about 5060 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 5070 Myr ago estimate of a maizewheat

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 5060 Myr as a reasonable estimate of the

maizewheat split.

Results

Cp Genome Data

The concatenated lengths of all known cp functional

protein-coding genes (Appendix 1) in the 12 sampledspecies (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-

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on positions (v2 test, p = 0.793 and 0.981) but not

o at the third codon positions (p < 0.000). The Gontent is particularly high at the first codon posi-

ions in all taxa and Marchantia much prefers the use

f 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-

ot (0.156) and the dicot (0.158) lineages is small.

These data are suggestive of stringent selective con-

traints on amino acid substitutions and correlate

well with the observed higher GC contents at the first

nd 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

t al. (1999). Figure 1B is a NJ tree reconstructed with

Ka values using Marchantia as the outgroup. The

opology of this tree strongly indicates that, to the

xclusion of the fern (Psilotum) lineage, the seed

lants form a monophyletic clade, within which the

onifer (Pinus) lineage and the angiosperms comprise

wo separate subgroups. The sampled angiosperms

re subdivided into two well-supported lineages, the

monocots and the eudicots. Both bootstrap and in-

erior 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 numbersof transitions and transversions (uncorrected P) were

plotted against corrected (Kimuras 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

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

odon 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

st 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

nd 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

rd 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

The start and stop codons were not included in analysis. Data on Marchantia are before the slash and the average of 11 sampled

acheopytes is after the slash and presented as mean SE (coefficient of variation).

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.

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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 ricewheat 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 ricewheat clade.

Phylogenetic Distribution of Cp Genes DuringTracheophyte 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 (ndhAK; 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-

Fig. 3. Relative branch lengths (based on Ka values) of monocots

and dicots, using Marchantia (A), Psilotum (B), and Pinus (C) asoutgroup, 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.

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ver, we found that the grass lineage has also recruited

ine novel genes; one of them,ycf68, is shared with the

ine lineage, and the remaining eight, ycf6976, are

nique. Functions of these genes are not known yet

nd they have no detectable homology to prokaryotic

enes (Martin et al. 2002). Except for spinach, all

ampled eudicots have lost the translational initiation

actor 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

ecome defunct in about 24 separate angiosperm lin-

ages, including almost all rosid species.

Nucleotide Substitution Rates

Before applying molecular calibration, we assessedhe assumption of rate constancy. Fig. 1B shows that

he branches from the calibration point C1 leading to

he Psilotum (fern) lineage and the Pinus lineage are

ot equal in length. The NJ trees in Figs. 3B, C, and

D, using Marchantia, Pinus, and Psilotum as the

utgroup, respectively, also indicate that the Ka rates

n the monocot and the dicot lineages are unequal.

The monocot lineage has evolved faster than the di-

ots, by 39.6, 37.3, and 32.3%, respectively, for the

hree outgroups. In Fig. 1B the branches from node

A leading to Arabidopsis, Spinacia, and Nicotiana are

trikingly shorter than those leading to the other

hree dicots and the monocots. Tajimas relative rate

est using rice, Marchantia, Psilotum, and Pinus as

utgroups, respectively, confirmed this observation

all ps < 0.001). However, exclusion of the above

hree slower dicot lineages (gray lines in Figs. 3BD)

ed to even higher estimates of divergence dates (data

ot shown). We therefore used the entire dataset.

By applying the equation, r = K/(2T), where K is

he distance and T is the divergence time between the

wo taxa compared, nonsynonymous rates were cal-

brated and are shown in Table 4. Based on the three

ivergence events, C1, C2, and C3, and the dataset

with all six dicots, the Ka rates are 0.2150.225 10)9,

.2320.257 10)9, and 0.1640.197 10)9 substi-

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 coniferangiosperms Ka rate is the

highest.

Dates of the MonocotDicot Divergence and the

Origin of Core Eudicots

Molecular Clock or Rate Constancy Method. The

date of the monocotdicot 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 monocotdicot divergence, 206 5217 6, 180 7 200 7, and 237 5285 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 Ksvalues 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

7185 8 and 149 2181 3 Myr ago (Table

4), respectively. These two estimates are close to each

other, and their average is 170 Myr ago.

