The Plant Journal (2013) 73, 941–951
doi: 10.1111/tpj.12089
Gene family evolution in green plants with emphasis on the
origination and evolution of Arabidopsis thaliana genes
Ya-Long Guo1,2,*
1
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences,
Beijing 100093, China, and
2
Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tu€ bingen, Germany
Received 22 May 2012; revised 1 November 2012; accepted 23 November 2012; published online 15 January 2013.
*For correspondence (e-mail [email protected] or [email protected]).
SUMMARY
Gene family size variation is an important mechanism that shapes the natural variation for adaptation in
various species. Despite its importance, the pattern of gene family size variation in green plants is still not
well understood. In particular, the evolutionary pattern of genes and gene families remains unknown in the
model plant Arabidopsis thaliana in the context of green plants. In this study, eight representative genomes
of green plants are sampled to study gene family evolution and characterize the origination of A. thaliana
genes, respectively. Four important insights gained are that: (i) the rate of gene gains and losses is about
0.001359 per gene every million years, similar to the rate in yeast, Drosophila, and mammals; (ii) some gene
families evolved rapidly with extreme expansions or contractions, and 2745 gene families present in all the
eight species represent the ‘core’ proteome of green plants; (iii) 70% of A. thaliana genes could be traced
back to 450 million years ago; and (iv) intriguingly, A. thaliana genes with early origination are under stronger purifying selection and more conserved. In summary, the present study provides genome-wide insights
into evolutionary history and mechanisms of genes and gene families in green plants and especially in
A. thaliana.
Keywords: Arabidopsis thaliana, evolutionary genomics, gene family, gene origination, green plants.
INTRODUCTION
Most genes can be grouped into gene families based on
sequence similarity. Gene family size is variable across different lineages and may have important functional outcomes related to adaptation or speciation (Lynch and
Conery, 2000; Nei and Rooney, 2005).
Various factors can alter the size of gene families (De
Bodt et al., 2005; Maere et al., 2005; Hahn et al., 2007;
Freeling, 2009; Proost et al., 2011; Tautz and DomazetLoso, 2011; De Smet and Van De Peer, 2012). First, gene
duplication or whole-genome duplication (polyploidy)
increases gene family size. Second, gene deletion
decreases gene family size. Third, the de novo creation of
genes creates new gene families. Gene duplication is an
ongoing contributor to genome evolution and is of the
same order of magnitude as the mutation rate per nucleotide site (Lynch and Conery, 2000, 2003; Gu et al., 2002;
McLysaght et al., 2002; Simillion et al., 2002; Doyle et al.,
2008; Flagel and Wendel, 2009; Van De Peer et al., 2009;
Van De Peer, 2011). While duplication and de novo creation
of genes increase the gene number, gene deletion
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd
decreases the gene number either by deletion of single
gene or several genes within segmental fragment.
Although variation in gene family has been the subject
of many studies, most analyses have focused on only one
or a few gene families across a small number of plant
genomes (Shiu et al., 2004; Chardon and Damerval, 2005;
Kim et al., 2006; Leseberg et al., 2006; Li et al., 2006; Porter
et al., 2009; Guo et al., 2011a) due to the limitation of available plant genomes or lack of robust pipelines. Recently,
two new methods have been developed to address the
evolutionary pattern of genes and gene families at the
whole-genome level, including the likelihood approach
implemented in CAFE (Computational Analysis of gene
Family Evolution) (De Bie et al., 2006) and the gene emergence approach (Domazet-Loso et al., 2007; Cai et al.,
2009; Zhang et al., 2010a; Banks et al., 2011). The likelihood approach uses a stochastic birth and death process
to model the evolution of gene family size over a phylogeny (Hahn et al., 2005, 2007; De Bie et al., 2006; Demuth
et al., 2006). In contrast, the gene emergence approach
941
942 Ya-Long Guo
maps genes to different phylogenetic ranks (internodes)
according to their emergence by similarity searches in the
representative genomes, called as ‘phylostratigraphy’,
naming phylogenetic ranks or internodes ‘phylostrata’
(Domazet-Loso et al., 2007; Prachumwat and Li, 2008; Cai
et al., 2009; Zhang et al., 2010b, 2011; Banks et al., 2011;
David and Alm, 2011). Recently, this approach has been
used to study the protein domain evolution and transcription-associated proteins as well (Lang et al., 2010; Kersting
et al., 2012; Zhang et al., 2012).
The comparison of gene families among closely related
and representative species of green plants (Plantae or
Viridiplantae), comprising land plants and green algae,
allows a comprehensive study of the genes and gene families evolution in green plants. Eight representative
genomes of green plants were compared, including two
Arabidopsis genomes (Figure 1). Here I have investigated
several fundamental questions regarding genes and gene
families evolution in green plants: (i) the distribution of the
gene family size; (ii) the rate of the gene gain and loss, the
rate of the expansion or contraction per gene family on
each lineage; and (iii) for A. thaliana genes in different
phylostrata, the variation of genes or gene families fixation
rate, the variation of functional annotation, of protein size,
of expression level, and the variation of selection strength.
