The Plant Cell, Vol. 27: 563573, March 2015, www.plantcell.org 2015 American Society of Plant Biologists.

. All rights reserved.

Coordinated Rates of Evolution between Interacting Plastid

and Nuclear Genes in Geraniaceae
Jin Zhang,a,1 Tracey A. Ruhlman,a Jamal Sabir,b J. Chris Blazier,a and Robert K. Jansena,b
a Department
b Department

of Integrative Biology, University of Texas, Austin, Texas 78712

of Biological Sciences, Biotechnology Research Group, Faculty of Science, King Abdulaziz University, Jeddah 21589,

Saudi Arabia

Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this
phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study
plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastidencoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and
rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic
evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN
(nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other
plastid/nuclear gene pairs, and identied the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2,
and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales
could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally
mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary
rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae.

INTRODUCTION
Although coevolution of gene sequences is a widely recognized
phenomenon in biological systems, it has rarely been studied
between the plastid and nuclear genomes of plants within
a well-established phylogenetic framework. Coevolution may
be detected within a single organism, such as gene pairs with
known physical interactions in Escherichia coli (Pazos and
Valencia, 2001), or between organisms, such as the correlated
change of sequences between viral and host genes (Lobo et al.,
2009). The coevolution of genes from organellar and nuclear
genomes may be considered an intermediate case, in which the
genes of interest are within the same organism but are encoded
in different cellular compartments. Given that there is an order of
magnitude higher mutation rate in nuclear genomes compared
with plastid genomes in plants (Wolfe et al., 1987; Drouin et al.,
2008), the detection of correlation in evolutionary rates, and how
that correlation is maintained, presents an interesting area of
study.
As gene function is expressed in amino acid sequences, coevolution between two genes is usually reected in the encoded
polypeptides. If mutual selective pressure exists between two
genes, changes to the amino acid sequences encoded in one
gene would be expected to cause corresponding changes in the
other gene to maintain normal biological activity (Pazos and

1 Address

correspondence to zj@utexas.edu.
The author responsible for distribution of materials integral to the ndings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Jin Zhang (zj@utexas.
edu).
www.plantcell.org/cgi/doi/10.1105/tpc.114.134353

Valencia, 2008). Similarly, coevolution between two genes can

be evaluated based on the rate of nonsynonymous substitutions
(dN), which are nucleotide mutations that cause a change in the
amino acid sequences. However, dN is also affected by local
rate heterogeneity or local background mutation rates, represented by the rate of synonymous substitutions (dS), which do
not result in amino acid changes. The correlation of dS between
two genes is more likely due to a shared mutation rate than an
indicator of coevolution.
Several factors can contribute to correlation of evolutionary
rates (Lovell and Robertson, 2010), such as obligate physical
interaction of gene products (Mintseris and Weng, 2005), shared
functional constraint (Zhang and Broughton, 2013), or gene
expression levels (Subramanian and Kumar, 2004). Because the
evolutionary rate of the mammalian mitochondrial genome is
much higher than that of the nuclear genome, studies of correlated evolution between organellar and nuclear genomes have
focused on proteins of enzyme complexes with subunits encoded
in each of these compartments. Using this approach, studies
have shown that some nuclear genes that encode products that
participate in mitochondrial-localized complexes have a corresponding higher evolutionary rate relative to cytosol targeted
nuclear gene products (Willett and Burton, 2004; Osada and
Akashi, 2012; Barreto and Burton, 2013; Zhang and Broughton,
2013).
The correlation of evolutionary rates between plastid and
nuclear genomes has rarely been studied because plastid
genome sequences are generally more highly conserved than
those of the nuclear genome (Wolfe et al., 1987; Drouin et al.,
2008), making it difcult to select appropriate taxa and genes for
analyses of correlated rate acceleration. Studies in Silene (Sloan
et al., 2014) identied elevated protein sequence divergence in

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organelle-targeted, but not cytosolic, ribosomal proteins in pairwise

comparisons of species with rapidly evolving mitochondrial and
plastid DNA, suggesting that coevolution occurs between different compartments. Like the Silene study, many investigations
have adopted pairwise species comparisons, an approach that
does not account for the effects of shared phylogeny on predictions of coevolution (Barreto and Burton, 2013).
Methods that incorporate a phylogenetic framework have
proven more accurate in detecting coevolution among interacting proteins than pairwise comparisons (Clark and Aquadro,
2010). Various methods have been developed that incorporate
the effects of phylogeny for detecting gene coevolution (Pazos
and Valencia, 2008; de Juan et al., 2013; Rao et al., 2014). The
mirror tree method (Pazos and Valencia, 2001) was originally
introduced to predict protein-protein interactions, and it quanties
rate correlations by estimating the similarities of corresponding
phylogenetic trees. For each gene tree, the evolutionary rates on
each branch are extracted to form a rate vector, and Pearson
correlation coefcients are calculated between the rate vectors of
two genes. Despite its popularity, the original mirror tree method
does not effectively account for underlying phylogenetic histories.
Different modications of this method were developed to remove
the effects of shared phylogeny by introducing a correction factor
(Pazos et al., 2005; Sato et al., 2005). A more recent likelihood
based approach evaluates the coevolution between genes using
normalized dN, or dN/dS (Clark and Aquadro, 2010). In this
method, the likelihoods of three models (null, correlated, and
free) are calculated and the correlation is quantied as the
proportional improvement of the likelihood of the correlated
model to the null model, with respect to the maximal possible
improvement gained by the free model over the null model.
Studies of coevolution of amino acids adopt a different set of
approaches, which assess coevolution between two sites by
detecting similar amino acid frequencies or substitution patterns
calculated from the multiple sequence alignment (Gbel et al.,
1994; Neher, 1994; Taylor and Hatrick, 1994). Dutheil and Galtier
(2007) developed an approach (CoMap) that examines the coevolution of given amino acid sites using the known phylogenetic history. In this method, the ancestral state of a given amino
acid site is inferred from the phylogenetic history and the sequence of changes that occur across time (branches on a phylogenetic tree) form a substitution vector. Structurally mediated
coevolution of any amino acid site is then evaluated using the
substitution vector and a cluster-based approach (Dutheil and
Galtier, 2007). Another approach studies the coevolved amino
acids by incorporating a continuous-time Markov process model
(Yeang and Haussler, 2007). Both of these approaches agree well
with experimental results; however, the latter approach is computationally demanding and therefore more feasible for studies of
small protein domains.
In plants, the plastid-encoded RNA polymerase (PEP) is
a multisubunit enzyme complex (Shiina et al., 2005) containing
subunits encoded by genes in both the plastid (RNAP: rpoA,
rpoB, rpoC1, and rpoC2) and nuclear genomes (SIG: sigma
factor 1-6). Studies in Geraniaceae have revealed highly elevated
evolutionary rates in the plastid genome, especially in rpoB, rpoC1,
and rpoC2 (Guisinger et al., 2008; Weng et al., 2012), and highly
divergent rpoA sequences in the genus Pelargonium (Chumley