LiTanimura 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 LiTanimura 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

able 4. Estimates of the monocotdicot divergence and the age of core eudicots based on the constant rate method

Outgroup Calibration event (fossil dates; Myr) Ka Rateb (10)9) Ka Time (Myr)

Monocotdicot divergence

Marchantia C1: Fernseed plant divergence (400420) Ka: 18.03 0.22 0.2150.225 Ka: 9.30 0.24 206 5217 6

silotum C2: Coniferangiospenn divergence (280310) Ka: 14.40 0.29 0.2320.257 Ka: 9.28 0.34 180 7200 7

inus C3: Maizewheat divergence (5060) Ka: 1.97 0.23 0.1640.197 Ka: 9.35 0.29 237 5285 9

Origin of core eudicots

inus C3: Maizewheat divergence (5060) Ka: 1.97 0.23 0.1640.197 Ka: 6.08 0.26 154 7185 8

Monocots C3: Maizewheat divergence (5060) Ks: 12.10 0.11 1.0081.210 Ks: 36.06 0.51 149 2181 3

K denotes the number of substitutions per 100 synonymous ( Ks) or nonsynonymous (Ka) sites between pair of taxa or groups.

Rate (r) is defined as the number of substitutions per site per year, r = K/(2T) (Li and Grauer 1991).

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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 137152 Myr ago, which is derivedfrom (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 104115 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

monocotdicot divergence (node A in Fig. 1 and Fig.

4) was estimated to be (400 or 420) (0.90 + 2.85)/10.4 = 144151 Myr ago, and the origin of core

eudicots was estimated to be (400 or 420) 2.85/10.4

= 110115 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

100115 Myr ago.

Discussion

Rate Variation Among Tracheophyte Lineages

Our phylogenetic analyses (Figs. 1B, 3, and 4) in-

dicate that nonsynonymous substitution rates of cp

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

Tajimas 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 (Tajimas relative rate test:

p < 0.001).

As revealed in Fig. 1B and Figs. 3BD, the Ka

rate in the grass lineage has evolved much fasterthan 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 Karates. 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-

Table 5. Ages of nodes (Myr) inferred from the phylogenetic tree

in Fig. 3 using the LiTanimura method (1987)

Node C1 (400420 Myr) C2 (280310 Myr)

C1 a 380421

C2 295309

A l44151 137152

B 110115 104115

a Nonapplicable.

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 monocotdicot 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.

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omes is quickly increasing, this trend may be re-

ested soon.

Significant rate variation in the cp genomes of the

racheophyte lineages is also consistent with the fin-

ing of P. S. Soltis et al. (2002), who studied one

uclear and three plastid genes using MP analyses. In

ummary, the molecular clock hypothesis does not

old for the Ka rates among the cp genomes of tra-

heophyte plants.

Reference Fossil Dates and the Phylogenetic Tree

Obtained

n the Data and Methods section we have carefully

ross-examined the three fossil dates by adopting

pdated phylogenies and documented fossil records.

Bremer (2000, pp 4709, 4710) suggested that in

hylogenetic dating rate calibration rather than

nequal substitution rates is the major source ofrror and is behind the discrepancies in earlier es-

imates of monocot and flowering plant evolution.

ndeed, in Table 4 the three calibrated rates based

n the molecular clock are discrete, and the ob-

ained dates for monocotdicot divergence do not

gree with one another. To evaluate if the fossil

ates and the cp Ka rates corresponded well with

ach other with respect to the two dating methods,

we also used the divergence rates and the Ka dis-

ances (Table 4) from the fernseed plant and con-

ferseed plant splits to date the others divergences.The rate constancy method led to an estimate of

50390 Myr ago for the former event and 320335

Myr ago for the latter. These two estimates differ

widely from the fossil records. In contrast, the di-

ergence times (Table 5) of these two events esti-

mated from the LiTanimura method are highly

ompatible with the paleobotanical data.

