Accelerated Evolution after Gene Duplication:

A Time-Dependent Process Affecting Just One Copy

Cinta Pegueroles,y ,1 Steve Laurie,y ,1 and M. Mar Alba`*,1,2
1
Evolutionary Genomics Group, Research Programme on Biomedical Informatics (GRIB), Hospital del Mar Research Institute
(IMIM), Universitat Pompeu Fabra (UPF), Barcelona, Spain
2
Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain
y
These authors contributed equally to this work.
*Corresponding author: E-mail: malba@imim.es.
Associate editor: Hideki Innan

Abstract
Gene duplication is widely regarded as a major mechanism modeling genome evolution and function. However, the
mechanisms that drive the evolution of the two, initially redundant, gene copies are still ill defined. Many gene duplicates
experience evolutionary rate acceleration, but the relative contribution of positive selection and random drift to the
retention and subsequent evolution of gene duplicates, and for how long the molecular clock may be distorted by these

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processes, remains unclear. Focusing on rodent genes that duplicated before and after the mouse and rat split, we find
significantly increased sequence divergence after duplication in only one of the copies, which in nearly all cases corre-
sponds to the novel daughter copy, independent of the mechanism of duplication. We observe that the evolutionary rate
of the accelerated copy, measured as the ratio of nonsynonymous to synonymous substitutions, is on average 5-fold
higher in the period spanning 412 My after the duplication than it was before the duplication. This increase can be
explained, at least in part, by the action of positive selection according to the results of the maximum likelihood-based
branch-site test. Subsequently, the rate decelerates until purifying selection completely returns to preduplication levels.
Reversion to the original rates has already been accomplished 40.5 My after the duplication event, corresponding to a
genetic distance of about 0.28 synonymous substitutions per site. Differences in tissue gene expression patterns parallel
those of substitution rates, reinforcing the role of neofunctionalization in explaining the evolution of young gene
duplicates.
Key words: gene duplication, evolutionary rate, positive selection, gene expression, indels, adaptive evolution.

Introduction Under the neutral theory of molecular evolution (Kimura

Gene duplication is one of the major mechanisms contribut- 1983), when the appearance of a new duplicate is not directly
ing to genome complexity, as well as to the generation of new advantageous (e.g., through increased gene-dosage advan-
Article

gene functions (Ohno 1970; Zhang et al. 2003). For instance,
in the human genome, between 15% and 38% of genes may
have arisen through gene duplication (Lynch and Conery
2000; Zhang et al. 2003). Unequal crossing-over during mei-
osis is the main contributor for tandem or closely located
gene duplicates, giving rise to a situation where the structure
of the progenitor (parent) and novel duplicated (daughter)
gene is expected to be initially equal, unless the gene was
disrupted during the process of duplication itself. On the
contrary, genes formed by retrotransposition (retrogenes)
will have a different structure from their parent copy, because
retrotransposition involves the random insertion of a
retrotranscribed cDNA. The structure of the daughter retro-
gene is characterized by a single exon, the presence of a polyA
tail, and a lack of regulatory regions (Hahn 2009). Segmental
duplications and whole-genome duplications (WGDs)
are other sources of gene duplicates, in which multiple
genes and their flanking regulatory regions are duplicated
simultaneously (Dehal and Boore 2005; Sharp et al. 2005).
tage), the likelihood of the duplicate becoming fixed is low
and can be estimated as 1/2N in diploid organisms, where N is
the effective population size. Two genes with identical func-
tions are unlikely to be maintained in the genome unless the
generation of extra gene product is beneficial (Zhang et al.
2003). Thus, most duplicates are expected to be lost to pseu-
dogenization rather than becoming fixed (Lynch and Conery
2000). Retrogenes are strong candidates to be pseudogenized,
because they are expected to be distantly located from the
promoter region of the parent copy. However, it has been
observed that duplicates formed through this mechanism can
be expressed (Marques et al. 2005; Zemojtel et al. 2010).
Several mechanisms have been proposed to explain the
maintenance of gene duplicates in the genome (reviewed in
Hahn [2009] and Innan and Kondrashov [2010]), which can
be broadly summarized into three main models: neofunctio-
nalization, subfunctionalization, and increased gene-dosage
advantage. In the neofunctionalization model, one copy is
maintained by purifying selection and retains the original