et al., 2006). The interaction of SIG and RNAP gene products

provides an attractive platform for the study of coevolution between the two genomes. Using transcriptomic and genomic data
from 27 species with a well-established phylogenetic framework,
the entire sigma factor gene family in Geraniaceae has been
characterized and a systematic correlation analysis of evolutionary
rates between plastid and nuclear genomes was conducted. Despite an order of magnitude difference in the mutation rate between these two genomes (Wolfe et al., 1987; Drouin et al., 2008),
we detected a correlation of evolutionary rates among 27 species
representing the entire family. Furthermore, analyses of interacting
amino acid pairs suggest that structurally mediated coevolution
plays a minimal role in maintaining the coordination of evolutionary
rates. The identication of rate correlations between RNAP and
SIG genes suggests a plausible explanation for the observed
plastome-genome incompatibility within Pelargonium and possibly other genera of owering plants.

RESULTS
Transcriptome sequencing and assembly for 27 species was
performed following Zhang et al. (2013). Sigma factor genes
were extracted and accession numbers are provided in
Supplemental Table 1. An amino acid maximum likelihood (ML)
tree was generated to infer phylogenetic relationships among
the 178 complete sigma factor (SIG) sequences identied from
the 27 species in Geraniales and Arabidopsis thaliana (Figure 1;
see Supplemental Data File 1 for alignments). The ML tree (-lnL =
265001.9) topology parsed the 178 sequences into six major
clades. Two additional alignment algorithms (see Methods) were
used and resulted in the same six major groups of sigma factor
genes (Supplemental Figure 1; see Supplemental Data File 1 for
alignments).
The copy number of individual SIG genes varied across different species (Supplemental Figure 2; see Supplemental Data
File 1 for alignments). A single copy of sig1 and sig2 was found
in all species except for Pelargonium transvaalense, Pelargonium tetragonum, and Geranium maderense, where two copies
of sig2 were identied. A complete sig3 sequence was identied
in all species except for P. tetragonum, Pelargonium myrrhifolium,
and Pelargonium nanum. The sig4 sequence was detected in all
Pelargonium and Geranium species, and, while a sig4 pseudogene
missing the start codon was detected in Melianthus villosus, sig4
was not found in Francoa sonchifolia, Erodium chrysanthum, and
Erodium gruinum. Two copies of sig5 and sig6 were identied in
various species (Supplemental Figures 2E and 2F). Multiple copies
of sig5 were identied in species of Geranium and Erodium and in
California macrophylla, while two copies of sig6 were found in C.
macrophylla and species of Erodium and Pelargonium. The sig6
gene of Hypseocharis bilobata contained multiple internal stop
codons. RT-PCR conrmed 18 out of 21 bioinformatically identied gene duplication and pseudogenization events (Supplemental
Table 1). Among the SIG gene families, 21 gene duplications
and 10 losses were inferred with Notung (Durand et al., 2006)
(Supplemental Figure 3 and Supplemental Table 2; see
Supplemental Data File 1 for alignments) on the branches leading
to the 27 species of Geraniaceae.

Plastid-Nuclear Genome Coevolution

Figure 1. Six Sigma Factor Families in Geraniales and Arabidopsis.

The unrooted amino acid-based ML tree was generated using 178 complete SIG sequences identied from 27 species of Geraniales and Arabidopsis. The ML tree (-lnL = 265001.9) topology parsed the 178 sequences
into six subgroups (enclosed in labeled circles). Scale bar represents the
number of amino acid substitutions per site. Numbers at nodes are bootstrap support values.

Evolutionary rates of each gene were estimated based on

alignments from MAFFT (Katoh and Standley, 2013). To avoid
biases of rates estimation specic to an alignment algorithm,
rates based on alignments from two other tools, MUSCLE and
ClustalW (Edgar, 2004; Larkin et al., 2007), were compared with
MAFFT (see Supplemental Data File 1 for alignments). The agreement between rate estimates from the three alignment methods
indicated that there was no or negligible bias due to the alignment
method (Supplemental Table 3). Thus, MAFFT was used for all
subsequent analyses.
Clade-specic rate acceleration was assessed for the four
plastid RNA polymerase (RNAP) subunits, six nuclear-encoded
SIG genes, and 20 control genes (Figure 2; Supplemental Figures
4 and 5). While 10 control genes from nuclear and plastid genomes
were selected for all coevolution analyses, two additional sets of
10 nuclear control genes were randomly selected from the APVO
database (see Methods) for the mirror tree analysis to reduce any
bias of nuclear control gene sampling (see Supplemental Data File
2 for detailed control gene information).
Although dN for the Pelargonium C clade was accelerated in
all four RNAP genes and two SIG genes (rpoA, rpoB, rpoC1,
rpoC2, sig1, and sig2) in an initial rank sum test, acceleration of
rates of rpoA/B/C1 only remain signicant after correction for
multihypothesis testing (Figure 2), while two control genes have
signicant acceleration of rates in the Geranium and Erodium
clades. Signicant acceleration of dS in the Pelargonium C clade
was observed for the rpoA gene alone (Supplemental Figure 4).
Elevated dS in sig6 and ve nuclear control genes was observed in Geranium, with no acceleration detected within other
clades (Supplemental Figure 4; see Supplemental Data File 1 for
alignments).