Sanderson and Doyle (2001) proposed that (1)

iases in the data or the statistical estimation

method used, (2) variation in rate across sites which

causes sequence divergences to be estimated in-

orrectly, and (3) incorrect phylogenies are the

nderlying sources of error in molecular dating. P.

. Soltis et al. (2002) added that inadequate sam-

ling of taxa...can compound the problem. The

ame concern could be raised about the results we

resent here. However, the effect of these problems

s likely to have been considerably reduced by the

ampling of 12 evolutionary successive land plants

Table 2; including all three living subclasses of

udicots), the use of 61 genes (>39,000 bp long)with different functions from the complete cp ge-

omes, and the highly reliable NJ tree (Fig. 1B),

which is consistent with the NJ tree of Goremykin

t al. (1997), inferred from concatenating 14,295

mino acids of cp genomes.

Comparison of Estimates from the Molecular Clock

and LiTanimura 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 LiTanimura 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 clockmethod 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

monocotdicot 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

monocotdicot split and the origin of core eudicots,

we concluded that the LiTanimura 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 LiTanimura method with

those from other methods. Goremykin et al. (1997)

used a very similar framework of the LiTanimura

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 1020 Myr

(Table 5). Based on our cp genome data (Figs. 1B and

3A), the Nicotiana lineage has the slowest Ka and Ksrates among the sampled dicots but was used as half

of the denominator by Goremykin et al. (1997) in

estimating the monocotdicot divergence time. Since

our estimates of the dates for the monocotdicotlineage 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

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group of terminal taxa to their common node (which

has a known fossil age) for calculating the change

rate (distance/age by Bremers 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 usedphylogenetic 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, Bremers estimate is compat-

ible with ours for the monocotdicot split (140150

Myr ago) and the core eudicot divergence (100115

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) andSanderson and Doyle (2001) estimated that the age

of crown angiosperms originated 160 and 140190

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), Wikstro m et al. (2001)

proposed the origin of the extant angiosperms to be

158179 Myr old and that of eudicots to be 131147

Myr old. These estimates are in good agreement

with ours, as the dicots we sampled are all eudicots.

Recent multigene analyses of angiosperm evolutionhave revealed that the monocotdicot 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 monocotdicot 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 100115 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 (9497 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)

(Magallo n 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 (110118 Myr ago [Magallo n 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 LiTanimura 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 LiTanimura method, we pro-

pose that the monocot lineage branched off from

dicots 140150 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 monocotdicot divergence postdate previous estimates based

on the molecular clock hypothesis by at least 50 Myr

(=200150 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.

434


12/18

p

gg

p

g

p

(

)

y

y

Taxon

Genea

Marc

hantia

Psilotum

Pinus

Triticum

Oryza

Zea

Lotus

Medica

go

Ara

bidopsis

Spinacia

Nicotiana

Oenot

hera

accD

951b

933

966

c

1,506

1,512

1,467

1,569

1,539

1,317

(ORF

316)

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

(ORF

320)

(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

(ORF

434)

(ycf10)

(ORF261)

(ORF230)

(ycf10)

(ORF229)

(ycf10)

chlB

1,542

1,533

(ORF

513)

chlL

870

876

(frxC)

chlN

1,398

1,404

(108667110064)

chlP

612

597

591

651

651

651

591

588

591

591

591

750

(ORF

203)

(ORF216)

cysA

1,113

(mbpX

)

cysT

867

(ORF

288)

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

(ORF

370i)

(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

(ORF

191)

Continued

435


13/18TableA1.