In summary, the analyses of gene families flanking the
eight representative genomes of green plants provide indepth insights into the researches of evolutionary genomics about green plants.
RESULTS
Gene family size distribution
All proteins from the eight genomes are used for the following analyses, since none of them is homologous to transposable elements. Markov Cluster Algorithm (MCL)
clustering of all these proteins resulted in 55 989 gene families; where 13.6% of genes are singletons, 4.6% are grouped
into two-gene families, and 81.7% are grouped into multigene families (Table 1, see Data S1 for the detail).
The clustering of genes in each genome indicates
that the number of gene families ranges from 8692
Figure 1. Phylogeny and divergence time (million years ago) of the eight green plants.
Table 1 Numbers of gene families (and genes) in green plants
Species
Singletonsa
Two-genesb
3c
Total gene familiesd
Max_gfe
Chlamydomonas reinhardtii
Physcomitrella patens
Selaginella moellendorffii
Oryza sativa
Sorghum bicolor
Populus trichocarpa
Arabidopsis lyrata
Arabidopsis thaliana
All eight genomes
9088 (9088)
11 513 (11 513)
1717 (1717)
13 597 (13 597)
7579 (7579)
6525 (6525)
6551 (6551)
5980 (5980)
38 330 (38 330)
890 (1780)
2081 (4162)
4348 (8696)
2201 (4402)
1850 (3700)
2806 (5612)
1964 (3928)
1689 (3378)
6548 (13 096)
697 (4293)
2115 (20 241)
2627 (24 262)
2589 (37 633)
2419 (24 308)
3465 (33 235)
2592 (21 990)
2054 (17 481)
11 111 (230 225)
10 675 (15 161)
15 709 (35 916)
8692 (34 675)
18 387 (55 632)
11 848 (35 587)
12 796 (45 372)
11 107 (32 469)
9723 (26 839)
55 989 (281 651)
132
4322
1541
2208
1258
335
226
204
4322
a
The number of singletons.
The number of gene families (genes) with two genes.
c
The number of gene families (genes) contain at least three genes.
d
The number of total gene families (genes).
e
The maximum gene family size.
b
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
Evolution of gene families in green plants 943
(S. moellendorffii) to 18 387 (O. sativa); and the number of
genes within gene families varies from 15 161 (C. reinhardtii) to 45 372 (P. trichocarpa) (Figure 2a and Table 1).
In addition, the proportion of singletons in each genome
ranges from 5% (S. moellendorffii) to 59.9% (C. reinhardtii); genes in two-gene families from 7.9% (O. sativa)
to 25.1% (S. moellendorffii); and genes in multigene families from 28.3% (C. reinhardtii) to 73.3% (P. trichocarpa).
The maximum gene family size varies from 132 (C. reinhardtii) to 4322 (P. patens). Interestingly, for the closely
related species within the same genus or family, the ratios
of genes within different classes are similar, such as
O. sativa and S. bicolor, A. lyrata and A. thaliana. Most
genes grouped into multigene families in each genome
except C. reinhardtii, in which singletons takes up more
than half of total genes (59.9%). However, only 2.4% (217/
9088) of singletons in C. reinhardtii can be annotated to
Gene Ontology (GO) terms, which are related with cellular
process, metabolic process, response to stimulus, developmental process, cellular component organization, and
multicellular organismal process.
Orphan genes and loss of entire gene families
in Arabidopsis
There are many orphan genes in each genome, which do
not have homologous in other genomes. The proportions
of orphan genes in different genomes are different, 61.5%
in C. reinhardtii, while other species have a much lower
proportion, especially two dicot species A. thaliana (5.3%)
(a)
and A. lyrata (12.3%) (Figure 2b and Table 2). The maximum orphan gene family size ranges from 17 (A. thaliana)
to 4322 (P. patens); the total number of orphan gene families varies from 1328 (A. thaliana) to 10 812 (O. sativa); and
the number of genes within orphan gene families varies
from 1430 (A. thaliana) to 19 351 (P. patens). Among the
eight genomes, 16.8% (S. moellendorffii) to 89.1% (A. thaliana) of orphan gene families are singletons; 4.8% (A. thaliana) to 31.3% (S. moellendorffii) are two-gene families;
and 6.2% (A. thaliana) to 51.9% (S. moellendorffii) are multigene families.
To understand the functional characteristics of the
orphan genes, the well studied model species A. thaliana
was selected as a representative to perform GO functional
annotation. 1042 genes (72.9%) of A. thaliana orphan
genes (1430) can be annotated to GO terms, though most
(93.0%) are classified as unknown biological processes;
other terms are other cellular processes, other metabolic
processes, protein metabolism, transcription, and response
to stress.
