GENE-37349; No. of pages: 8; 4C:
Gene xxx (2012) xxx–xxx
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Gene
journal homepage: www.elsevier.com/locate/gene
Short Communication
Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis
relatives (Brassicaceae)
Chi-Chun Huang a, 1, Kuo-Hsiang Hung b, 1, Wei-Kuang Wang a, 1, Chuan-Wen Ho a, Chao-Li Huang a,
Tsai-Wen Hsu c, Naoki Osada d,⁎, Chi-Chuan Hwang e,⁎, Tzen-Yuh Chiang a,⁎⁎
a
Department of Life Sciences, National Cheng-Kung University, Tainan 701, Taiwan
Graduate Institute of Bioresources, National Pingtung University of Science & Technology, Pingtung 912, Taiwan
Taiwan Endemic Species Research Institute, Nantou 552, Taiwan
d
Department of Population Genetics, National Institute of Genetics, Yata, Mishima, Shizuoka 411–8540, Japan
e
Department of Engineering Science, National Cheng-Kung University, Tainan 701, Taiwan
b
c
a r t i c l e
i n f o
Article history:
Accepted 20 February 2012
Available online xxxx
Keywords:
Arabidopsis
Evolutionary rates
Generation time
Nonsynonymous substitutions
Populus
Synonoymous substitutions
a b s t r a c t
Recovering the genetic divergence between species is one of the major interests in the evolutionary biology.
It requires accurate estimation of the neutral substitution rates. Arabidopsis thaliana, the first whole-genome
sequenced plant, and its out-crossing relatives provide an ideal model for examining the split between sister
species. In the study, rates of molecular evolution at markers frequently used for systematics and population
genetics, including 14 nuclear genes spanning most chromosomes, three noncoding regions of chloroplast
genome, and one intron of mitochondrial genome, between A. thaliana and four relatives were estimated.
No deviation from neutrality was detected in the genes examined. Based on the known divergence between
A. thaliana and its sisters about 8.0–17.6 MYA, evolutionary rates of the eighteen genes were estimated.
Accordingly, the ratio of rates of synonymous substitutions among mitochondrial, chloroplast and
nuclear genes was calculated with an average and 95% confidence interval of 1 (0.25–1.75): 15.77
(7.48–114.09): 74.79 (36.27–534.61). Molecular evolutionary rates of nuclear genes varied, with a range of
0.383–0.856×10− 8 for synonymous substitutions per site per year and 0.036–0.081×10− 9 for nonsynonymous
substitutions per site per year. Compared with orthologs in Populus, a long life-span tree, genes in
Arabidopsis evolved faster in an order of magnitude at the gene level, agreeing with a generation time hypothesis.
The estimated substitution rates of these genes can be used as a reference for molecular dating.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: atpB, ATP synthase beta subunit; rbcL, large subunit of the ribulosebisphosphate carboxylase; cpDNA, chloroplast DNA; Adh, alcohol dehydrogenase; CHS,
chalcone synthase; CTAB, cetyltrimethylammonium bromide; trnE, tRNA-Glu; trnT,
tRNA-Thr; trnL, tRNA-Leu; nad, NADH dehydrogenase; mtDNA, mitochondrial DNA;
nrDNA, nuclear DNA; AG, AGAMOUS; CA, gamma carbonic anhydrase; CHI, chalcone
isomerase; CO2, CONSTANS-LIKE 2; Cry2, cryptochrome 2 gene; Dfr, dihydroflavonol
4-reductase; EFE, ethylene forming enzyme; F3h, flavanone-3-hydroxylase; Fah, ferulic
acid 5-hydroxylase; PIS, PISTILLATA; ZIP, Fe-regulated transporter-like protein; nrITS,
nuclear ribosomal internal spacers; TAIR, The Arabidopsis Information Resource database; UTR, untranslated region; EMBL, The European Molecular Biology Laboratory;
PCR, polymerase chain reaction; MYA, million years ago; μ, evolutionary rate; K, the
number of substitutions; T, the divergence time; L, Arabidopsis lyrata; M, Arabidopsis
morrisonensis; H, Arabidopsis halleri; G, Arabidopsis gemmifera; T, Arabidopsis thaliana;
Ks, the synonymous substitutions; Ka, nonsynonymous substitutions.
⁎ Corresponding authors.
⁎⁎ Corresponding author. Tel.: + 886 6 2757575x65525; fax: + 886 6 2742583.
E-mail addresses: [email protected] (N. Osada), [email protected]
(C.-C. Hwang), [email protected] (T.-Y. Chiang).
1
These authors equally contributed to this work.
Ever since the ‘neutrality’ was proposed for elucidating most
evolutionary changes at the molecular level and the maintenance of
genetic polymorphisms within species/ populations, molecular dating
of evolutionary events or lineage split based on a molecular clock has
been frequently practiced. The neutral theory of molecular evolution
predicts that the rate of substitution in nucleotide or amino acid
sequences equals to the mutation rate when the mutations are
selectively neutral. Hence, if the mutation rate per year is constant
over time, the rate of substitution would become clock-like (Graur
and Li, 2000). Nevertheless, various evolutionary forces, such as
functional constraints, can cause rate heterogeneity across genes.