Continued

Taxon

Genea

Marc

hantia

Psilotum

Pinus

Triticum

Oryza

Z

ea

Lotus

Medicago

Ara

bidopsis

Spinacia

Nicotiana

Oenot

hera

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

(ORF

169)

(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

(ORF

37)

(petG)

(petG)

(petG)

(petG)

(petG)

(petG)

(petG)

(petG)

(petG)

petL

96

96

189

96

96

96

96

96

96

96

96

96

(ORF

31)

(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

(ORF

36b)

(ORF36)

psaJ

129

129

135

129

135

129

135

135

135

135

135

132

(ORF

42b)

(ORF44)

psaM

99

99

93

(ORF

32)

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

(ORF

74)

psbI

111

111

158

111

111

111

111

111

111

111

111

111

(ORF

36a)

(83988508)

psbJ

123

123

123

123

123

123

123

123

123

123

123

123

(ORF

40)

(ORF40)

psbK

168

177

180

186

186

186

186

186

180

180

186

180

(ORF

55)

(ORF98)

psbL

117

117

117

117

117

117

117

117

117

117

117

117

(ORF

38)

436


14/18psbM

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

(sec

X)

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

51

3e

516

510

513

510

513

381

381

381

498

507

477

(ORF167)

(ORF169)

(ORF170)

(ORF170)

(37

8)

ycf4

555

555

555

558

558

558

603

576

555

555

555

558

(ORF184)

(ORF184)

(ORF185)

(ORF185)

Continued

437


15/18TableA1.

Continued

Taxon

Genea

Marchantia

Psilotum

Pinus

Triticum

Oryza

Zea

Lotus

Medicago

Ara

bidopsis

Spinacia

Nicotiana

Oenot

hera

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)

(140818141021)

(ORF77)

(9084891039)

ycf66

408

(ORF135)

ycf68

228

435

402

405

(ORF75a)

(9299193425)

(ORF133)

(ORF133)

ycf69

177

216

177

396

(124696124872)

(ORF72)

(ORF58)

(ORF131)

ycf70

129

270

210

(1453814666)

(ORF91)

(ORF69)

ycf71

153

249

225

(8077380925)

(ORF82)

(ORF75)

ycf72

414

414

414

(8104881461)

(ORF137)

(ORF137)

ycf73

750

750

522

(8375884507)

(ORF249)

(ORF173)

ycf74

150

330

150

(9446794616)

(ORF109)

(ORF49)

ycf75

192

192

(ORF63)

(ORF63)

ycf76

255

258

258

(124382124636)

(ORF85)

(ORF85)

TotalNo.genes

87

81

73

84

85

86

77

74

79

79

80

78

Totallength

71,509

68,355

60,470

58,095

58,677

58,581

61,908

60

,296

63,543

67,839

64,551

70,110

TotalNo.genes9

8

Averagelength,63,661

4,764

a

GenenamesfollowthoseofMartinetal.(2002)and

Swiss-ProtProteinKnowlegebase(2003)andeachNCBIaccessionofagiv

entaxon(refertoTable2).

b

Genelength(bp

)isgivenaftereachgenenameunder

eachspecies.Withinparenthesesare

thepositionranges(wherenoannotationwasavailablebutaputativelyres

pectivegenehomologuewas

detectedusingtheBLASTXinNCBI),ororiginalgenenames,orORFnamesinagiventaxon,respectively.

c

Absenceofthegeneinagivenchloroplastgenome.

d

Pseudogene.

e

Thegenelength

weusedwasdifferentfromtheNCBIannotationofagivenspeciesdueto

anearlierstoporlongerreadingfram

edetected.

f

Martinetal.(20

02)consideredthatthisgeneisnotrelatedtoprokaryoticgenesanddesign

atedityc

f78.

g

AFtsH-likepro

teingenedesignatedyc

f77byMartin

etal.(2002).

438


16/18

References

APG (Angiosperm Phylogeny Group) (1998) An ordinal classifi-

cation for the families of flowering plants. Annal Mo Bot Gard

85:531533

asinger JF, Dilcher DL (1984) Ancient bisexual flowers. Science

224:511513

ousquet J, Strauss SH, Doerksen AH, Price RA (1992) Extensive

variation in evolutionary rate of rbcL gene sequences among

seed plants. Proc Natl Acad Sci USA 89:78447848

owe LM, Coat G, dePamphilis CW (2000) Phylogeny of seed

plants based on all three genomic compartments: Extant gym-

nosperms are monophyletic and Gnetales closest relatives are

conifers. Proc Natl Acad Sci USA 97:40924097

randl R, Mann W, Sprintzl M (1992) Estimation of the monocot

dicot age through t-RNA sequences from the chloroplast. Proc

R Soc Lond B 249:1317

remer K (2000) Early Cretaceous lineages of monocots flowering

plants. Proc Natl Acad Sci USA 97:47074711

hase MW, et al. (1993) Phylogenetics of seed plants: An analysis

of nucleotide sequences from the plastid gene rbcL. Annal Ma

Bot Gard 80:528580

hase MW, et al. (2000) Higher-level systematics of the mono-cotyledons: An assessment of current knowledge and a new