Another class of interesting genes represents those with
homologues between one Arabidopsis species and at least
one of other six species but not in another Arabidopsis
species, which could have been deleted from the latter
Arabidopsis species. There are 98 gene families (174
genes, the minimum is 1, the maximum is 8, the average is
1.7755) only have A. lyrata genes; and 117 gene families
(163 genes, minimum 1, maximum 7, average 1.3932) only
have A. thaliana genes. Based on the unique genes in each
Arabidopsis species but shared with other species, the
gene extinction rate for the entirely lost gene families can
be calculated. Assuming the divergence time of A. thaliana
and A. lyrata to be 13 million years ago (Beilstein et al.,
2010; Guo et al., 2011b) (Figure 1) and normalized to the
total gene number, extinction rate in A. thaliana is 0.00050
(174/13 9 27 025) extinctions/million year/gene; in A. lyrata, 0.00038 (163/13 9 32 670) extinctions/million year/gene.
Gene family expansion or contraction
(b)
Figure 2. Proportions of the genes that are singletons, in two-gene families,
or in multigene families.
(a) Gene family sizes at whole-genome level.
(b) Gene family sizes of orphan genes.
The likelihood approach assumes that there is at least one
ancestral gene in the most recent common ancestor
(MRCA), thus only those gene families that are shared
between C. reinhardtii and at least one of other seven species are included for the analysis. Of the 3296 families
present in the MRCA of green plants, 551 gene families
have been lost completely in at least one species. It is 2745
gene families that are present across all the eight species
most probably represent the ‘core’ proteome of green
plants.
Assuming that each species has the same k value, the
average rate of gene turnover is estimated to be 0.001379
(gains and losses/gene/million years), the rate of gene family to either expand or contract over time. The estimated
rate of gene gains and losses implies that within a single
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
944 Ya-Long Guo
Table 2 Number of orphan gene families (and genes) in green plants
Species
Singletonsa
Two-genesb
3c
Total gene familiesd
Max_gfe
Chlamydomonas reinhardtii
Physcomitrella patens
Selaginella moellendorffii
Oryza sativa
Sorghum bicolor
Populus trichocarpa
Arabidopsis lyrata
Arabidopsis thaliana
6619 (6619)
9001 (9001)
1356 (1356)
9534 (9534)
3600 (3600)
4667 (4667)
2279 (2279)
1274 (1274)
443 (886)
700 (1400)
1263 (2526)
775 (1550)
394 (788)
688 (1376)
272 (544)
34 (68)
317 (1817)
368 (8950)
600 (4184)
503 (4961)
265 (3663)
343 (2055)
180 (1165)
20 (88)
7379 (9322)
10 069 (19 351)
3219 (8066)
10 812 (16 045)
4259 (8051)
5698 (8098)
2731 (3988)
1328 (1430)
68
4322
129
562
1258
82
99
17
a
The number of singletons.
The number of gene families (genes) with two genes.
c
The number of gene families (genes) contain at least three genes.
d
The number of total gene families (genes).
e
The maximum gene family size.
b
genome like A. thaliana, there are approximately 37.267
new duplicates and 37.267 new losses fixed every million
years (0.001379 gains and losses/gene/million years 9
27 025 genes).
The average expansions and contractions among different lineages are different (Figure 3). Along the phylogenetic ranks, most lineages expanded, including
embryophyte, tracheophyte, angiosperm, and rosid. In
contrast, lineages of C. reinhardtii, grass, and Arabidopsis
contracted, and Arabidopsis genus is the most contracted
lineage (average contraction size is 0.547).
The likelihood approach can identify individual gene
families evolving at the rate of gain or loss significantly
higher than the genome-wide average (Hahn et al., 2005).
Of the 3296 gene families, 41 are evolved non-randomly at
P < 0.0001. Of the 41 rapidly evolved gene families, only 37
contain A. thaliana genes, and 34 of which can be annotated to GO terms. Rapidly evolving gene families are
associated with various biological processes, including
other metabolic processes, other cellular processes,
response to stress, protein metabolism, other biological
processes, and response to abiotic or biotic stimulus
(Tables S1 and S2). Nine of the 41 rapidly evolving gene
families have significantly changed (P < 0.0001) on the terminal branch to A. thaliana. All of the nine gene families
significantly contracted (P < 0.0001), most of which are
affiliated with nucleotide binding, protein binding, DNA or
RNA binding, hydrolase activity, and structural molecular
activity (Table S2).
Among nine significantly changing gene families on the
A. thaliana terminal, eight of them have A. thaliana genes
and in total 43 genes, of them 34 have the orthologues in
A. lyrata. The average of dN/dS for these genes is 0.1505
(median 0.0466), lower than all proteins of A. thaliana
within the gene families (average 0.2799, median 0.1786)
(Wilcoxon rank sum test, P = 4.525 9 10 6); the average of
dS is 0.1480 (minimum 0.0427, maximum 0.3557, median
0.1398), also a little lower than all proteins of A. thaliana
within the gene families (average 0.2167, median 0.1448)
(P = 0.3325). The results indicate there is not any correlation between the gene copy variation and the sequence
divergence.