Besides, previous studies have shown that the rates of molecular
evolution at synonymous sites, which by definition are considered
as selectively nearly neutral, may vary greatly among organisms
(Wolfe et al., 1989). In plants, Soria-Hernanz et al. (2008) found
that annual species tend to have higher molecular evolution rates
than their perennial relatives. One of the hypotheses explaining the
heterogeneity of mutation rate among taxa is the generation time
0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2012.02.037
Please cite this article as: Huang, C.-C., et al., Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives
(Brassicaceae), Gene (2012), doi:10.1016/j.gene.2012.02.037
2
C.-C. Huang et al. / Gene xxx (2012) xxx–xxx
effect. Since most of mutations passing to the next generation are
supposed to occur during the formation of germ line cells, mutation
rate per generation, instead of per year, would be constant among
taxa, and mutation rate per year would become negatively correlated
with the generation time of organisms. Therefore, investigating rates
of molecular evolution among species with different life spans would
provide additional insights into the neutral evolution.
Over the past decades, botanists generated numerous phylogenetic
hypotheses based on molecular data with organelle noncoding spacers
or nuclear DNA introns (Slowinski and Page, 1999; Savolainen et al.,
2000; Chiang et al., 2006; Hung et al., 2009). Besides, multilocus
analyses that assess the genetic divergence within and between sister
species also provide genealogical information for inferring the
phylogeography (Stadler et al., 2005; Wright and Gaut, 2005; Zhu et
al., 2007; Zou et al., 2008; Wang et al., 2010). In addition to the
bifurcate topologies that interest the evolutionists, molecular dating
provides much useful information (Wikstrom et al., 2001). For example, the genetic variation at the atpB-rbcL spacer of cpDNA in Taiwan
populations of Pinus luchuensis is about ten times of that of the
mainland populations due to the possession of an additional clade
(Chiang et al., 2006), likely implying that Taiwan's populations might
be more ancestral than the ones in mainland. Molecular dating,
however, revealed that those clades diverged about eight million
years ago, a time long predating the formation of Taiwan. This analysis
suggested that the maintenance of the genetic polymorphisms is likely
due to secondary mergence over the glacial maxima in the island
populations. Likewise, molecular dating recovered long paraphyly
between sections of Cycas (Chiang et al., 2009).
Absolute evolutionary rates of both nuclear and organelle genes
have mostly been estimated at higher ranks, such as gymnosperms
and angiosperms (Wolfe et al., 1987; Wolfe et al., 1989; Gaut,
1998), and between genera (Gaut et al., 1996; Kay et al., 2006).
Gaut et al. (1996) and Kay et al. (2006) found that the evolutionary
rates can be highly variable among genes and between taxa.
Nevertheless, the rates were usually estimated with a limited variation range at genes, e.g., 2–8 × 10 − 9 and 1–3 × 10 − 9 synonymous
substitutions/year/synonymous site for nuclear and cpDNA genes,
respectively (Wolfe et al., 1987; Wolfe et al., 1989), with exceptions
in nuclear genes between spinach and Silene (15.8–31.5× 10− 9
substitutions/ year/ synonymous site (Wolfe et al., 1987). On the
other hand, Koch et al. (2000) estimated the rate of molecular
evolution in Arabidopsis as 1.5 × 10 − 8 substitutions/ year/ synonymous
site in Adh and CHS genes. It has been known that the mutation rates
may greatly vary among genes. Applying this fixed rate to other
molecular markers would inevitably lead to biased estimations in
molecular dating.
As the first whole-genome sequenced higher plant, Arabidopsis
thaliana (L.) Heynh. can be used to gain insights of the mechanism
of molecular evolution among related species or genera of the
Brassiccaceae (Clauss and Koch, 2006). Besides, closely related A.
lyrata (L.) O'Kane and Al-Shehbaz and A. halleri (L.) O'Kane and AlShehbaz diverging from A. thaliana (Koch et al., 2000) provide a
desirable system for estimating the evolutionary rates of molecular
markers. In addition, another model woody plant of cottonwood
Populus trichocarpa Torr. & Gray (Tuskan et al., 2006), which belongs
to a genus of the Salicaceae that contains 25–35 species of deciduous
plants, and its relatives were further chosen to compare the rate heterogeneity between trees and herbs. Tuskan et al. (2006) suggested
that nucleotide substitution rates were lower in Populus than in
Arabidodpsis, a similar trend, though with a greater difference, also
found in this study. Here we estimated the substitution rates of
molecular markers from nuclear, mitochondrial, and chloroplast
genomes of Arabidopsis. In total, four noncoding regions of organelle
DNAs, and thirteen genes that span most chromosomes and one noncoding region of nuclear DNA of Arabidopsis were selected. These
genes and noncoding regions have been widely used for phylogenetic
inferences in angiosperms. Several ortholog sequences in Populus were
also acquired from the public database to compare with the Arabidopsis
dataset. In this paper, we provide a reference of molecular evolutionary
rates in Arabidopsis. In addition, the rate difference between herbs and
trees is compared to test the generation time hypothesis.