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1830 Mol. Biol. Evol. 30(8):18301842 doi:10.1093/molbev/mst083 Advance Access publication April 26, 2013
Neofunctionalization of Gene Duplicates . doi:10.1093/molbev/mst083
function, whereas the redundant copy is free to evolve and
potentially acquire new gene functions (Ohno 1970; Walsh
1995; Force et al. 1999). Following gene duplication, the evo-
lutionary rate is expected to be accelerated in the copy freed
from purifying selection, resulting in asymmetry between the
two copies. If mutations lead to adaptive functional changes
then the new copy is expected to evolve by positive selection.
In the subfunctionalization model (complementation
degeneration model), both duplicates accumulate mutations
through genetic drift, and consequently, their evolutionary
rates increase symmetrically relative to the original copy,
and the ancestral function(s) may be split between duplicates
(Force et al. 1999; Lynch and Force 2000). Subsequently,
purifying selection is expected to maintain the two function-
ally distinct copies. A related model is that of escape from
adaptive conflict, in which a protein with multiple functions
can optimize each function independently after gene dupli-
cation, following release from antagonistic pleiotropy
(Hughes 1994; Des Marais and Rausher 2008). Under the
increased gene-dosage advantage model, gene duplication is
itself beneficial due to the increased amount of gene product,
and thus, both duplicates are expected to become rapidly
fixed (Konrad et al. 2011). In this case, the evolutionary rate
should be symmetrical between duplicates but not signifi-
cantly different to that of the ancestral copy.
Discrimination between these three models can theoreti-
cally be achieved by the comparison of the evolutionary pace
of duplicates and the ancestral copy. Several studies have at-
tempted to quantify the fraction of duplicated gene pairs that
evolve asymmetrically. Conant and Wagner (2003) reported
that this figure is about 2130% of the duplicated gene pairs
from Saccharomyces cerevisiae, Schizosaccharomyces pombe,
D. melanogaster, and Caenorhabditis elegans. Similarly, an
analysis of recently duplicated human genes has shown that
about one-fifth of pairs evolve asymmetrically (Panchin et al.
2010). In addition, the percentage of mammalian young gene
duplicates affected by positive selected has been estimated to
be about 10% (Hahn 2009). Both Cusack and Wolfe (2007)
and Jun et al. (2009) reported that tandem duplicates tend to
evolve symmetrically, whereas distant duplicates, including
retrogenes, tend to evolve asymmetrically. However, a study
of the angiogenin gene family in mouse found evidence of
neutral, purifying, and positive selection in different branches
of this family of six tandemly duplicated genes (Codoner et al.
2010). Although these studies support neofunctionalization
for a fraction of the gene families, a global overview is still
missing.
Another way to examine the evolution of duplicated gene
pairs is through functional studies. So far, most studies have
focused on the comparison of gene expression patterns.
These studies support the idea that duplication leads to
rapid acquisition of divergent tissue-expression patterns
(Huminiecki and Wolfe 2004; Farre and Alba` 2010; Huerta-
Cepas et al. 2011). The main source of expression data to
date has been microarray-based analyses, despite the fact
that cross-hybridization takes place at a high rate in such
experiments (Blanc and Wolfe 2004) and is likely to be parti-
cularly problematic in the case of duplicates. RNAseq
MBE
technology appears to be more appropriate for studying
expression in duplicates because it negates the cross-
hybridization problem. However, there is currently a paucity
of data for most species, and where data are available, it is
only for a limited number of tissues and time points, which
may lead to underdetection of differences in gene expression
between duplicates.
The aim of this study is to investigate the evolutionary
pace of recently duplicated genes, tracking their nonsynony-
mous to synonymous substitution rate ratio (dN/dS) during
different time intervals, and to evaluate functional conse-
quences through the analysis of patterns of positive selection
and gene expression. Estimation of evolutionary rate in young
duplicates can be a difficult task, due to the short period of
time available for changes to accumulate. In addition, when
few changes have accumulated, it may not be possible to
detect significant differences in the speed of evolution be-
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tween the two copies. Thus, we focus on rodent duplicates,
as the molecular evolutionary rate in this order is two to
three times higher than the rate in other mammalian
orders such as primates (Waterston et al. 2002; Toll-Riera
et al. 2011). We examine the behavior of evolutionary rate
focusing on three different situations: comparing dN/dS
before and after gene duplication, within different time in-
tervals, and between duplicated gene pairs. By combining
data from 404 duplicated gene copies generated at different
times during rodent evolution, we conclude that evolution-
ary rate is not constant through time, with younger dupli-
cates having higher rates. Asymmetry is a general trend
following duplication, and the novel daughter copy generally
evolves more rapidly both in terms of substitution rate and in
the incorporation of short indel events. We observe that rate
acceleration is limited to a relatively short period of time
following the duplication event, and its effect is erased com-
pletely approximately 40.5 My later. Positive selection ap-
pears to be prevalent after the duplication event, acting
mainly on the accelerated copy, and tissue-expression pat-
terns are better conserved between the single-copy human
ortholog gene and the slower evolving duplicate than with
the faster evolving duplicate.
Results
Initial Characterization of the Duplicated Gene Set
We obtained a comprehensive set of protein families formed
by genes undergoing a single duplication event in Mus
musculus (mouse), Rattus norvegicus (rat), or their common
ancestor, using paralogy annotations from Ensembl Compara
(Vilella et al. 2009). The trees were further validated using
maximum likelihood (ML)-based tree reconstruction (see
Materials and Methods). The set was split into two groups,
according to whether duplication occurred before or after the
speciation event separating mouse and rat. These groups
were termed the prespeciation duplication set (Pre-SD) and
postspeciation duplication set (Post-SD), respectively (fig. 1).
The Pre-SD set comprised 35 families including 140
duplicated gene sequences (fig. 1A), whereas the Post-SD
set comprised 117 families including 264 duplicated gene
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FIG. 1. Schematic representation of the gene duplication data sets employed in this study. (A) Prespeciation duplication set (Pre-SD), 35 families.
(B) Postspeciation duplication set (Post-SD) with one duplication in mouse, 73 families. (C) Postspeciation duplication set (Post-SD) with one
duplication in rat, 29 families. (D) Postspeciation duplication set (Post-SD) with one duplication in mouse and one in rat, 15 families. Black circles
indicate a duplication event and grey circles a speciation event. Rn, Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens; Bt, Bos taurus; Cf, Canis
familiaris; Ec, Equus caballus; Fc, Felis catus.