Figure 3. Expansions and contractions of gene
families in green plants. The numbers nearby
branches show the average expansion (mean
number of genes gained or lost per family,
where ‘–’ expansion is a net contraction), and
the values inside the brackets are the number
of families with expansions and contractions,
respectively.
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
Evolution of gene families in green plants 945
Origination of Arabidopsis thaliana genes
The 26 839 A. thaliana genes within gene families are
assigned to different phylogenetic ranks (phylostrata) (Figure 4a and Table 3). More than 70% genes are assigned to
Viridiplantae (40.6%) and Embryophyte (30.1%) internodes.
In addition, fixation rates of gene or gene family for A. thaliana genes, which is the rate of genes (or gene families)
appearance on different phylogenetic ranks, are calculated,
respectively (Figure 4b and Table S3). A. thaliana internode has the highest gene fixation rate (110.000 genes/
MY), while angiosperm internode has the lowest gene fixation rate (6.267 genes/MY).
For A. thaliana genes assigned to different phylostrata,
the proportion of A. thaliana genes annotated to GO terms
is 86.7% (23 282/26 839) (Figure 4c and Table S4). Among
internodes, the proportion ranges from 92.1% (rosid internode) to 57.2% (A. thaliana internode). For the unknown
function process, along the phylostrata, Viridiplantae has
the lowest proportion, but A. thaliana has the highest proportion. For most other biological processes, Viridiplantae
has the highest proportion; A. thaliana has nearly the lowest proportion (Figure S1 and Table S5). Within A. thaliana
internal, the most abundant terms are unknown biological
processes, followed by other cellular processes, other metabolic processes, and transcription.
Along phylostratigraphy from Viridiplantae to A. thaliana, the medians of protein sizes of the A. thaliana genes
in different phylostrata reduced from 413 (Viridiplantae) to
77 (A. thaliana) (Figure 4d and Table S6). While the median
of protein size for all A. thaliana proteins within the gene
families is 351 (minimum 29, maximum 5337, average
407.8). In addition, along phylostratigraphy, the proportion
of expressed genes reduced from 90.1% (Viridiplantae
internode) to 37.1% (A. thaliana internode) (Figure 5a and
Table S7); and the median of expression levels reduced
(a)
(b)
(c)
(d)
Table 3 Number of A. thaliana gene families (and genes) assigned
to each phylostratum
Phylostratum internode
Genes (%)a
Gene families
(min, max, avg)b
1 Viridiplantae
2 Embryophyte
3 Tracheophyte
4 Angiosperm
5 Rosid
6 Arabidopsis
7 A. thaliana
10 897 (40.601)
8074 (30.083)
741 (2.761)
1617 (6.025)
1076 (4.009)
3004 (11.193)
1430 (5.328)
3007 (1, 193, 3.6239)
2216 (1, 204, 3.6435)
290 (1, 28, 2.5552)
732 (1, 25, 2.2090)
484 (1, 106, 2.2231)
1666 (1, 134, 1.8031)
1328 (1, 17, 1.0768)
a
The total number of genes assigned to each phylostratum in
A. thaliana genome, and their percentage of whole genome given
in parentheses.
b
The total number of gene families assigned to each phylostratum. Within parentheses, min indicates the minimum gene family
size; max indicates the maximum gene family size; avg indicates
the average of gene family size.
Figure 4. Phylostratigraphic analysis of A. thaliana genes.
(a) Gene appearance.
(b) Gene fixation rate.
(c) Gene ontology annotation. A dashed line indicates the fraction of A. thaliana genes within gene families that can be annotated to GO biological process terms.
(d) Protein sizes. A dashed line indicates the median protein size of A. thaliana genes within gene families. Significances of the deviations from the
median protein size of A. thaliana genes within gene families using Wilcoxon rank sum test are shown in the P-value panel.
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
946 Ya-Long Guo
from 6.985 (Viridiplantae) to 2.841 (A. thaliana) (Figure 5b
and Table S7). In total, 79.2% A. thaliana genes are
expressed (21 248/26 839); and the median of expression
level is 5.988 (min 1.344, max 14.050, average 5.866).
In order to understand the divergence variation along
phylostratigraphy from Viridiplantae to A. thaliana, the
divergence between A. thaliana genes and their orthologues in A. lyrata were calculated. The medians of dN/dS
increased from 0.1389 (Viridiplantae) to 0.4556 (Arabidopsis), and the medians of dS increased from 0.1407 (Viridiplantae) to 0.1553 (Arabidopsis) (Figure 6 and Table S8).
The median of dN/dS for all proteins of A. thaliana within
the gene families is 0.1786, and the median of dS is 0.1448.