2. Materials and methods
2.1. Sampling and experiments
For examining the genetic divergence, four wild relatives of
A. thaliana, i.e., A. halleri ssp. gemmifera, A. halleri ssp. halleri, A. lyrata
ssp. morrisonensis, and A. lyrata ssp. lyrata were included in the genetic
analysis. A single sample of each taxon, all being diploid, was chosen: A.
lyrata ssp. lyrata (North America), A. lyrata ssp. morrisonensis (Taiwan),
A. halleri ssp. halleri (Europe), and A. halleri ssp. gemmifera (Japan) (cf.
Wang et al., 2010). Young, healthy leaves were collected and dried in
silica gel.
Leaf tissue was ground to powder in liquid nitrogen and stored at
−70 °C. Genomic DNA was extracted following a CTAB procedure
(Doyle and Doyle, 1987). Using the genome sequence of A. thaliana as
a reference, widely used molecular markers from three different
genomes were selected. In total, three noncoding regions of cpDNA,
i.e., trnE-trnT, trnT-trnL spacers, and trnL intron (Baumel et al., 2002;
Taberlet et al., 2007; Melotto-Passarin et al., 2008), nad intron of
mtDNA (Soria-Hernanz et al., 2008), and fourteen regions of nuclear
DNA (thirteen genes and one nrDNA spacer) were amplified and sequenced. Genes AGAMOUS (AG), gamma carbonic anhydrase (CA),
chalcone isomerase (CHI), chalcone synthase (CHS), CONSTANS-LIKE
2 (CO2), cryptochrome 2 gene R (Cry2), dihydroflavonol 4-reductase
(Dfr), ethylene forming enzyme (EFE), flavanone-3-hydroxylase
(F3h), ferulic acid 5-hydroxylase (Fah), LEAFY, PISTILLATA (PIS), Feregulated transporter-like protein (ZIP) and nuclear ribosomal internal
spacers (nrITS) (El-Assal et al., 2001; Olsen et al., 2004; Ramos-Onsins
et al., 2004; Masuzaki et al., 2006; Flowers et al., 2009; Hung et al.,
2009). The annotated A. thaliana genome from The Arabidopsis Information Resource database [TAIR; (Rhee et al., 2003)] was used to design primers to amplify the targeted regions. To avoid super gene
families or duplicated genes in nuclear genome, all forward and reverse
primers of nuclear genes were designated to be located at 5′ UTR and 3′
UTR. Furthermore, potential paralogs were excluded from data analyses based on a criterion of the nucleotide divergence for silent sites
among Arabidopsis speices no more than 7% as a rule of thumb (Zhou
et al., 2007).
PCR amplification was carried out in 100 μL reaction using 10 ng of
template DNA, 10 μL of 10X reaction buffer, 10 μL MgCl2 (25 mM),
10 μL dNTP mix (8 mM), 10 pmole of each primer, and 5 U of Taq polymerase (Promega, Madison, USA). The reaction was programmed on an
MJ Thermal Cycler (PTC 100) as one cycle of denaturation at 95 °C for
2 min, 30 cycles of 35 s denaturation at 94 °C, 45 s annealing at 52 °C,
and 1 min 30s extension at 72 °C, followed by 10 min extension at 72 °C.
PCR products were purified by electrophoresis in a 1.0% agarose
gel using 1X TAE buffer. The gel was stained with ethidium bromide
and the desired DNA bands were excised and eluted using QIAquick
Gel Kit (Qiagen, Chatsworth, USA). Purified products were ligated to
a pGEM-T easy vector system (Promega, Madison, USA). A single
clone was chosen for nucleotide sequencing. Purified cloned products
were sequenced in both directions using BigDye chemistry (Perkin
Elmer) on ABI 3730 automated sequencer (Applied Biosystems).
Primers for sequence determination were T7-promoter and SP6promoter located on pGEM-T easy Vector termination site.
2.2. Sequence alignment and phylogenetic analyses
Nucleotide sequences were aligned with the program ClustalW
and visually examined (Larkin et al., 2007). After the alignment, the
Please cite this article as: Huang, C.-C., et al., Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives
(Brassicaceae), Gene (2012), doi:10.1016/j.gene.2012.02.037
C.-C. Huang et al. / Gene xxx (2012) xxx–xxx
3
dating of Sterck et al. (Sterck et al., 2005) suggested that poplar
sections split 8–13 MYA. Accordingly, P. tremula (section Populus)
and P. trichocarpa (section Tacamahaca) may have diverged some 10
MYA (Unneberg et al., 2005). We calculated the evolutionary rate
(μ) based on the number of substitutions (K) and divergence time
(T) between two homologous sequences (μ = K/2 T) (Graur and Li,
1999).
indels were coded as missing data. Nucleotide sequences have been
deposited in the EMBL (accession numbers HE616534–HE616549)
and GenBank (accession numbers JQ180270–JQ180381) libraries.