sequences. Post-SD families had a single duplication event
(fig. 1B and C) except for a few cases in which the gene had
independently duplicated in mouse and rat (fig. 1D).
We next investigated whether the set of duplicated
genes showed any functional biases with respect to the rest
of the genes in the genome. Gene Ontology (GO) term en-
richment analysis of the 152 families from the Post-SD and
Pre-SD sets, using the human ortholog to identify function,
revealed an over-representation of olfactory receptor and
mitochondrial genes. For the molecular function ontology,
we found an enrichment in olfactory receptor activity
(GO:0004984) in 10.53% of the genes list (P < 0.05 using a
two-tailed Fishers exact test and adjusting for multiple
comparisons). Fifty percent of these genes were also defined
as melanocortin receptor activity (GO:0004977). For the bio-
logical process ontology, we found an enrichment in several
terms related to sensory perception of smell (GO:0007600,
GO:0007606, and GO:0007608), in 11.18% of the gene list
(P < 0.05 using a two-tailed Fishers exact test and adjusting
for multiple comparisons). The cellular component ontology
was enriched in mitochondrial genes (GO:0044429), involving
8.55% of the gene list (P < 0.05 using a two-tailed Fishers
exact test and adjusting for multiple comparisons), 38.5%
of which were specifically involved in the respiratory
chain (GO:0070469).
Comparison of the distribution of nonsynonymous to
synonymous substitution rates (dN/dS) in the branches
before and after the duplication event (PreDupl vs.
PostDupl, fig. 1) showed a significant tendency toward an
increase in evolutionary rate following duplication
(P < 0.001 and P < 108, for Pre-SD and Post-SD, respectively,
Wilcoxons test; fig. 2A and B, respectively). This acceleration
diminished over time, as we detected a significant decrease
in dN/dS in the Pre-SD set by the time the mouse and rat
lineages have segregated (P < 0.01, PostDupl vs. PostSp
Wilcoxons test, fig. 2A).
Asymmetric Evolution of Gene Duplicates
To investigate whether acceleration in evolutionary rate
affects each of the duplicates differently, we estimated the
percentage of asymmetry in evolutionary rate between the
two copies. As our aim was to compare different branches
from the same family, we only used families in which all
branches had passed the quality filters for dN and dS estima-
tion (see Materials and Methods). We found that 65.38% and
20.41% of the pairs were evolving at a significantly different