565

The values of dN and dS for each gene from all branches were
used to analyze the rate correlation between gene pairs from
RNAP, SIG, and control genes. The highest average values for
dN were found in SIG genes followed by RNAP genes (Figure
3A; Supplemental Figure 5). Four plastid genes (cemA, matK,
rpl14, and rps2) and one nuclear gene (rh22) had similar average
dN values to the RNAP genes, and the other nuclear genes had
slightly lower values. The lowest average dN values were found
in the remaining plastid genes, which represent ATP synthase
and photosynthetic genes. The dS values were similar among
genes from the same cellular compartment (Figure 3B). The average dS values of nuclear genes were much higher than those of
plastid genes except for rpoA, which had the highest dS value
among plastid genes.
Correlation of dN and dS was evaluated for each gene pair by
three variations of the mirror tree method, each of which adopts
a different approach for removing the effect of shared phylogeny
prior to tree similarity estimation (Pazos et al., 1997, 2005; Pazos
and Valencia, 2001): average by all, average by separation, and
principal component analysis (PCA; see Methods for detailed
description). The sequences of sig1, sig2, and sig5 were grouped
together for rate correlation analysis using complete sequences (Figure 4) or conserved domains (Supplemental Figure 6).
Due to their absence in different species, the genes sig3, sig4,
and sig6 were analyzed separately using complete sequences
(Supplemental Figure 7) or conserved domains (Supplemental
Figure 8). In addition to the initial 10 plastid and nuclear control

Figure 2. Shared Clade-Specic Nonsynonymous Rate (dN) Acceleration in Geraniaceae.

RNAP and SIG genes are highlighted in the key in blue and orange, respectively. Blue numerals on the constraint tree indicate shared dN acceleration in RNAP genes of the Pelargonium C clade. For a more detailed
version of the constraint tree, see Supplemental Figure 5. Numbers at
nodes indicate accelerated dN in corresponding gene from the key at left
(0 to 14, plastid genes; 15 to 29, nuclear genes). Scale bar represents the
number of nucleotide substitutions per codon. The long branch leading to
E. chrysanthum and E. gruinum was interrupted for ease of visualization.

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The Plant Cell

Figure 3. Nonsynonymous (dN) and Synonymous (dS) Substitution Rates for Individual Genes.
Box plots represent the distribution of dN (A) or dS (B) value for each branch on the constraint tree (Supplemental Figure 5). While the dS values (B)
were similar among genes from the same cellular compartment, the highest average values for dN (A) were found in SIG genes (orange) followed by
RNAP genes (blue). The scale for dN and dS is different to facilitate visualization of the results.

genes, two additional sets of nuclear control genes were added to

the sig1, sig2, and sig5 analyses (Supplemental Figures 9 and 10).
A cutoff of 0.6 for the Pearson correlation coefcient was used
as an indicator of strong rate correlation (Sato et al., 2005). After
removing the effects of shared phylogeny (see Methods), correlation of dN was detected between RNAP and sig1/2 genes
(orange rectangle in Figures 4A and 4B), but not between RNAP/
SIG and the control genes. The mirror tree methods did not
detect a dN correlation between RNAP and sig3, sig4, or sig6
(Supplemental Figure 7). The correlation of dS was sensitive to
the average method used in the analyses (Figures 4A and 4B).
Correlation of dS was identied between certain plastid or nuclear gene pairs but not between the two groups when the average by all method was employed (Figure 4A), and only one pair
of nuclear genes had correlated dS when the average by separation method was used (Figure 4B). Application of the PCA
method produced correlations of dN and dS that were similar to
those from the average by all method except that correlation of
dN was detected between rpl14/rpoA and sig1 (Figure 4C).
None of the three methods identied correlation of dS between
RNAP and SIG genes (Figures 4A to 4C), suggesting that the
correlation of dN was not due to the effects of background
mutation rates. The number of gene pairs with positive rate
correlations is shown in Table 1. Similar rate correlations were detected with the additional nuclear control genes (Supplemental
Figures 9 and 10).
The correlation of dN and dS between RNAP and SIG genes
using conserved domains was similar to that seen using the
entire sequences; however, more gene pairs of RNAP and SIG
were identied as correlated for dN (Supplemental Figures 6 and
8). The number of rate correlations of all gene pairs is shown in
Supplemental Table 4.
The rate correlation coefcient between each individual gene
and the RNAP genes was compared (Table 2). The dN correlation coefcients of RNAP and RNAP/sig1/sig2 genes were
ranked signicantly higher (P < 0.05 after correction for