Nucleotide substitution models were determined with the Akaike
Information Criterion by ModelTest 3.7 (Posada and Crandall, 1998;
Posada and Buckley, 2004). Accordingly, Kimura's 2-parameter
model (Kimura, 1980) was the most optimal model and was thereby
used to estimate the genetic divergence in non-coding sequences (K).
Numbers of synonymous and nonsynonymous substitutions of the
nuclear genes were also obtained with the Li's (1993) method, an analog to Kimura's 2-parameter model. P values for differences between
groups were calculated using Mann–Whitlney U-test. NeighborJoining trees were reconstructed based on each gene or DNA
fragment (Saitou and Nei, 1987) with MEGA 5.0 (Tamura et al.,
2011). Confidence of the clade reconstruction was tested by bootstrapping with 1,000 replicates.
3. Results and discussion
3.1. Evolutionary rates of genes in three genomes in Arabidopsis
In this study, we estimated the evolutionary rates of 18 widely
used molecular markers of chloroplast, mitochondrial, and nuclear
DNAs. Thirteen selected nuclear genes contain at least one coding
exon, while organelle markers and nrITS are noncoding spacers or introns. All these genes were PCR amplified from each sample of four
Arabidopsis taxa: A. lyrata ssp. lyrata, A. lyrata ssp. morrisonensis,
A. halleri ssp. halleri, and A. halleri ssp. gemmifera. The nucleotide
sequence of A. thaliana (the Colombia ecotype) obtained from the
GenBank was used as an outgroup. Herein, we designated five
Arabidopsis taxa as lyrata (L), morrisonensis (M), halleri (H), gemmifera (G), and thaliana (T). The genetic divergence between A. thaliana
and its relatives at genes of chloroplast, mitochondrial, and nuclear
DNAs was examined. Despite the fact that the phylogenetic relationship between outcrossing relatives remains unclear due to interspecific gene flow and hybridization (Wang et al., 2010), the
divergence from each outcrossing species to A. thaliana was not affected by such genetic exchanges. In addition, because the occurrence
of the most recent common ancestors of the four out-crossing species
are much more recent than the speciation between A. thaliana and A.
lyrata-halleri, gene sequences from a single individual suffice the estimation of molecular evolution rates. As expected, A. thaliana is the
taxon most distant from other four taxa. To further test the neutrality
hypothesis, we examined the rate heterogeneity by looking at the
synonymous (Ks) and nonsynonymous substitutions (Ka) per site between speices in a pairwise manner using A. thaliana as an outgroup.
Approximately equal rates between all pairs of lineages were found in
the markers of organelle genome, indicating no deviation from the
neutrality between species (Tajima's relative rate test; P > 0.05). Besides, Ks and Ka of the nuclear genes between lineages also follow
neutrality (All P > 0.05).
The average of genetic divergence (Kimura's distance, K) between
A. thaliana and its relatives varied among three genomes. The intron
sequence of nuclear DNA (average K = 0.158) had higher nucleotide
divergence than non-coding cpDNA (average K = 0.0295)
(P = 0.003; Mann–Whitney U-test), and the cpDNA showed higher
divergence than non-coding mtDNA (Table 1 and 2). No significant
difference between the K values of introns and the Ks of exon regions
in the nuclear genome (Table 3) was found (P = 0.550; Mann–
Whitney U-test), agreeing with neutral evolution of synonymous
sites. In contrast, the values of Ka of exon regions are significantly
lower than Ks values (P b 0.001; Mann–Whitney U-test). Ka/Ks values,
which have been widely applied for assessing the selective
constraints on coding sequences (Li, 1997; Hurst, 2002), varied in a
range of 0.011–0.257 (average = 0.103) among nuclear genes.
2.3. Testing of a molecular clock
To test the heterogeneity of molecular evolutionary rates, the
divergence between A. halleri (ssp. gemmifera and ssp. halleri) and
A. lyrata (ssp. lyrata and ssp. morrisonensis) lineages was examined
using the relative rate test of Tajima (1993) implemented in the
MEGA 5.0 (Tamura et al., 2011), which determines whether there is
unequal evolution rates among different lineages (Broughton et al.,
2001). Tajima's relative rate test was conducted between species in
a pairwise manner using A. thaliana as an outgroup. The statistics of
Tajima's relative rate test is asymptotically chi-square distributed,
and can be used to specify whether the number of nucleotide differences in two lineages is significantly different. The confidence
intervals of the ratio of evolutionary rates between mitochondrial,
chloroplast, and nuclear introns were estimated by re-sampling the
same number of codons within each concatenated sequence.