classification. In: Wilson KL, Morrison DA (eds) Monocots:

Systematics and evolution. Commonwealth Scientific and In-

dustrial Research Organization, Collingwood, Australia, pp 3

16

haw SM, Zharkikh HA, Sung HM, Lau TC, Li WH (1997)

Molecular phylogeny of extant gymnosperms and seed plant

evolution: analysis of nuclear 18S rRNA sequences. Mol Biol

Evol 14:5668

haw SM, Parkinson CL, Cheng Y, Vincent TM, Palmer JD

(2000) Seed plant phylogeny inferred from all three plant ge-

nomes: Monophyly of extant gymnosperms and origin of

Gnetales from conifers. Proc Natl Acad Sci USA 97:40864091legg MT, Gaut BS, Learn GH Jr, Morton BR (1994) Rates and

patterns of chloroplast DNA evolution. Proc Natl Acad Sci

USA 91:67956801

rane PR (1988) Major clades and relationships in higher

gymnosperms. In: Beck CB (ed) Origin and evolution of gym-

nosperms. Columbia University Press, New York, pp 218272

repet WL, Feldman GD (1991) The earliest remains of grasses in

the fossil record. Am J Bot 78:10101014

ronquist A (1988) The evolution and classification of fowering

plants, 2nd ed. New York Botanical Garden, Bronx, NY

Darwin C, Darwin F, Seward AC (eds) (1903) More letters from

Charles Darwin. D. Appleton, New York

Delevoryas T, Hope RC (1973) Fertile coniferophyte remains from

the Late Triassic Deep River Basin, North Carolina. Am J Bot

60:810818

Doyle JA (1992) Revised palynological correlations of the lower

Potomac Group (USA) and the Cocobeach sequence of Gabon

(Barremian-Aptian). Cretaceous Res 13:337349

Doyle JA (1998) Molecules, morphology, fossils, and the rela-

tionship of angiosperms and Gnetales. Mol Phylogenet Evol

9:448462

Doyle JA, Donoghue MJ (1987) The origin of angiosperms: a

cladistic approach. In: Friis EM, Chaloner WG, Crane PR (eds)

The origins of angiosperms and their biological consequences.

Cambridge University Press, Cambridge, pp 1749

Gallois JL, Achard P, Green G, Mache R (2001) The Arabidopsis

chloroplast ribosomal protein L21 is encoded by a nuclear gene

of mitochondrial origin. Gene 274:179185

Gantt JS, Baldauf SL, Caline PJ, Weeden NF, Palmer JD (1991)

Transfer of rpl22 to the nucleus greatly preceded its loss from

the chloroplast and involved the gain of an intron. EMBO

J 10:30734078

Gaut BS, Muse SV, Clark WD, Clegg MT (1992) Relative rates of

nucleotide substitution at the rbcL locus of monocotyledonous

plants. J Mol Evol 35:292303

Gaut BS, Muse SV, Clegg MT (1993) Relative rates of nucleotide

substitution in the chloroplast genome. Mol Phylogenet Evol

2:8996

Gaut BS, Clark LG, Wendel JF, Clegg MT, Muse SV (1997)

Comparisons of the molecular evolutionary process at rbcL and

ndhF in the grass family (Poaceae). Mol Biol Evol 14:769777

Goremykin VV, Hansmann S, Martin WF (1997) Evolutionary

analysis of 58 proteins encoded in six completely sequenced

chloroplast genomes: Revised molecular estimates of two seed

plant divergence times. Pl Syst Evol 206: 337351

Goremykin VV, Hirsch-Ernst KI, Wolfl S, Hellwig FH (2003)