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FIG. 2. Boxplot representation of dN/dS for the Pre-SD (A) and Post-SD (B) sets. After filtering out branches with dS < 0.01, for the Pre-SD set, we had
34, 62, and 139 pre-, postduplication, and postspeciation branches, respectively, and for the Post-SD set, we had 115 and 128 pre- and postduplication
branches, respectively. Differences between the PreDupl and PostDupl branches were significant at P < 0.001 for the Pre-SD set (A) and P < 108 for the
Post-SD set (Wilcoxons test). The difference between the PostDupl and PostSp branches in (A) was also significant at P < 0.01.
rate after the duplication (i.e., asymmetrically, P < 0.05 using
a Fishers exact test and adjusting for multiple comparisons)
in the Pre-SD and Post-SD sets, respectively. It should be
noted that the low number of absolute changes in individual
genes could lead to an underdetection of significant differ-
ences within families, especially in the Post-SD set, which is
composed of younger duplicates. In fact, when classifying the
two rodent duplicates from the Post-SD set into slower and
faster evolving, we observed that even in those cases classified
as symmetrical, the dN/dS of the faster evolving copy was
on average 3-fold higher than that of the slower evolving
copy, and the difference in the distribution of dN/dS values
between the faster and slower copies pooled together was
significant (P < 106, Wilcoxons test). A similar trend was
observed in the Pre-SD set where, even when the pair had
been classified as symmetrical, the faster copy was evolving on
average 2.5 times more rapidly, though in this case, differences
were not significant because the sample size was reduced to
just six families.
The aforementioned observations prompted us to con-
sider the faster and slower evolving branches independently.
This revealed that dN/dS for the slower evolving branch
(PostDuplS) was not significantly different from the dN/dS
of the preduplication branch (PreDupl), in both the Pre-SD
and Post-SD sets (fig. 3A and B, respectively). In contrast, the
faster evolving branch (PostDuplF) was on average 4.7 and 2.5
times accelerated with respect to the preduplication branch,
for the Post-SD and Pre-SD sets, respectively (PostDuplF
against PostDuplS in figure 3A and B; P < 1012, Wilcoxons
test). In the Pre-SD set, the speed of evolution of the faster
evolving copy decelerated after speciation, as shown by a
significant decrease in dN/dS values in this more recent
period when compared with the period immediately after
the duplication (fig. 3A, PostDuplF against PostSpF;
P < 0.002, Wilcoxons test).
MBE
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We investigated whether the duplicate gene acceleration
observed in our sets was due primarily to the effect of
retrogenes. Analysis of the genomic location of duplicated
genes showed that retrogenes tended to be located on a
different chromosome from the parent gene, contrary to
what is observed for nonretrogene copies. Considering the
Pre-SD and Post-SD sets together, 69% of duplicates located
on different chromosomes were retrogenes, compared with
just 11% of duplicates located on the same chromosome.
Most of the duplicates located on the same chromosome
were found within 300 kb of each other (83%), in agreement
with the predominance of DNA-based tandem duplication.
Analysis of dN/dS values for the two types of duplicated gene
indicated that acceleration is not exclusive to retrogenes. In
the Post-SD set, asymmetry between fast and slow evolving
copies remained significant when excluding retrogenes from
the comparison (P < 106, Wilcoxons test) and when con-
sidering only duplicates located on the same chromosome
(P < 105, Wilcoxons test). Furthermore, fast evolving copies
of the retrogene containing families were not significantly
accelerated when compared with the nonretrogene families
(P = 0.308, Wilcoxons test) nor was there a difference be-
tween fast copies for families located on the same chromo-
some versus those found on different chromosomes
(P = 0.770, Wilcoxons test).
Deceleration of Evolutionary Rates after the Initial
Increase
In an attempt to better define the observed pattern of evo-
lutionary rate deceleration, duplicates of the Pre-SD and
Post-SD sets were classified into younger and older by sub-
tracting the value of dS for the preduplication branch from
the dS of the postduplication branch (or the mean of both
postduplication branches when available). For this analysis,
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FIG. 3. dN/dS for slower and faster duplicated gene copies for the Pre-SD (A) and Post-SD set (B). Differences between the faster postduplication branch
(PostDuplF) branch and all other branches were highly significant for both sets (P < 1010). Differences between the preduplication (PreDupl) and
faster postspeciation (PostSpF) or slower postspeciation (PostSpS) branches were not significant (see supplementary data, Supplementary Material
online, for all pairwise comparisons).