DISCUSSION
Family size distribution
Markov Cluster (MCL) algorithm, a method for clustering
proteins into groups based on similarity, rigorously tested
(a)
and validated on a number of databases (Van Dongen,
2000; Enright et al., 2002), has been used for gene family
analysis in many taxa, including human (Lander et al.,
2001), vertebrate and invertebrate (Prachumwat and
Li, 2008), and Drosophila (Drosophila 12 Genomes Consortium, 2007). In this study, using the MCL algorithm, most
genes in each genome are grouped into multigene families
except C. reinhardtii, in which singletons make up more
than half of total genes (59.9%). The predominant number
of singletons in C. reinhardtii suggests that the C. reinhardtii genome has a lower gene duplicability than other
genomes, possibly due to functional constraints or that
some genes of C. reinhardtii are too young to have accumulated duplicates like the case of vertebrate-specific
genes (Prachumwat and Li, 2008), or there are probably
some annotation artifacts in C. reinhardtii. However, only
2.4% of singletons in C. reinhardtii can be annotated to a
GO term, thus functional constraint is unlikely the case
here. For S. moellendorffii genome, in order to sufficiently
(a)
(b)
(b)
Figure 5. Expression variation of A. thaliana genes among phylostrata.
(a) Expressed genes. A dashed line indicates the expression fraction level of
A. thaliana genes within gene families.
(b) Expression level. A dashed line indicates the median expression level of
A. thaliana genes within gene families. Significances of the deviations from
the median expression level of A. thaliana genes within gene families using
Wilcoxon rank sum test are shown in the P-value panel.
Figure 6. Divergence variation of A. thaliana genes among phylostrata, estimated between A. thaliana and A. lyrata. (a) dN/dS. (b) dS.
A dashed line indicates the median dN/dS or dS of A. thaliana genes within
gene families, respectively. Significances of the deviations from the median
dN/dS and dS of A. thaliana genes within gene families using Wilcoxon
rank sum test are shown in the P-value panel, respectively.
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
Evolution of gene families in green plants 947
represent the S. moellendorffii genome, all gene models
instead of the data set with only one of the two haplotypes
are used in this study, thus in the present study for this genome, genes in two-gene families are inflated, and genes in
singletons are reduced at the same time accordingly.
In total, 2745 gene families are shared among all the
eight species, which must be the ‘core’ proteome of green
plants. This number is consistent with another recently
published study (Van Bel et al., 2012), in which 2928 core
gene families are found via a parsimony method from 25
genomes, although these two studies used different strategy to call ‘core’ proteome. Among the 2928 gene families
found in the previous study, 2207 (75%) are found in the
present study, when match the representative A. thaliana
genes of each core gene family reported in the previous
study (Van Bel et al., 2012) to the present study. Interestingly, two recent studies have indicated across green
plants, there is a relatively large and well conserved core
set of protein domains as well (Kersting et al., 2012; Zhang
et al., 2012). In addition, phylogenetic comparative analysis
indicated the expansion of transcription-associated proteins is positively correlated with the increase in morphological complexity (Lang et al., 2010).
Orphan genes are protein-coding genes that do not have
recognizable homologues in other species. Various factors
can lead to rise of the orphan genes (Domazet-Loso and
Tautz, 2003; Demuth et al., 2006; Hahn et al., 2007). Theoretically, all of these factors can be classified into six classes: (i) true loss of all functional genes; (ii) the de novo
evolution of new genes; (iii) rapid protein evolution in previously existing genes so that they are no longer identified
as being part of a pre-existing family; (iv) horizontal gene
transfer; (v) annotation artifacts; and (vi) artifacts of the
clustering process. Such genes might be involved
frequently in specific ecological adaptations or be the raw
fuel for micro-evolutionary divergence (Carroll et al., 2001;
Domazet-Loso and Tautz, 2003; Yang et al., 2009). Not surprisingly, in C. reinhardtii and P. patens, more than half of
the genes within these two genomes are orphan genes,
given some genes in C. reinhardtii and P. patens are probably not required for vascular plants. Recently, in Drosophila, it has been demonstrated that orphan genes can
quickly become essential and play an essential role in
development (Chen et al., 2010).
Similar gene gain/loss rate between green plants
and animals
The likelihood method implemented in CAFE version 2.0 (De
Bie et al., 2006) is a robust pipeline to study gene family
evolution, and has been used widely in genome analyses,
including mammalian genomes (Demuth et al., 2006), Drosophila genomes (Hahn et al., 2007), and rhesus macaque
genome (Rhesus Macaque Genome Sequencing and Analysis Consortium, 2007). Of the 3296 families used for the
likelihood analysis, along the phylogenetic ranks, gene
families in each lineage expands to different extent except
for C. reinhardtii, grass, and Arabidopsis. Consistent with
the contraction of A. thaliana at the whole-genome level,
all nine rapidly evolving gene families contract on the terminal branch to A. thaliana. In Drosophila, it has been
demonstrated that some gene families (defense response,
proteolysis, and trypsin activity) are evolving rapidly both
in copy number and sequence level (Drosophila 12
Genomes Consortium, 2007; Hahn et al., 2007). However,
in Arabidopsis, there is not any correlation between the
copy number variation and sequence divergence for the
genes of rapidly evolving gene families, which suggested
that the relationship between copy number variation and
sequence divergence is much more complex.