Analysis of the Amborella trichopoda chloroplast genome se-

quence suggests that Amborella is not a basal angiosperm. Mol

Biol Evol 20:14991505

GPWG (Grass Phylogeny Working Group) (2001) Phylogeny and

subfamilial classification of the grasses (Poaceae). Ann Mo Bot

Gard 88:373373

Gu Z, Cavalcanti ARO, Chen FC, Bouman P, Li WH (2002) Ex-tent of gene duplication in the genomes of Drosophila, nema-

tode, and yeast. Mol Biol Evol 19:256262

Hallick RB, Bairoch A (1994) Proposals for the naming of chlo-

roplast genes. III. Nomenclature for open reading frames

encoded in chloroplast genomes. Plant Mol Biol Rep 12:S29

S30

Hart JA (1987) A cladistic analysis of conifers: Preliminary results.

J Arnold Arbor 68:296307

Herendeen PS, Crane, PR (1995) The fossil history of the mono-

cotyledons. In: Rudall PJ, Cribb PJ, Cutler DF, Humphries CJ

(eds) Monocotyledons: Systematics and evolution. Royal Bo-

tanic Gardens, Kew, pp 121

Hiratsuka J, et al. (1989) The complete sequence of the rice ( Oryzasativa) chloroplast genome: Intermolecular recombination be-

tween distinct tRNA genes accounts for a major plastid DNA

inversion during the evolution of the cereals. Mol Gen Genet

217:185194

Hughes NF (1994) The enigma ofangiosperm origins. Cambridge

University Press, Cambridge

Hupfer H, Swiatek M, Hornung S, Hermann RG, Maier RM, Chiu

WL, Sears B (2000) Complete nucleotide sequence of the Oe-

nothera elata plastid chromosome, representing plastome I of

the five distinguishable euoenothera plastomes. Mol Gen Genet

263:581585

Ikeo K, Ogihara Y (2000) Triticum aestivum chloroplast, complete

genome (unpublished)Katayama H, Ogihara Y (1996) Phylogenetic affinities of the

grasses to other monocots as revealed by molecular analysis of

chloroplast DNA. Curr Genet 29:572581

Kato T, Kaneko T, Sato S, Nakamura Y, Tabata S (2000) Com-

plete structure of the chloroplast genome of a legume, Lotus

japonicus. DNA Res 7:323330

Kenrick P, Crane PR (1997) The origin and early evolution of land

plants. Nature 389:3339

Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA 2: Mo-

lecular evolutionary genetics analysis software. Arizona State

University, Tempe

Laroche J, Li P, Bousquet J (1995) Mitochondrial DNA and

monocotdicot divergence time. Mol Biol Evol 12:11511156

Li WH, Graur D (1991) Fundamentals of molecular evolution.

Sinauer Associates, Sunderland, MA

Li WH, Tanimura M (1987) The molecular clock runs more slowly

in man than in apes and monkeys. Nature 326:9396

439


17/18

Lin S, Wu H, Jia H, Zhang P, Dixon R, May G, Gonzales R, Roe

BA (2000) Medicago truncatula variety Jema Long A-17 chlo-

roplast, complete sequence (unpublished)

Lockhart PJ, Howe CJ, Barbrook AC, Larkum AWD, Penny D

(1999) Spectral analysis, systematic bias, and the evolution of

chloroplasts. Mol Biol Evol 16:573576

Magallo n S, Sanderson MJ (2001) Absolute diversification rates in

angiosperm clades. Int J Org Evol 55:17621780

Magallo n S, Crane PR, Herendeen PS (1999) Phylogenetic pattern,

diversity, and diversification of eudicots. Ann Mo Bot Gard

86:297372

Maier RM, Neckermann K, Igloi GL, Kossel H (1995) Complete

sequence of the maize chloroplast genome: Gene content, hot-

spots of divergence and fine tuning of genetic information by

transcript editing. J Mol Biol 251:614628

Martin W, Gierl A, Saedler H (1989) Molecular evidence for pre-

Cretaceous angiosperm origin. Nature 339:4648

Martin W, Lagrange T, Li YF, Bisanz-Seyer C, Mache R (1990)

Hypothesis for the evolutionary origin of the chloroplast ri-

bosomal protein L21 of spinach. Curr Genet 18:553556

Martin W, Lydiate D, Brinkmann H, Forkmann G, Saedler H,

Cerff R (1993) Molecular phylogenies in angiosperm evolution.