multihypothesis testing) than all other pairs by average by

separation and PCA methods. Using the average by separation
method, correlation coefcients of RNAP genes and sig6 were
also ranked signicantly higher than other pairs. No signicantly
higher rank was detected between RNAP and the plastid or
nuclear control genes by any method. Synonymous substitution
rate correlation coefcient ranking produced no signicant result
for any of the gene groups. The same tests were performed with
rates calculated from the conserved domains of selected genes
as described (Supplemental Table 5). Similar to the results
generated using the entire sequences, the dN correlation coefcients of RNAP and RNAP/sig1/sig2/sig6 were ranked signicantly higher than any other pairs by average by separation
and PCA methods. The correlation coefcients of dN for RNAP
and sig5 were ranked signicantly higher using PCA method.
The rank of dS correlation coefcients was the same as that
using the entire sequences with no signicant highly ranked
gene groups detected.
Correlation of normalized dN (dN/dS ratio) was evaluated with
the proportional improvement method (Clark and Aquadro,
2010). Since the proportional improvement dN/dS test is more
appropriate when dS is unsaturated (Clark and Aquadro, 2010),
saturation was tested for each of the genes examined. To examine saturation of synonymous sites, values of dN and dS
were plotted and linear/quadratic models were used to t the
data. If dS is saturated, the quadratic model with a concave
curve should t the data better. The two models were compared
with the improvement of sum of squares explained by these
models (Fares and Wolfe, 2003; Weng et al., 2014). Of the 30
genes tested, only rbcL showed signicant improvement (P <
0.05) of sum of squares (Supplemental Table 6); however, rbcL is
known to be a conservative gene with low dS values (dS < 0.15
for all branches) (Figure 3B).
Strong correlation (proportional improvement > 0.6) of dN/dS
was identied between rpoB/C1/C2 and sig1/5/6 genes, between rpoC1/C2 and sig2 genes, and between rpoB/C2 and

Plastid-Nuclear Genome Coevolution

567

Figure 4. Strong Correlation of Nonsynonymous (dN) but Not Synonymous (dS) Substitution Rates between sig1/2/5 and RNAP Genes Using Three
Methods of Analysis.
The entire sequence of each gene was used in this analysis (see Methods). The correlation of dN and dS values were calculated by modications of the
mirror tree method average method (all) (A), average method (separation) (B), and PCA (C). All interaction pairs with a correlation coefcient of higher
than 0.6 were considered signicant and shown with a blue square. RNAP and SIG genes, highlighted in blue and orange fonts, respectively, show
strong correlation of dN but not dS (highlighted in orange shaded box). Little to no correlation of dN between RNAP/SIG genes and the control genes (in
black font) was detected. Gene names and cellular locations corresponding to each number are given below the diagram.

sig3 genes (Figure 5). Correlation of dN/dS was also identied

among RNAP (between rpoB and rpoC1/C2; rpoC1 and rpoC2)
and among SIG (between sig1 and sig2/5/6; sig2 and sig5)
genes (Figure 5). A correlation of RNAP/SIG and control genes
was lacking between most interaction pairs except for rpoB and
three nuclear control genes (OXase, ppr, and nprb7) and sig1/2
and nprb7 genes (Figure 5). Compared with the mirror tree
methods, more interaction pairs (proportional improvement, 13;
average by all, 2; average by separation, 4; PCA, 6) were identied with strong rate correlation between RNAP and SIG genes,
while fewer or comparable interaction pairs (proportional improvement, 5; average by all, 20; average by separation, 5; PCA,
2) were identied between RNAP/SIG and control genes.
To investigate the role of structurally mediated coevolution in
the correlation of evolutionary rates between RNAP and SIG
genes, CoMap (Dutheil and Galtier, 2007) was used to predict
coevolved amino acid pairs by comparing the substitution
vectors, weighted by the different amino acid properties (volume, charge, and polarity) at given positions (see Methods for
more details). Since the b9 subunit of the cyanobacterial ancestor was split in the lineage leading to plants (Shiina et al.,
2005), the relevant residues from the b9 and b99 subunits in
plants were combined for comparison with the b9 subunit in E.
coli. The interaction sites between SIG and RNAP subunits were
predicted by contact map analysis (Sobolev et al., 2005) and by
estimation of the physical distance between two interacting
residues. More interaction sites were predicted by contact map

analysis than by distance estimation (Supplemental Table 7);

however, few (0 to 20%) of the predicted coevolving amino acid
sites overlapped with interaction sites (Supplemental Table 7).
The analysis of distance distributions across the coevolved
amino acid pairs suggested that among the 4223 residue pairs
predicted to be involved in structurally mediated coevolution by
CoMap, only one pair had a distance of <5 . The analyses of
structurally mediated coevolution within amino acid pairs showed
a minimal overlap between coevolved and interacting amino acid
sites (Supplemental Table 7), with few of the coevolved residues
in close physical proximity (<5 ; Supplemental Figure 11).
DISCUSSION
Duplication and Loss of Sigma Factor Genes
Twenty-one duplicated SIG genes were identied across the
Geraniaceae (Supplemental Figures 2 and 3 and Supplemental
Table 2). Duplications of SIG genes have been documented in
several angiosperms, including maize (Zea mays), rice (Oryza
sativa), and poplar (Populus trichocarpa) (Lerbs-Mache, 2011).
These duplicate copies may be functionally diversied to regulate gene expression at different developmental stages or under
changing environmental conditions as seen for the duplicated
sig1 of maize that is differentially expressed in etiolated leaves
(Tan and Troxler, 1999; Lerbs-Mache, 2011). The pattern of
SIG gene duplication in Geraniaceae could have arisen in

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Table 1. The Number of Interaction Pairs with a Rate Coefcient of over

0.6 within Corresponding Genes Estimated by Three Mirror Tree
Methods
dN

dS

Interactionsa

rava

ravs

rpca

rava

ravs

rpca

RNAP-RNAP (6)
RNAP-sig1 (4)
RNAP-sig2 (4)
RNAP-sig3 (4)
RNAP-sig4 (4)
RNAP-sig5 (4)
RNAP-sig6 (4)
RNAP-control (80)
sig1-control (20)
sig2-control (20)
sig3-control (20)
sig4-control (20)
sig5-control (20)
sig6-control (20)