2.4. Orthologous sequences of populus from GenBank
For comparing the rate heterogeneities between trees and herbs,
seven genes of Populus species orthologous to the Arabidopsis genes
were obtained from the NCBI GenBank. The accession numbers
are listed as below: CA gene: BU870620 (P. trichocarpa), DN496774
(P. tremula); CO gene: XM_002324900 (P. trichocarpa), EU823371
(P. tremula); CHI gene: BU868620 (P. trichocarpa), BU891967 (P. tremula);
CHS gene: XM_002337712, (P. trichocarpa), EU752711 (P. tremula);
Dfr gene: XM_002307631 (P. trichocarpa), BU890711 (P. tremula); EFE
gene : XM_002320451 (P. trichocarpa), DN493993 (P. tremula); Fah
gene: AJ010324, (P. trichocarpa), DN498422 (P. tremula).
Phylogenetically, A. lyrata and A. halleri are closely related with
divergence from the model species A. thaliana about 5.1–5.4 MYA
(Koch et al., 2000) or 4.2–10.9 MYA (Wright et al., 2002). Recently,
Beilstein et al. (2010) proposed a much older divergence time
between A. thaliana and A. lyrata at 13 MYA (95% highest probability
density: 8.0–17.9 MYA), which is adopted herein. In contrast, the
divergence time of the genus Populus is less clear than Arabidopsis
because of the lack of fossil. Eckenwalder (1996) estimated the divergence time between sections Populus and Tacamahaca around
5.2–23.3 MYA based on the fossil records. In contrast, molecular
Table 1
The length (L), Kimura distance (K), and molecular rate of organelle noncoding spacer/intron selected of Arabidopsis species.
Genome
Gene
Accession no. in A. thaliana
L
K
Ratea (× 10− 8)
REF
Mitochondrial
Choloroplast
nad
trnL intron
trnE-trnT
trnT-trnL
trnF-trnL
X98301
DQ313521
AP000423
AP000423
957
562
904
790
679
0.00175 ± 0.0015
0.0155 ± 0.00191
0.04175 ± 0.01063
0.03125 ± 0.00206
0.02645
0.005–0.011
0.044–0.097
0.119–0.261
0.089–0.195
This study
This study
This study
This study
Beck et al. (2008)
a
Substitutions/site/year × 10− 8
Please cite this article as: Huang, C.-C., et al., Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives
(Brassicaceae), Gene (2012), doi:10.1016/j.gene.2012.02.037
4
C.-C. Huang et al. / Gene xxx (2012) xxx–xxx
Table 2
The length (L), Kimura distances (K) and molecular rates of noncoding spacer/intron of nuclear genes of Arabidopsis species.
Gene
Accession no. in A. thaliana
Function
L
K
ratea
AG
CA
CHI
CHS
CO2
Cry2
Dfr
EFE
F3h
Fah
ITS
LEAFY
PIS
ZIP
average
AT4g18960
AT5g66510
AT5g66220
AT5g13930
AT3g02380
AY576266
AT5g42800
AT1g05010
AJ493133
AT4g36220
ATU43224
AT5g61850
AT5g20240
AT4g33020
Floral homeotic gene encoding a MADS domain transcription factor
Encodes mitochondrial gamma carbonic anhydrase.
Chalcone isomerase
Secondary metabolism. Glucosinolate biosynthesis and possible insect resistance
Homologous to the flowering-time gene CONSTANS encoding zinc-finger proteins
Cryptochrome 2 gene
Secondary metabolism. Encodes an enzyme of the phenylpropanoid pathway
Encodes 1-aminocyclopropane-1-carboxylate oxidase
Secondary metabolism. Encodes an enzyme of the phenylpropanoid pathway
Secondary metabolism. Encodes an enzyme of the phenylpropanoid pathway
Nuclear ribosomal DNA
Encodes transcriptional regulator that promotes the transition to flowering
Floral homeotic gene encoding a MADS domain transcription factor
Member of Fe(II) transporter isolog family
1141
769
408
93
143
355
179
363
90
484
559
487
1534
663
519
0.141 ± 0.002
0.136 ± 0.007
0.178 ± 0.044
0.144 ± 0.000
0.138 ± 0.027
0.208 ± 0.034
0.116 ± 0.027
0.172 ± 0.003
0.150 ± 0.019
0.133 ± 0.020
0.055 ± 0.001
0.196 ± 0.002
0.075 ± 0.002
0.366 ± 0.019
0.1577 ± 0.073
0.401–0.882
0.386–0.850
0.506–1.113
0.409–0.900
0.392–0.863
0.591–1.300
0.330–0.725
0.489–1.075
0.426–0.938
0.379–0.831
0.156–0.344
0.557–1.225
0.213–0.469
1.040–2.288
0.448–0.986
a
Substitutions/site/year × 10− 8
Besides, given all Ka/Ks values lower than 1, the evolution of these nuclear genes tend to be governed by a purifying selection (Nekrutenko
et al., 2002; Fay and Wu, 2003).