Mol Biol Evol 10:140162Martin W, Stoebe B, Goremykin V, Hansmann S, Hasegawa M,

Kowallik KV (1998) Gene transfer to the nucleus and the

evolution of chloroplasts. Nature 393:162165

Martin W, et al. (2002) Evolutionary analysis of Arabidopsis, cy-

anobacterial, and chloroplast genomes reveals plastid phylog-

eny and thousands of cyanobacterial genes in the nucleus. Proc

Natl Acad Sci USA 99:1224612251

Mathews S, Donoghue MJ (1999) The root of angiosperm phy-

logeny inferred from duplicate phytochrome genes. Science

286:947950

Matsuoka Y, Yamazaki Y, Ogihara Y, Tsunewaki K (2002) Whole

chloroplast genome comparison of rice, maize, and wheat: im-

plications for chloroplast gene diversification and phylogeny ofcereals. Mol Biol Evol 19:20842091

Millen RS, Olmstead RG, Adams KL, Palmer JD, Lao NT, Heggie

L, Kavanagh TA, Hibberd JM, Gray JC, Morden CW, Calie

PJ, Jermiin LS, Wolfe KH (2001) Many parallel losses of infA

from chloroplast DNA during angiosperm evolution with

multiple independent transfers to the nucleus. Plant Cell

13:645658

Miller Jr CN (1977) Mesozoic conifers. Bot Rev 43:217280

Miller Jr CN (1988) The origin of modern conifer families. In: Beck

CB (ed) Origin and evolution of gymnosperms. Columbia

University Press, New York, pp 448486

Muse SV, Gaut BS (1997) Interlocus comparisons of the nucleotide

substitution process in the chloroplast genome. Genetics146:393399

Nicholas KB, Nicholas HB Jr (1997) GeneDoc: Analysis and vis-

ualization of genetic variation. http://www.cris.com/Ketchup/

genedoc.shtml

Nicholas KJ, Tiffney BH, Knoll AH (1983) Patterns in vascular

land plant diversification. Nature 303:614616

Nickrent DL, Parkinson CL, Palmer JD, Duff RJ (2000) Multigene

phylogeny of land plants with special reference to bryophytes

and the earliest land plants. Mol Biol Evol 17:18851895

Ogihara Y, Isono K, Kojima T, Endo A, Hanaoka M, Shiina T,

Terachi T, Utsugi S, Murata M, Mori N, Takumi S, Ikeo K,

Gojobori T, Murai R, Murai K, Matsuoka Y, Ohnishi Y, Tajiri

H, Tsunewaki K (2002) Structural features of a wheat plastome

as revealed by complete sequencing of chloroplast DNA. Mol

Gen Genomics 266:740746

Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S,

Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi

H, Ozeki H (1986) Chloroplast gene organization deduced from

complete sequence of liverwort Marchantia polymorpha chlo-

roplast DNA. Nature 322:572574

Palmer JD (1985a) Comparative organization of chloroplast ge-

nomes. Annu Rev Genet 19:325354

Palmer JD (1985b) Evolution of chloroplast and mitochondrial

DNA in plants and algae. In: MacIntyre RJ (ed) Molecular

evolutionary genetics. Plenum Press, New York, pp 131240

Parkinson CL, Adams KL, Palmer JD (1999) Multigene analyses

identify the three earliest lineages of extant flowering plants.