5
1
1
0
0
0
0
0
0
0
8
11
0
1

3
2
2
0
0
0
0
0
0
0
5
0
0
0

5
3
3
0
0
0
0
0
1
0
1
0
0
0

3
0
0
0
0
0
0
10
4
5
3
5
8
10

0
0
0
1
0
0
0
0
0
0
5
1
0
0

2
0
0
0
0
0
0
8
3
4
3
2
5
9

RNAP contains rpoA, rpoB, rpoC1, and rpoC2. Results of the average by
all method (rava), average by separation method (ravs), and PCA method
(rpca) are shown.
a
The number in parentheses is the total number of interaction pairs within
corresponding genes.

several ways, including whole-genome duplication followed

by elimination of some copies. Polyploidy is widespread across
Geraniaceae (Widler-Kiefer and Yeo, 1987; Yu and Horn, 1988;
Touloumenidou et al., 2007) and has likely contributed to duplication of SIG genes. However, an alternative explanation, that
multiple, small-scale gene duplications have occurred (Davis
and Petrov, 2004; Li et al., 2006) was supported by the pattern of
SIG gene duplications observed in Geraniaceae (Supplemental
Figures 2 and 3).
Multiple gene losses were detected in the SIG gene family in
Geraniales. While it is possible that these genes are so lowly
expressed as to fall below the level of detection, the high depth
of coverage in transcriptome sequencing (Zhang et al., 2013)
and the fact that the same genes were identied in transcriptomes
of closely related species make this unlikely. The Sig4 protein
specically recognizes the promoter of ndhF, which encodes a
subunit of NADH dehydrogenase in the plastid (Endo et al., 1999;
Favory et al., 2005). The lack of sig4 transcripts in E. chrysanthum
and E. gruinum is plausible given the loss of ndh genes from the
plastid genomes of these species (Chris Blazier et al., 2011). The
identication of a sig4 pseudogene in M. villosus also correlates
with a recent loss of ndh genes in that species (Weng et al., 2014).
The loss of both sig4 and ndh genes provides an explicit example
of coevolution between the plastid and nuclear genomes.
Coevolution of Plastid and Nuclear Genomes
Genome coevolution is expected to produce correlated evolutionary rate changes between different genes. Studies of coevolution usually focus on protein sequences from multisubunit
enzyme complexes. Correlated change of evolutionary rates has
been widely observed in various organisms (Campo et al., 2008;

Pazos and Valencia, 2008; Lovell and Robertson, 2010). A previous study showed that nuclear genes encoding subunits of
enzyme complexes that assemble in the mitochondria with
subunits encoded in the organelle have signicantly higher
evolutionary rates than genes whose products are targeted to
the cytosol (Barreto and Burton, 2013). Likewise, analyses of
coevolution between organellar and nuclear genomes have
mainly focused on genes from mitochondrial and nuclear genomes (Zhang and Broughton, 2013; Barreto and Burton, 2013).
A recent study of Silene (Sloan et al., 2014) suggested that
coevolution occurred between plastid and nuclear genomes
based on the observation of elevated protein sequence divergence in organelle targeted, but not cytosolic, ribosomal proteins for species with fast evolving mitochondrial and plastid
genomes. The unusually high substitution rates of genes in the
plastid genomes of Geraniaceae (Chumley et al., 2006; Guisinger
et al., 2008; Weng et al., 2012) provide an attractive system for the
study of coevolution. Using both transcriptome and genome data
and a well-characterized phylogenetic framework, this systematic
analysis revealed the existence of correlation of evolutionary
rates, evidence of coevolution between plastid and nuclear genomes at different levels (dN and dN/dS).
Correlation of dN but not dS was detected in gene pairs between RNAP and SIG genes, suggesting that the correlation
of dN was not due to shared background mutation rates. The
absence of correlation of dN between RNAP/SIG and control
genes indicates that the rate correlation between RNAP and SIG
genes is likely due to coevolution between plastid and nuclear
genome or a local functional constraint acting on RNAP and SIG
genes, rather than a global constraint on dN of all genes. The
case of dN correlation between rpoA and rpl14, detected exclusively by the PCA method, may be a result of factors other than
direct physical interaction, such as a common functional role in
gene expression in plastids (transcription for sig1 and translation
for rpl14) (Agraoti et al., 2005; Chen and Dokholyan, 2006).

Table 2. Rank Sum Test of Rate Correlation Coefcient

dN

dS

Interactions

rava

ravs

rpca

rava

ravs

rpca

RNAP-RNAP
RNAP-sig1
RNAP-sig2
RNAP-sig3
RNAP-sig4
RNAP-sig5
RNAP-sig6
RNAP-pt
RNAP-nu

+
2
2
2
2
2
2
2
2

+
+
+
2
2
2
+
2
2

+
+
+
2
2
2
2
2
2

2
2
2
2
2
2
2
2
2

2
2
2
2
2
2
2
2
2

2
2
2
2
2
2
2
2
2

The entire sequence of each gene was used in the analysis. Correlation
coefcients ranked signicantly higher among all interaction pairs are
indicated with + sign. Coefcients that are not ranked signicantly
higher are indicated with 2 sign. pt is the group of control genes from
the plastid genome, and nu is the group of control genes from the
nuclear genome. Results of the average by all method (rava), average by
separation method (ravs), and PCA method (rpca) are shown. Results from
average method were estimated in two ways (all/separate; see Methods).

Plastid-Nuclear Genome Coevolution

Figure 5. Strong Correlation of dN/dS between RNAP and SIG Genes.

The correlation of dN/dS was quantied using proportional improvement
(Clark and Aquadro, 2010). Interaction pairs with a proportional improvement of higher than 0.6 were considered signicant and shown with a blue
square in the gure. RNAP and SIG genes, highlighted in blue and orange
fonts, respectively, show strong correlation of dN/dS (highlighted in orange
shaded box). Little to no correlation of dN/dS between RNAP/SIG genes
and the control genes (in black font) was detected. Gene names corresponding to each number are given below the diagram.