The nuclear introns evolved faster than cp- and mtDNAs, with a
ratio (average plus 95% bootstrap confidence intervals) of evolutionary rates between mitochondrial, chloroplast, and nuclear introns of
1(0.25–1.75): 15.77 (7.48–114.09): 74.79 (36.27–534.61) (Tables 1
and 2), which apparently agrees with a trend between organelle
and nuclear genomes in the previous studies (Palmer, 1985; Wolfe
et al., 1989; Drouin et al., 2008). Nevertheless, Drouin et al. (2008) estimated this ratio as 1:3:10 in seed plants, 1:2:4 in gymnosperms but
1:3:16 in angiosperms, while up to 1:3:20 in basal angiosperms.
Wolfe et al. (1987) suggested that the average synonymous rates in
mt, cp- and nuclear genes were 0.2–1.1 × 10 − 9, 1.1–2.9 × 10 − 9 and
5.8–31.5 × 10 − 9, respectively. In this study, cp- and nuclear DNA
genes evloved with rates approximating previous estimations in
plants, seemingly indicating that mtDNA genes of Arabidopsis evolved
slower than those of other angiosperms (cf. Mower et al., 2007).
3.2. Gene flow or/ and lineage sorting causing systematic inconsistency
Gene trees of the eighteen loci were reconstructed using all
informative sites (Figs. 1 and 2). If considering intron/noncoding
regions only, only five intron trees (27.8%) that were supported
with high bootstrap values (>50%) were consistent with the taxonomic hypothesis, i.e., the putative species tree of [(KM)(HG)]T
(Wang et al., 2010), while other introns yielded various phylogenies
(Fig. 1). As there existed very few informative sites in the mtDNA
nad intron (4 out of 957 bp), all out-crossing Arabidopsis remained
unresolved phylogenetically, i.e., (KMHG)T (Fig. 1r). Likewise, all
cpDNA introns failed in recovering a consistent tree (Fig. 1o-q). In
contrast to organelle DNAs, five nuclear intron/noncoding regions
(35.7%), including nrITS (Fig. 1n), recovered the putative species phylogeny. Furthermore, when considering coding regions only, seven
nuclear genes (53.8%) with higher bootstrap support (>50%)
displayed topologies agreeing with the hypothetical species tree
(Fig. 2). There exists a trend that as long as the intron regions are
able to recover the species phylogeny, the exon regions likely yield
consistent topologies, reflecting concordant evolutionary trends
between exons and introns.
In plant phylogenetics, one of the most striking issues is the
confidence of the phylogenetic trees retrieved from the three plant
genomes. In this study, fewer than half of the selected molecular
markers, eight out of eighteen (44.4%), particularly only nuclear genes,
recovered phylogenies consistent with the putative species tree.
There are several explanations for the systematic inconsistency
among loci. First, incorrect genealogies can be obtained simply by
insufficient data and/or abundant recurrent mutations. Second, the
lineage sorting of ancestral polymorphisms segregated at the time
of speciation has not been complete. And third, an inter-species
gene flow, i.e., hybridization after speciation, may distort the genealogy (Zhou et al., 2007). Furthermore, in organelle genomes, paternal
leakage, i.e., incident passage of paternal chloroplast or mitochondrial
genome to offspring, can also occur. This phenomenon has been well
documented in some plants, e.g., mitochondria in banana and chloroplasts in Silene vulgaris (McCauley et al., 2007; Pearl et al., 2009),
Table 3
The length (L), synonymous (Ks), nonsynonymous (Ka) substitutions and molecular rates of exons of nuclear genes of Arabidopsis species.