Table 1. dN/dS Values for Preduplication and Postduplication Branches.

My 70 to 43 43 to 16 16 to 8 8 to Present
Older Pre-SD (16) Younger Pre-SD (17) Older Post-SD (19) Younger Post-SD (55)
PreDupl PostDupl PostSp PreDupl PostDupl PostSp PreDupl PostDupl PreDupl PostDupl
Mean 0.31 0.31 0.27 0.19 0.47 0.21 0.31 0.39 0.16 0.43
Median 0.19 0.28 0.26 0.10 0.31 0.16 0.13 0.36 0.09 0.30
Standard deviation 0.39 0.31 0.15 0.30 0.35 0.15 0.67 0.23 0.16 0.38
NOTE.Estimated million years are shown at the top. Number of families in parentheses.

we considered all families having dS > 0.01 in both the
preduplication branch and at least one of the two postdu-
plication branches, comprising 33 families for the Pre-SD set
and 74 families for the Post-SD set. Positive values, indicating
that the postduplication branch is longer than the predupli-
cation branch (dS PostDupl > dS PreDupl, see branches in
fig. 1), were classified as older duplicates and negative values
as younger duplicates (table 1). Assuming that the primate
and rodent lineages split approximately 70 Ma and the
mouse and rat lineages approximately 16 Ma (Douzery
et al. 2003), the mid point of the rodent ancestral branch
is 43 Ma. From this, we can infer that the mean age of the
older Pre-SD duplicates is approximately 56.5 Ma and of the
younger Pre-SD duplicates approximately 29.5 Ma. Doing
similar calculations for the Post-SD duplicated gene set,
the mean age of the older Post-SD duplicates is approxi-
mately 12 Ma and of the younger Post-SD duplicates
approximately 4 Ma (table 1).
When considering the dN/dS values (table 1), we observe
that the postduplication branches are evolving significantly
faster than the preduplication branches in both older and
younger Post-SD duplicates (P = 0.001 and P < 106, respec-
tively, Wilcoxons test). For the younger Pre-SD duplicates, the
rate initially increases by about 23-fold (P < 103 PreDupl
vs. PostDupl) and subsequently experiences a similar decrease
(P = 0.006 PostDupl vs. PostSp). In the older Pre-SD duplicates,
there are no significant differences between the PreDupl,
PostDupl, and PostSp branches. The same result is obtained
when we consider only the faster evolving copy of each pair
(supplementary data, Supplementary Material online). Thus,
older duplicates in the Pre-SD set (~56.5 My mean age)
appear to have already returned to their initial preduplication
dN/dS values before speciation (~16 Ma), which means that
in rodents 40.5 My may be sufficient for purifying selection to
erase signs of duplication driven acceleration. Using the same
rationale, because we detect significant differences between
the pre- and postduplication branches of the Post-SD set, we
may conclude that 12 My of rodent evolution is not sufficient
to return to similar values of dN/dS similar to those of before
the duplication event.
The increase in purifying selection after the initial acceler-
ation period was confirmed by comparisons at the intrafamily
level. We calculated normalized dS and dN/dS for older and
younger Pre-SD and Post-SD duplicates as the average
postduplication value divided by the sum of the average
postduplication and preduplication values. Using this for-
mula, values have a range from 0 to 1; dN/dS values close
to 0.5 indicate that post- and preduplication branches have

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FIG. 4. Normalized dS and dN/dS values older and younger for older and younger Pre-SD and Post-SD duplicates. Normalized values were calculated as

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average postduplication branch values divided by the sum of average postduplication and preduplication branch values (A). Pre-SD set: differences in dS
and dN/dS between younger and older duplicates were significant (P < 108 for dS and P = 0.01 for dN/dS, respectively, Wilcoxons test). Normalized
dN/dS in older duplicates was not significantly different from the corresponding value in postspeciation branches (dN/dS Older-PostSp, P = 0.792 and
dN/dS Younger-PostSp, P = 0.002, Wilcoxons test). (B) Post-SD set: only differences in normalized dS were significant (P < 109).