In the present study, the rate of gene gains and losses is
about 0.001379 (gains and losses/gene/million years). This
rate is a little lower than some previous estimates. For
instance, the average duplication rate of a eukaryotic gene
was estimated to be on the order of 0.01/gene/million
years and 0.002 for A. thaliana (Lynch and Conery, 2000,
2003). However, this rate is similar to that in Drosophila –
0.0012 (Hahn et al., 2007) and Mammalian – 0.0016
(Demuth et al., 2006). As this result is consistent with the
average rate of duplication of a eukaryotic gene, and also
eight representative genomes of green plants are used,
thus the estimated rate is reliable for A. thaliana relatives
and green plants as well.
One important factor that affects the estimation of gene
gains or losses rate in certain lineages is polyploidy/wholegenome duplication. Especially for most green plants,
including a few angiosperm species studied here, are paleopolyploidy, and whole-genome duplication is normally
followed by gene loss and diploidization (Simillion et al.,
2002; Blanc and Wolfe, 2004; Adams and Wendel, 2005;
Doyle et al., 2008; Soltis et al., 2009; Koh et al., 2010; Jiao
et al., 2011; Proost et al., 2011). Therefore, at the green
plants level, whole-genome duplication likely raises the
rate of gene gains and losses. However, with the availability of more genomes from various plant lineages, it would
be interesting to address how these paleopolyploidy
events affects gene gains or losses rate in some specific
plant lineages that experienced whole-genome duplication.
Origin of Arabidopsis thaliana genes
The gene emergence approach has been used to study the
evolutionary emergence of whole-genome genes in individual species or lineage at different phylogenetic levels
(Domazet-Loso et al., 2007; Domazet-Loso and Tautz, 2008,
2010a,b; Prachumwat and Li, 2008; Zhang et al., 2010a,b,
2011; Banks et al., 2011). The significant difference of gene
emergence on the phylostratigraphic map supports the
assumption that some of these genes have retained a
signal of their evolutionary history. In this way, the
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
948 Ya-Long Guo
phylostratigraphy can imply a picture of evolutionary history using extant species. In this study, more than 70% of
A. thaliana genes are traced back to 450 million years ago.
Interestingly, the A. thaliana internode also has the highest
gene fixation rate, which might be due to high gene origination rate, or more complete annotation since much more
expression data can be used for annotation. Transposition
in certain families is a possible cause of the high gene
origination rate. A recent study indicates that specific gene
families can transpose at specific points in evolutionary
time, especially after whole-genome duplication events in
the Brassicales (Woodhouse et al., 2011).
The fraction of expressed genes, expression level, and
protein size reduced along the phylostratigraphy from
Viridiplantae to A. thaliana. In yeast, there is an inverse
relationship between protein size and protein expression
level (Warringer and Blomberg, 2006). Conversely, in
A. thaliana the oldest genes are much longer and also
highly expressed. In addition, the comparison between
A. thaliana and A. lyrata indicates transposable elements
and small RNAs contribute to gene expression divergence
(Hollister et al., 2011). Most probably, different lineage or
species has different regulatory mechanism. For example,
even comparing to its sister species A. lyrata, A. thaliana
has much higher intron loss rate (Fawcett et al., 2012) and
deletion rate, and also tend to delete longer fragments (Hu
et al., 2011).
Interestingly, Viridiplantae internode has the lowest dN/
dS, while Arabidopsis internode has the highest. This
probably suggests that older genes have been under stronger purifying selection, since older genes are mostly fundamental genes for green plants to survive. Accordingly,
the older genes are more conserved with a slow evolution
rate (synonymous substitution rate). Evolution rate can be
shaped by various factors (Alba and Castresana, 2005; Larracuente et al., 2008; Singh et al., 2009; Liao et al., 2010;
Gaut et al., 2011; Yang and Gaut, 2011). Recent comparisons between two available Arabidopsis genomes provide
more robust results. The rates of protein evolution are
correlated mainly with expression level and specificity
(Slotte et al., 2011); and rates at synonymous sites vary on
genomic scale and might be a function of recombination
rate and evolution rates at non-synonymous sites, strongly
correlated with expression pattern and whether a gene is
retained after a whole-genome duplication or not (Yang
and Gaut, 2011). In addition, 1939 annotated protein-coding genes with little evidence of expression in A. thaliana
are shorter and evolved with a two-fold higher rate of nonsynonymous substitutions (Yang et al., 2011). This finding
is consistent with what is found here, that younger genes
are shorter, higher in the non-synonymous substitution
rate, and lowly expressed.