Gene
L
Ks
Ks ratea
Ka
Ka ratea
Ka/Ks
AG
CA
CHI
CHS
CO2
Cry2
Dfr
EFE
F3h
Fah
LEAFY
PIS
ZIP
Average
759
810
264
1167
1044
1833
346
972
489
1054
795
627
1035
861
0.071 ± 0.008
0.072 ± 0.004
0.222 ± 0.045
0.184 ± 0.002
0.124 ± 0.007
0.130 ± 0.008
0.188 ± 0.019
0.120 ± 0.006
0.117 ± 0.035
0.134 ± 0.005
0.116 ± 0.003
0.148 ± 0.007
0.152 ± 0.014
0.137 ± 0.042
0.198–0.444
0.201–0.450
0.620–1.388
0.514–1.150
0.346–0.775
0.363–0.813
0.525–1.175
0.335–0.750
0.329–0.731
0.374–0.838
0.324–0.725
0.413–0.925
0.425–0.950
0.383–0.856
0.005 ± 0.001
0.019 ± 0.003
0.025 ± 0.002
0.005 ± 0.001
0.015 ± 0.001
0.010 ± 0.002
0.017 ± 0.003
0.011 ± 0.002
0.001 ± 0.001
0.006 ± 0.001
0.019 ± 0.004
0.023 ± 0.003
0.017 ± 0.005
0.013 ± 0.008
0.014–0.031
0.053–0.119
0.070–0.156
0.014–0.031
0.042–0.094
0.028–0.063
0.047–0.106
0.031–0.069
0.003–0.006
0.017–0.038
0.053–0.119
0.064–0.144
0.047–0.106
0.036–0.081
0.072 ± 0.007
0.257 ± 0.030
0.114 ± 0.032
0.026 ± 0.008
0.118 ± 0.007
0.075 ± 0.021
0.090 ± 0.023
0.091 ± 0.010
0.011 ± 0.001
0.041 ± 0.006
0.166 ± 0.035
0.158 ± 0.033
0.113 ± 0.028
0.103 ± 0.0655
a
substitutions/site/year × 10− 8
Please cite this article as: Huang, C.-C., et al., Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives
(Brassicaceae), Gene (2012), doi:10.1016/j.gene.2012.02.037
C.-C. Huang et al. / Gene xxx (2012) xxx–xxx
5
a) AG
g) Dfr
m) ZIP
b) CA
h) EFE
n) ITS
c) CHI
i) F3h
o) trnL
d) CHS
j) Fah
p) trnE-trnT
e) CO2
k) LEAFY
q) trnT-trnL
f) CRY2
l) PIS
r) nad
Fig. 1. Neighbor-joining trees of eighteen introns of Arabidopsis species. (a)–(n), nuclear introns. (o)–(q), chloroplast introns. (r), mitochondrial intron. Numbers at nodes indicate
bootstrap values that are above 50%. G: A. halleri ssp. gemmifera; H: A. halleri; M: A. lyrata ssp. morrisonensis; L: A. lyrata; T: A. thaliana.
whereas being rare in Arabidopsis (Azhagiri and Maliga, 2007).
Paternal leakage consequently increases the occurrence of the latter
two processes.
Apparently, the first case occurred in the mitochondria nad intron,
which had very few informative sites (0.42% only), inevitably resulting in an unresolved tree of (KMHG)T. In contrast, three noncoding
fragments of cpDNA failed in recovering a consistent phylogeny,
although possessing a similar number of informative sites with the
nrDNA ITS. Hence, the insufficient variation would be less likely the
reason for the systematic inconsistency. Because chloroplast DNA
lacks genetic recombination, if paternal leakage or interspecies
hybridization occurred with the whole chloroplast transferred, the
three noncoding markers would display similar phylogenies. However, in our data, the phylogenies varied across the three noncoding
markers (Fig. 1), indicating paternal leakage or interspecific hybridization less likely. The most plausible explanation for the systematic
Please cite this article as: Huang, C.-C., et al., Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives
(Brassicaceae), Gene (2012), doi:10.1016/j.gene.2012.02.037
6
C.-C. Huang et al. / Gene xxx (2012) xxx–xxx
a) AG
g) Dfr
b) CA
h) EFE
c) CHI
i) F3h
d) CHS
j) Fah
e) CO2
k) LEAFY
f) Cry2
l) PIS
m) ZIP
Fig. 2. Neighbor-joining trees of thirteen nuclear exons of Arabidopsis species. Numbers at nodes indicate bootstrap values that are above 50%. G: A. halleri ssp. gemmifera;
H: A. halleri; M: A. lyrata ssp. morrisonensis; L: A. lyrata; T: A. thaliana.
inconsistency tends to be incomplete lineage sorting, which refers to
the random sorting of ancestral alleles into descendent species during
speciation. Intuitively, lineage sorting is usually the primary factor
determining patterns of allelic distribution, especially for recentdiverged species (Chiang et al., 2004).
In contrast to organelle DNAs, half of the molecular markers of nuclear DNA recovered the putative species phylogeny. As the exon regions of nuclear DNA displayed consistent patterns with the intron
regions, natural selection may not be the main reason for systematic
inconsistency, as further indicated by approximating Ka/Ks values
across genes. As described above, in addition to incomplete lineage
sorting, inter-species gene flow has to be aroused to explain the incongruent phylogenies between nuclear genes. As the inter-species
gene flow takes place frequently in plants (Lexer et al., 2005;
Heuertz et al., 2006), Arabidopsis species are less likely to be exceptional. In a multi-locus research of wild Arabidopsis, inter-specific
gene flow was observed between A. lyrata and A. halleri (Wang
et al., 2010). Given frequent gene introgression between Arabidopsis
Please cite this article as: Huang, C.-C., et al., Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives
(Brassicaceae), Gene (2012), doi:10.1016/j.gene.2012.02.037
C.-C. Huang et al. / Gene xxx (2012) xxx–xxx
species, various phylogenies among genes would not be unexpected.