Because correlation coefcients of gene pairs with known

interactions are signicantly higher than unrelated sequences
(Clark et al., 2012), correlation coefcients between each SIG
gene and the four RNAP genes should be higher than those
between any control genes and the four RNAP genes. The signicantly highly ranked correlation coefcients of dN between
RNAP and sig1/2 by any method supports the conclusion that
there is a strong rate correlation between RNAP and sig1/2
genes (Table 2). The signicantly highly ranked rate correlation
detected between RNAP and sig5/6 genes using conserved
domain sequences in both average and PCA methods suggests
that there might be correlation between RNAP and sig5/6 genes
but that it is weaker than those between RNAP and sig1/2
(Supplemental Table 4).
A correlation analysis of normalized dN (dN/dS ratio) was performed (Clark and Aquadro, 2010), and this approach identied
additional strong rate correlations between RNAP (rpoB/C1/C2)
and sig3/5/6. The low number (5/400) of strong rate correlation
pairs (Figure 5) between RNAP/SIG and control genes is the result
of either weaker correlation or false discoveries.
Across all methods, strong rate correlations (dN and/or dN/dS)
are present for RNAP and all SIG except sig4. Rate correlations

569

among interacting genes are affected by several factors (Lovell

and Robertson, 2010), such as physical interaction (Mintseris and
Weng, 2005), functional constraint (Zhang and Broughton, 2013),
or gene expression levels (Fraser et al., 2004). The sig4 gene is
involved in the transcription of ndhF (Favory et al., 2005). Knockout
studies of sig4 in Arabidopsis revealed no observable phenotypes
(Lerbs-Mache, 2011), and the loss of it and the corresponding
plastid encoded ndh genes (Chris Blazier et al., 2011) in multiple
species in Geraniaceae suggests that sig4 is dispensable. Relaxed
functional constraint may contribute to the absence of coevolution
between RNAP and sig4 genes.
Possible phenomena that could underlie the rate correlations
between RNAP and SIG genes include: (1) a cause-and-effect
relationship between rate variation of RNAP and SIG genes or (2)
a common factor affecting rates of both RNAP and SIG genes,
such as relaxed functional constraint acting on both gene sets
(Subramanian and Kumar, 2004; Mintseris and Weng, 2005;
Lovell and Robertson, 2010; Zhang and Broughton, 2013). If the
rst explanation were correct, rate correlations between RNAP
and SIG genes could be due to structurally mediated compensatory evolution. However, results suggest that structurally mediated
coevolution plays a minor role in maintaining rate correlations between SIG and RNAP subunits and other factors contributing to
compensatory evolution could not be excluded. Shared functional
constraint is another agent that may be maintaining the observed
correlations. Additional study is required to further elucidate the
contribution of functional constraints, gene expression, or other
factors in the correlation of evolutionary rates of PEP subunits in
Geraniaceae.
Plastome-genome incompatibility (PGI), which was rst
documented in Pelargonium species (Smith, 1915), is observed
across owering plants (Schmitz-Linneweber et al., 2005;
Greiner et al., 2008; Weihe et al., 2009; Greiner et al., 2011).
Various mechanisms have been proposed for PGI, including
impaired interactions between cis-elements and their cognate
nuclear factors involved in transcription and/or transcript stability. In Oenothera, perturbations of photosystem II activity,
presumed to be caused by changes in transcription of the psbB
gene, contributes to PGI (Greiner et al., 2008). Likewise, steady
state RNA levels of three PEP-controlled genes were severely
reduced in leaf sections taken from variegated, interspecic
hybrids of Zantedeschia (Yao and Cohen, 2000). The two nuclear genes included in the Zantedeschia study also evidenced
low levels of mRNA; expression of cab and rbsS is known to be
regulated by retrograde plastid to nuclear signals and would
therefore be susceptible to the PGI phenotype (Ruckle et al.,
2007). Correlation of accelerated nucleotide substitution rates
between SIG and RNAP genes provides a plausible explanation
for PGI in Geraniaceae (Weihe et al., 2009; Greiner et al., 2011).
Specically, the high evolutionary rates and rate correlation
between SIG and RNAP genes within species could lead to interspecic incompatibilities, and such incompatibilities would
reduce efciency or even cause dysfunction of the PEP holoenzyme, impairing the transcription of essential plastid genes.
The role of sigma factors in transcription initiation through ciselement binding and polymerase recruitment suggests similarity
between the Geraniaceae, Zantedeschia, and Oenothera PGI
systems.