Furthermore, it would be interesting to study dN/dS ratio
in other species pairs, similar comparisons had been made
between A. thaliana and popular and between two conifers
(Picea sitchensis and Pinus taeda) (Buschiazzo et al., 2012),
which indicates low rates of diversification in conifers but
much higher dN/dS, possibly due to positive selection. It
would also be interesting to do a lineage-specific study about
dN/dS ratio, which could be informative for understanding
the mechanism of lineage-specific evolutionary events.
Although many other plant species’ genome sequences
are available now, the eight genomes here are good representative of green plants with clear phylogeny and divergence times estimated, especially assembled with longer
and high quality reads at high coverage, which are
believed to be very important to address evolutionary
questions at whole-genome level (Demuth and Hahn, 2009;
Schneider et al., 2009; Milinkovitch et al., 2010). Therefore,
the eight representative green plants’ genomes are sufficient to address those interesting questions at whole-genome level. The comparison of the eight genomes, revealed
the evolutionary pattern of green plants’ genes and gene
families at an unprecedented resolution. The rate of gene
gains and losses is similar to that in animals, some gene
families are rapidly evolved and the 2745 gene families are
‘core’ proteome of green plants. Most genes of A. thaliana
are biased toward ancient genes, and genes with early
origination are under stronger purifying selection and
more conserved; and gene families of Arabidopsis lineage
contract a lot, especially A. thaliana, which could be correlated with the shaping of the difference of morphology,
physiology, and metabolism among species.
EXPERIMENTAL PROCEDURES
Genome data sets
Protein sequences from eight genomes of green plants were analysed, including A. thaliana (TAIR8) (27 025 proteins) (The Arabidopsis Genome Initiative, 2000), A. lyrata (JGI-v1.0, FM6) (32 670
proteins) (Hu et al., 2011), Populus (Populus trichocarpa, JGI-v1.1)
(45 555 proteins) (Tuskan et al., 2006), rice (Oryza sativa, v5.0)
(56 278 proteins) (International Rice Genome Sequencing Project,
2005; Ouyang et al., 2007), Sorghum (Sorghum bicolor, JGI-v1.0)
(35 899 proteins) (Paterson et al., 2009), Selaginella moellendorffii
(JGI-v1.0) (34 697 proteins) (Banks et al., 2011), Physcomitrella
patens ssp patens (JGI-v1.1) (35 938 proteins) (Rensing et al.,
2007), and Chlamydomonas reinhardtii (JGI-v3.1) (15 256 proteins)
(Merchant et al., 2007). The phylogeny and divergence times of
€m
the eight genomes were clarified clearly (Kellogg, 2001; Wikstro
et al., 2001; Gaut, 2002; Wellman et al., 2003; Magallon and Sanderson, 2005; Zimmer et al., 2007; Beilstein et al., 2010; Lang
et al., 2010; Guo et al., 2011b) (Figure 1 and Table S9 for the detail
of the divergence time at each node). In addition, the sampled
genomes are representative, flanking the most important events
in the green plants leading to A. thaliana. In order to get rid of
possible transposable element sequences within the proteins data
sets, all proteins were searched against the repeat library database (repeat masker database version 20090604) using tblastn
(E value <1 9 10 5), and length of hit must be at least half of the
query sequence. The putative transposable elements are deleted
from the dataset in the following analyses.
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
Evolution of gene families in green plants 949
Homology searches and gene family construction
Markov Cluster Algorithm (mcl-06-058 package; http://micans.org/
mcl/src/) was used to cluster all the eight genomes’ protein
sequences into gene families with default parameters ( I 2, S 6).
There are two steps for clustering analysis: (i) blastp was used to
match each protein against all other proteins (E value <1 9 10 5)
to produce a raw NCBI BLAST output; and (ii) MCL was used to
build Markov Matrix using the parsed similarity data converted
from raw NCBI BLAST output, and then generated the final gene
families. The clustering results were imported into MySQL database for the following analyses. Genes (gene families) that do not
have recognizable homologues in other species are called as
orphan genes (gene families).
Gene family size analysis
Genes were classified into one of the following three groups by its
family size in the clustering according to Prachumwat and
Li (2008): (i) a singleton is a single-copy gene; (ii) a two-gene
family has two copies; and (iii) a multigene family has 3 copies.