Curr Biol 9:14851488

Price RA, Thomas J, Strauss SH, Gadek PA, Quinn CJ, Palmer JD

(1993) Familial relationships of the conifers from rbcL sequence

data. Am J Bot 80:172

Pryer KM, Schneider H, Smith AR, Cranfill R, Wolf PG, Hunt JS,

Sipes SD (2001) Horsetails and ferns are a monophyletic group

and the closest livingrelatives to seed plants. Nature 409:618622

Qiu YL, Lee J, Bernasconi-Quadroni F, Soltis DE, Soltis PS, Zanis

M, Chen Z, Savolainen V, Chase MW (1999) The earliest an-

giosperms: Evidence from mitochondrial, plastid and nuclear

genomes. Nature 402:404407

Rai HS, OBrien HE, Reeves PA, Olmstead RG, Graham SW

(2003)1 Inference of higher-order relationships in the cycadsfrom a large chloroplast data set. Mol Phylogenet Evol 29:350

359

Ramshaw JAM, Richardson DL, Meatyard BT, Brown RH, Ri-

chardson M, Thompson EW, Boulter D (1972) The time of

origin of the flowing plants determined by using amino acid

sequence data of cytochrome C. New Phytol 71:773779

Renzaglia KS, Johnson TH, Gates HD, Whittier DP (2001) Ar-

chitecture of the sperm cell ofPsilotum. Am J Bot 88:11511163

Rost B (1999) Twilight zone of protein sequence alignments. Pro-

tein Eng 12:8594

Saito N, Nei M (1987) The neighbor-joining method: A new

method for reconstructing phylogenetic trees. Mol Biol Evol

4:406425Sanderson MJ (1997) A nonparametric approach to estimating

divergence times in the absence of rate constancy. Mol Biol

Evol 14:12181231

Sanderson MJ, Doyle JA (2001) Sources of error and confidence

intervals in estimating the age of angiosperms from rbcL and

18S rDNA data. Amer J Bot 88:14991516

Sato S, Nakamura Y, Kaneko T, Asamizu E, Tabata S (1999)

Complete structure of the chloroplast genome of Arabidopsis

thaliana. DNA Res 6:283290

Schmitz-Linneweber C, Maier RM, Alcaraz JP, Cottet A, Herr-

mann RG, Mache R (2001) The plastid chromosome of spinach

(Spinacia oleracea): Complete nucleotide sequence and gene

organization. Plant Mol Biol 45:307315

Shinozaki K, et al. (1986) The complete nucleotide sequence of

tobacco chloroplast genome: Its gene organization and ex-

pression. EMBO J 5:20432049

Soltis DE, et al. (2000) Angiosperm phylogeny inferred from 18S

rDNA, rbcL, and atpB sequendes. Bot J Linn Soc 133:381461

Soltis PS, Soltis DE, Chase MW (1999) Angiosperm phylogeny

inferred from multiple genes: A research tool for comparative

biology. Nature 402:402404

Soltis PS, Soltis DE, Savolainen V, Crane PR, Barraclough TG

(2002) Rate heterogeneity among lineages of tracheophytes:

Integration of molecular and fossil data and evidence for

molecular living fossils. Proc Natl Acad Sci USA 99:4430

4435

Stebbins GL (1981) Coevolution of grasses and herbivores. Ann

Mo Bot Gard 68:7576

Stebbins GL (1987) Grass systematics and evolution: Past, present

and future. In: Sonderstrom TR, Hilu KH, Campbell CS,

440


18/18

Varkworth ME (eds) Grass systematics and evolution.

Smithsonian Institution Press, Washington, DC, pp 359367

tewart WN, Rothwell GW (1993) Paleobotany and the evolution

of plants, 2nd ed. Cambridge University Press, Cambridge

toebe B, Martin W, Kowallik KV (1998) Distribution and no-

menclature of protein-coding genes in 12 chloroplast genomes.

Plant Mol Biol Rep 16:243255

un G, Ji Q, Dilcher DL, Zheng S, Nixon KC, Wang X (2002)

Archaefructaceae, a new basal angiosperm family. Science

296:899904

wiss-Prot Protein Knowledgebase (2003) List of chloropl

Documents

Dating the Monocot–Dicot Divergence and the Origin of Core Eudicots Using Whole Chloroplast Genomes