570

The Plant Cell

METHODS
RNA Isolation, Transcriptome Sequencing, and Assembly
Total RNA was isolated from newly emerged leaves of 26 species in
Geraniales (Supplemental Figure 5), and four tissues (emergent and expanded leaves, roots, and owers) of Pelargonium 3 hortorum following
the protocols described by Zhang et al. (2013). Transcriptome sequencing
was performed on the HiSequation 2000 platform, and the sequence data
were preprocessed and assembled as described by Zhang et al. (2013).
Identication of Sigma Factors
SIG sequences were extracted from transcriptome assemblies with reciprocal BLAST as described by Zhang et al. (2013). The orthologous
genes of each class of SIG were determined by reciprocal BLAST and
single-gene phylogeny. RT-PCR was performed to verify the problematic
SIG gene sequences with internal stop codons or missing 59 or 39 ends.
For any species in which multiple gene sequences were identied as the
same class of sigma factor, RT-PCR was performed to verify the existence
of all the gene sequences (for primers, see Supplemental Table 8). All RTPCR products were subjected to Sanger sequencing to conrm the result.
Phylogenetic Analysis
Multiple sequence alignments were done using MAFFT (Katoh and
Standley, 2013), MUSCLE (Edgar, 2004), and ClustalW (Larkin et al., 2007)
with default parameters in Geneious 6.0 (Biomatters, http://www.
geneious.com/) (see Supplemental Data File 1 for alignments). Amino
acid-based ML trees for all SIG genes were constructed by RAxML
(Stamatakis, 2006) with parameters raxmlHPC-PTHREADS-SSE3 -f a -x
12345 -p 12345 -T 12 -m PROTGAMMAJTT -N 100. A Perl script (http://
sco.h-its.org/exelixis/web/software/raxml/hands_on.html) was used to examine all protein models and the model with the best likelihood score
(JTT) was selected. ML trees of each class of SIG genes were constructed
by RAxML (Stamatakis, 2006) with parameters raxmlHPC-PTHREADS-SSE3
-f a -x 12345 -p 12345 -T 12 -m GTRGAMMAI -N 100. Bootstrap values
were generated using RAxML with 100 replicates and the above settings.
Evolutionary Rate Estimation
PAMLs codeml (Yang, 2007) was used to estimate dN, dS, and dN/dS
using the codon frequencies model F3X4. Gapped regions were excluded
with parameter cleandata = 1. The constraint tree was generated by
RAxML using 12 plastid genes (atpA, atpB, atpI, ccsA, cemA, matK, petA,
rbcL, rpoB, rpoC1, rpoC2, and rps2) with a total length of 21,500 bp.
Bootstrap values were generated using RAxML with 100 replicates and
the above settings. Ten plastid genes (rbcL, cemA, atpA, atpB, matK,
petB, psaA, psbC, rpl14, and rps2) from different functional groups and 30
nuclear genes with three different subcellular targeting sites (plastid,
mitochondria, and other), which are orthologous to genes in the APVO
database (Duarte et al., 2010), were used as negative control groups. The
APVO database was separated into three groups based on their subcellular locations (plastid, mitochondria, and other), and an approximately
equal number of genes were selected randomly from each group. The
plastid genes were extracted from the annotated plastid genome assemblies as described by Weng et al. (2014). Thirty Arabidopsis thaliana
nuclear genes were downloaded from TAIR (Lamesch et al., 2012), and the
corresponding accession numbers are in Supplemental Data File 2.
Analysis of Correlation of Evolutionary Rate
Rates along branches leading to and within Pelargonium A, B, and C
clades, Monsonia, Geranium, and Erodium I and II clades were grouped

separately for clade specic rate acceleration analysis. The rank sum test
was performed to test clade-specic rate accelerations, and a P value of
<0.05 was considered signicant. Correction for multihypothesis testing
was performed by adjusting the original P value with the false discovery
rate correction method using the built-in p.adjust function (method=fdr)
in the R software package (http://www.r-project.org). After correction, the
false discovery rate among the signicantly accelerated clades within
each gene is <5%.
Correlation coefcients of evolutionary rates dN and dS between each
gene pair were estimated using modied mirror tree methods (Pazos et al.,
1997, 2005; Pazos and Valencia, 2001). Specically, the evolutionary
rates, dN or dS, on each branch of a given gene tree were collected to
form a rate vector. The rate correlation was quantied using the Pearson
correlation coefcient between the rate vectors of different genes. The
rate vector of each gene pair was adjusted via vector projection by
a correction vector representing the shared phylogenetic effects prior to
the comparison. The correction vector was generated with different
modications of the mirror tree method, the average method, and PCA
(Sato et al., 2005). In the average method, the correction vector was
determined in two ways, the average by all and the average by separation,
in which either the correction vector was dened as the average of rate
vectors of all genes or two different correction vectors for nuclear and
plastid genes were dened separately, as the average of rate vectors of
corresponding gene groups. In PCA, the correction vector was calculated
as the rst principle component of the rate matrix formed by rate vectors
of all genes.
The correlation of dN/dS ratio of most (447/450) interaction pairs,
quantied as proportional improvement as described (Clark and
Aquadro, 2010), was analyzed using HYPHY (Pond et al., 2005) with batch
scripts downloaded from http://mbg.cornell.edu/cals/mbg/research/
aquadro-lab/software.cfm (Clark and Aquadro, 2010). Specically, the
evolutionary rates and the likelihood of three (correlated, null, and free)
models for the estimated rates of each gene pair were evaluated with
HYPHY, and the proportional improvement method estimates correlation
of dN/dS ratio by calculating the proportional improvement of likelihood of
the correlated model over the null model, with respect to the maximal
possible improvement gained by the free model over the null model (Clark
and Aquadro, 2010). The remaining interaction pairs (3/450, psbC and
psaA, psbC/psaA and nprb7) were analyzed using the same batch script
template with modications so that the likelihoods of the correlation
model with different start points (20.8, 1, and 1.3) were optimized separately rather than sequentially. The test for dS saturation was performed
as described by Fares and Wolfe (2003) and Weng et al. (2014).
The conserved domains of RNAP and SIG genes were predicted by
NCBI CDD (Marchler-Bauer et al., 2013). The predicted conserved domains were used for rate analysis, except for rpoC1 and rpoC2, because
conserved domains were predicted to comprise the entire sequence for
both of these genes. Rate corrections were done by custom python
scripts and are available as Supplemental Data File 3. The Pearson
correlation coefcients were calculated using the built in function in the
python scipy module. A correlation coefcient value of 0.6 or above was
used to indicate a positive rate correlation (Sato et al., 2005). The rate
correlation of each gene with itself was removed from all analyses. The
one-side Wilcoxon Rank Sum test and correction for multihypothesis
testing was performed using R software package as described above.
Structurally mediated coevolution within groups of amino acids was
evaluated for 28 plant species (27 Geraniales from this study plus Arabidopsis) and Escherichia coli using CoMap (Dutheil and Galtier, 2007). To
evaluate structurally mediated coevolution between amino acids from
different genes, three different features (volume, charge, and polarity) of
amino acids were considered and amino acid substitutions at specic sites
on each branch of the gene tree were quantied based on these features.
For each gene, the changes of amino acids on all branches at each site were