Likelihood analysis of gene gain and loss
version 2.0 (De Bie et al., 2006), a tool for the statistical analysis of the evolution of the gene family size, was used to study the
evolution of gene families. One can estimate gene gain and loss
rate among these plant genomes; calculate the average expansion
or contraction per gene family on each branch; and identify the
gene family that has larger-than-expected contractions or expansions. The below input files and parameters are used. First, the
input tree structure file, which is a Newick description of a rooted
and bifurcating phylogenetic tree (including branch lengths in
units of time) (Figure 1). Second, the data file consists of gene
family sizes of each gene family across eight species. Third, k, the
probability of both gene gains and losses per gene per unit time
in the phylogeny (CAFE assumes that gene birth and death are
equally probable), was calculated by CAFE given the gene families
in the data file (the estimated k is the globally most probable
value across all families in the input file or the different lineages’
k with related options of t). Here I calculated the k across all the
eight species. Fourth, two other parameters were set as P-value
threshold (0.01) and number of random samples used the default
value (1000). Fifth, the default Viterbi method was employed to
identify the branch with the most unlikely amount of change. For
gene families that are evolving at rates of gains and losses significantly higher than the genome-wide average with a P-value below
P < 0.0001 (Hahn et al., 2005), Viterbi method was used to identify
the branch that is the most likely cause of deviation from the random model significant at P < 0.005 (Hahn et al., 2007).
CAFE
Phylostratigraphic analyses of Arabidopsis thaliana genes
Phylostratigraphic analysis was performed according to previous
study (Domazet-Loso et al., 2007). All genes of A. thaliana were
assigned into seven phylogenetic ranks (phylostrata) of different
ages according to the emergence in the reference genomes on the
phylogeny based on the similarity clustering via MCL. The seven
gene groups were: (1) A. thaliana, genes that arose along the
A. thaliana; (2) Arabidopsis, genes were present in the MRCA of
Arabidopsis genus; (3) rosid, genes were present in the MRCA of
Arabidopsis genus and P. trichocarpa; (4) angiosperm, genes were
present in the MRCA of rosid and grass (S. bicolor and O. sativa);
(5) Tracheophyte, genes were present in the MRCA of angiosperm
and S. moellendorffii; (6) Embryophyte, genes were present in the
MRCA of Tracheophyte and P. patens; and (7) Viridiplantae, genes
were present in the MRCA of Embryophyte and C. reinhardtii.
Genes (or gene families) fixation rates were calculated according
to the formula, r = N/T: r is the number of genes (or gene families)
per million years; N is the number of genes (or gene families) in
the phylostratum; T is duration of the interval (million years).
Divergence and polymorphism
There are several steps to estimate the divergence of orthologs:
(i) the orthologous gene pairs between A. thaliana and A. lyrata
were extracted from a whole-genome alignment analysis (Hu
et al., 2011); (ii) a codon-based alignment of orthologous coding
sequences was performed with PAL2NAL (Suyama et al., 2006);
(iii) dS, the number of synonymous substitutions per synonymous
site, and dN/dS ratios, the ratio of the number of non-synonymous
substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site, were calculated for
orthologue pairs using the codeml program implemented in phylogenetic analysis by maximum likelihood version 3.15 package
(Yang, 1997).
GO annotation and gene expression
Gene ontology annotation was performed using GO implemented
at TAIR (October 2009) (http://www.arabidopsis.org/tools/bulk/go/
index.jsp) for A. thaliana (Gene Ontology Consortium, 2004); for
other species, blast2go was used to do the GO annotation (Conesa
et al., 2005). GO Biological Processes were used to estimate the
abundance of GO terms. Gene expression data from AtGen
Express Arabidopsis expression atlas (Schmid et al., 2005), which
was generated on the widely available Affymetrix ATH1 array platform, were averaged across all tissues as an estimate of expression level of A. thaliana genes.
ACKNOWLEDGEMENTS
I thank Matthew W. Hahn for suggestions on CAFE analysis; Tomislav Domazet-Loso for suggestions on phylostratigraphy analysis;
Manyuan Long, Yong Zhang, Jie Guo, and anonymous reviewers
for comments about this work; and Tina Hu for proofreading and
improving the manuscript. Especially, I am very grateful to Detlef
Weigel and Song Ge for their long-term support and also valuable
comments on this work. This work was supported by 100 talents
program of Chinese Academy of Sciences and the Max Planck
Society.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article.
Data S1. The gene family clustering among all proteins of the
eight genomes.
Figure S1. GO annotation of A. thaliana genes in each phylostratum.
Table S1. GO annotation of 41 rapidly evolved gene families.
Table S2. Functional annotation of 41 rapidly evolved gene families.
Table S3. Fixation rate of A. thaliana genes or gene families in
each phylostratum (genes/duration of interval or gene families/
duration of interval).
Table S4. The fraction of A. thaliana genes in each phylostratum
that can be annotated to GO biological process.
Table S5. GO annotation of A. thaliana genes in each phylostratum in terms of proportion of major biological processes (%).
Table S6. Protein sizes of A. thaliana genes in each phylostratum.
Table S7. Expression of A. thaliana genes in each phylostratum.
© 2012 The Author
The Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 941–951
950 Ya-Long Guo
Table S8. Divergences between A. thaliana genes of each
phylostratum and their orthologous genes in A. lyrata.
Table S9. Divergence time of each node on the phylogeny of
green plants.
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