Besides, genetic recombination with varying rates across chromosomes, which are correlated with the mutation rates (Yang and
Gaut, 2011), could also result in phylogenetic inconsistency between
genes.
3.3. Rate heterogeneities of nucleotide substitution between genes, and
between taxa
In this study, we estimated the substitution rates per year of molecular markers of chloroplast, mitochondrial, and nuclear genomes
in Arabidopsis. In this study, we adopted the split time between A.
thaliana and wild relatives of 13 MYA (95% highest probability density: 8.0–17.9 MYA) (Beilstein et al., 2010). The divergence time was
used to calibrate the substitution rates at various genomes and various loci of the study. The estimated molecular rates varied with a
range of 0.044–0.261 × 10 − 8 substitutions per site per year in chloroplast DNA, which are about 9.29–24.29 times of those of mtDNA
(0.005–0.011 × 10 − 8) (Table 1). In nuclear DNAs, the K values for introns as well as Ks and Ka values for exons were calculated separately.
The average rates per year at noncoding and synonymous sites varied
with a range of 0.448–0.986 × 10 − 8 and 0.383–0.856 × 10 − 8, respectively, and both were faster than those of nonsynonymous sites
(0.036 × 10 − 10–0.081 × 10 − 9) (Tables 2 and 3).
Across the 13 nuclear functional genes, the molecular rates (μ) at
the synonymous sites varied with a range of 0.383–0.856 × 10 − 8 synonymous substitutions per site per year, nearly 3-fold difference
among genes. The estimated rate variation is relatively greater than
that of previous studies which yielded only 2.5-fold differences of
synonymous rates among genes (Wolfe et al., 1989; Gaut, 1998).
Here, given nearly 3-fold differences among genes, only selecting a
specific gene with a well-estimated rate to infer the molecular
dating can reduce the variance of molecular dating. For example,
without knowing the synonymous substitution rates of CHI gene
(0.620–1.388 × 10 − 8 substitutions per site per year), simply based
on an average rate for nuclear genes of 0.383–0.856 × 10 − 8 substitutions per site per year (Gaut, 1998), genetic divergence between CHI
lineages is likely to be overestimated up to three times.
Seven nuclear genes from cottonwood Populus species, a woody
model species (Tuskan et al., 2006), were chosen to compare with
Arabidopsis orthologs (Table 4). Taking 10 MYA as the divergent
time between P. trichocarpa and P. tremula, μ values for synonymous
sites of four selected nuclear genes in Populus were estimated with
a range from 0.200 to 0.515 × 10 − 8 substitutions per site per year, significantly different from those in Arabidopsis (Mann–Whitney U-test;
P = 0.029) (Table 4). The μ values for synonymous sites of Populus
species are ca. 2.90–7.48 times lower than those of Arabidopsis species
(Table 4), agreeing with the previous studies that found heterogeneous DNA substitution rates in nuclear between annuals and perennials (Andreasen and Baldwin, 2001; Smith and Donoghue, 2008).
Table 4
Molecular rates of synonymous in orthologous genes in Arabidopsis and Populus.
Arabidopsis
CA
CHI
CHS
CO2
Dfr
EFE
Fah
a
Populus
Ks
Ks ratea
Ks
Ks ratea
0.072
0.222
0.184
0.124
0.188
0.12
0.134
0.201–0.450
0.620–1.388
0.514–1.150
0.346–0.775
0.525–1.175
0.335–0.750
0.374–0.838
0
0.103
0.04158
0.06972
0.04274
0.04
0.04092
–
0.515
0.208
0.349
0.214
0.2
0.205
Substitutions/site/year × 10− 8
7
Although only seven orthologs were compared, the differences of
substitution rate were consistent among genes. This suggests that
the difference is likely to reflect the genome-wide difference between
Arabidopsis and Populus. In addition, if we apply 5 MYA divergence
time between A. thaliana and A. lyrata-halleri (Koch et al., 2000), the
difference became the order of magnitudes. The differences in synonymous substitution rate per year between Arabidopsis and Populus are
probably associated with the generation time, i.e., the time from seed
germination to the production of seeds (Ohta, 1993). The flowering
time of Arabidopsis and Populus species are about 1 and 8–20 years,
respectively (Weigel and Nilsson, 1995; Bohlenius et al., 2006;
Clauss and Koch, 2006). Apparently, the rates of molecular evolution
are low in long-life Populus species compared to short-life
Arabidopsis. The generation time hypothesis predicts different
frequencies of cell replication between plants with different life
spans. As the difference in generation time could affect substitution
rates, Arabidopsis species flowering annually may accumulate more
mutations than Populus with long juvenile, causing lineage effects
on molecular evolutionary rates between Arabidopsis and Populus
(Gaut et al., 1996; Smith and Donoghue, 2008).
Acknowledgments
This study was granted by the grants of the National Cheng Kung
University and the National Science Council, Taiwan; and KAKENHI,
Grant-in-Aid for Young Scientists (A) (22687021).
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