Plastid-Nuclear Genome Coevolution

extracted to form a site-specic substitution vector. The site-specic

substitution vectors from two different genes were compared with nd
structurally mediated coevolved changes (i.e., volume increase at site X
of gene A and volume decrease at site Y of gene B) of sites from different
genes. The interacting amino acid pairs between SIG and RNAP subunits were predicted with CMA (Sobolev et al., 2005) and by mapping
the amino acid pairs with distance between them of <5 (Hu and Yan,
2009) in RNA polymerase of E. coli to those of Geraniales using custom
python scripts (Supplemental Data File 3). The distribution of distances
of the coevolved amino acid pairs identied in the structurally mediated
evolutionary analysis and the overlap between coevolved and the interacting amino acid pairs were executed with custom python scripts
(Supplemental Data File 3).

Accession Numbers
All sequences have been submitted to GenBank/EMBL data libraries
under accession numbers KJ916247 through KJ917105, and KM461126
through KM461665.

Supplemental Data

571

Supplemental Table 4. The Number of Interaction Pairs with a Rate

Coefcient over 0.6 within Corresponding Genes.
Supplemental Table 5. Rank Sum Test of Rate Correlation Coefcient
Using Conserved Domains.
Supplemental Table 6. Test of dS Saturation of 30 Genes.
Supplemental Table 7. Analysis of Interaction Sites and Overlap
between the Coevolving and Interaction Sites.
Supplemental Table 8. Primer Pairs Used for Amplication of Sigma
Factor Genes.
The following materials have been deposited in the DRYAD repository
under accession number http://dx.doi.org/10.5061/dryad.m02v7.
Supplemental Data File 1. Alignments of Different Genes by Different
Tools.
Supplemental Data File 2. List of Plastid and Nuclear Control Genes.
Supplemental Data File 3. Custom Scripts Used in This Study.

ACKNOWLEDGMENTS

Supplemental Figure 5. Maximum likelihood tree of 27 species from

Geraniales and Arabidopsis.

We thank Mao-Lun Weng for helpful discussions on rate analyses and

the graphic icon photograph, Scott Hunicke-Smith and Heather Deiderick
of the University of Texas GSAF for assistance with Illumina sequencing,
Claus Wilke for suggestions on correction of multihypothesis testing, Mario
Fares for suggestions on dS saturation test, Chen-Hsiang Yeang for
suggestions on analysis of coevolution of amino acids, Nancy Moran,
Ahmed Bahieldin, Greg Clark, and three anonymous reviewers for comments on an earlier version of the article, and Texas Advanced Computing
Center for supercomputer access. Support was provided by the National
Science Foundation (IOS-1027259 to R.J.K. and T.A.R.) and from Vice
President for Educational Affairs Abdulrahman O. Alyoubi at King Abdulaziz
University, Jeddah, Saudi Arabia, to J.Z. and J.S.

Supplemental Figure 6. Strong Correlation of Nonsynonymous (dN)

but Not Synonymous (dS) Substitution Rates between sig1/2/5 and
RNAP Genes by Three Methods Using Conserved Domains.

AUTHOR CONTRIBUTIONS

Supplemental Figure 1. Phylogeny of the Sigma Factor Families in

Geraniales and Arabidopsis.
Supplemental Figure 2. Copy Number of the Six SIG Genes Varies
across Geraniales.
Supplemental Figure 3. Multiple Gene Duplication and Loss Events in
Geraniales.
Supplemental Figure 4. Shared clade-specic synonymous rate (dS)
acceleration in Geraniaceae.

Supplemental Figure 7. Little to No Correlation of Nonsynonymous

(dN) or Synonymous (dS) Substitution Rate between sig3/4/6 and
RNAP Genes by Three Methods.
Supplemental Figure 8. Little to No Correlation of Nonsynonymous
(dN) or Synonymous (dS) Substitution Rate between sig3/4/6 and
RNAP Genes by Three Methods Using Conserved Domains.
Supplemental Figure 9. Strong Correlation of Nonsynonymous (dN)
but Not Synonymous (dS) Substitution Rates between sig1/2/5 and
RNAP Genes by Three Methods Using the Entire Sequences and
a Second Set of 10 Nuclear Control Genes.
Supplemental Figure 10. Strong Correlation of Nonsynonymous (dN)
but Not Synonymous (dS) Substitution Rates between sig1/2/5 and
RNAP Genes by Three Methods Using the Entire Sequences and
a Second Set of 10 Nuclear Control Genes.
Supplemental Figure 11. The Distribution of Distance between Amino
Acid Pairs Predicted to Be Involved in Structurally Mediated
Coevolution.
Supplemental Table 1. Summary of Accession Numbers, RT-PCR
Results, and Voucher Information for All Species Examined.
Supplemental Table 2. Summary of Gene Duplication and Loss
Events.
Supplemental Table 3. Pairwise Comparison of Evolutionary Rates
from Different Alignment Methods.

J.Z., T.A.R., and R.K.J. designed the research. J.Z. performed research
and analyzed the data. T.A.R. isolated RNA and assisted with experiments. J.C.B. contributed plastid genome data. J.Z. wrote the article.
T.A.R., J.S., J.C.B., and R.K.J. critically revised the article. All authors
read and approved the article.

Received November 19, 2014; revised January 28, 2015; accepted

February 12, 2015; published February 27, 2015.

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Coordinated Rates of Evolution between Interacting Plastid and Nuclear Genes in Geraniaceae
Jin Zhang, Tracey A. Ruhlman, Jamal Sabir, J. Chris Blazier and Robert K. Jansen
Plant Cell 2015;27;563-573; originally published online February 27, 2015;
DOI 10.1105/tpc.114.134353
This information is current as of April 16, 2015
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