similar dN/dS, whereas for values larger than 0.5, dN/dS of

the postduplication branch is higher. For comparison, in the
Pre-SD set, we also calculated the average postspeciation
value divided by the sum of the average postspeciation and
preduplication values. The results are displayed in figure 4.
In both sets, older duplicates displayed significantly higher
normalized dS values than younger duplicates, confirming
that the two groups represent duplicates of very distinct
age (P < 108 both sets). For dN/dS, clear differences between
younger and older can be detected for the Pre-SD set,
with younger duplicates showing significantly higher values
(P = 0.01, fig. 4A). In contrast, the younger and older dupli-
cates in the Post-SD set show no significant differences
(P = 0.611, fig. 4B).
Identification of Positive Selection
To further investigate whether acceleration of one of
the copies was related to adaptive processes, we tested for
positive selection in each of the branches of those families in
which all branches had passed the filters for dN and dS
estimation (26 families for the Pre-SD set and 49 families
for the Post-SD set) using the branch-site test (see Materials
and Methods). We observed a similar increase (~3.75-fold) in
cases showing positive selection following duplication in
both Pre-SD and Post-SD families (table 2). No significant
differences were detected when comparing the adjusted
P-value distributions of branches PreDupl and PostSp
(P = 0.514, Wilcoxons test), whereas comparisons between
PostDuplPreDupl and PostDuplPostSp were both signifi-
cant (P = 0.0005 and P = 0.001, respectively, Wilcoxons test).
In most cases, the branch showing positive selection was the
accelerated one (60% in the Pre-SD set, and 93.3% in the Post-
SD set). Table 3 lists several examples of positively selected
genes from each group.
Table 2. Percentage of Branches Showing Significant Positive
Selection.
Sets PreDupl PostDupl PostSp
Pre-SD 7.69 28.85 4.81
Post-SD 4.08 15.31 n.a.
Total 5.89 22.08 4.81
NOTE.P < 0.05 Benjamini and Hochberg false discovery rate correction.
Positive selection at the molecular level is usually indicated
by a dN/dS ratio higher than 1. However, only 4 branches
out of the 12 with dN/dS >1 tested significant for positive
selection using the branch-site test, which could be due to the
previously reported conservativeness of this test (Zhang et al.
2005; Han et al. 2009; Toll-Riera et al. 2011). The authors of
the branch-site test claim that the method is sensitive to
sequence and alignment errors (Yang and dos Reis 2011).
Given that the rat genome used here is of lower quality
than that of mouse, in terms of coverage, number of genes
and genes with supporting evidence (Kasprzyk et al. 2004),
positive selection may be expected to be overestimated in
spite of the careful filtering criteria employed here. However,
our results do not appear to be biased because there were no
significant differences in the proportion of mouse and rat
branches under positive selection.
As genomic context can influence both evolutionary rate
and selective processes, where possible, paralogs were classi-
fied into parent and daughter copies (see Materials and
Methods). For the Post-SD set, we could unambiguously
assign the parentdaughter relationship to 23 families, repre-
senting 47% of the total set, and in 96% of these cases (22
of 23 families), the daughter copy was the faster evolving
duplicate. Five of the 23 families tested significant for positive
selection, and in all of them, the gene undergoing selection

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Table 3. Selected Gene Duplicates Undergoing Positive Selection after Duplication Event.
Set Gene ID Description P*
ENSMUSP00000023655 Myxovirus (influenza virus) resistance 2.26  109
ENSMUSP00000009058 ATP-binding cassette, subfamily B 3.64  106
ENSMUSP00000028986 BPI fold containing family A 6.88  104
ENSMUSP00000130225 Pyroglutamylated RFamide peptide receptor 2.06  104
Pre-SD ENSMUSP00000045229 Fucosyltransferase 2 9.11  103
ENSMUSP00000031693 Sperm adhesion molecule 1 4.12  103
ENSMUSP00000082947 Zinc finger protein 1.86  103
ENSMUSP00000050330 GTPase, IMAP family 1.67  103
ENSMUSP00000038931 Glutathione S-transferase, pi 2 8.20  108
ENSMUSP00000077984 Kinesin family member C5B 4.86  107
ENSMUSP00000040485 Kininogen 1 2.20  107
ENSRNOP00000062817 Type I keratin KA11 3.28  106
Post-SD ENSRNOP00000020952 Olfactory receptor 68 1.46  105
ENSMUSP00000058779 Olfactory receptor 1033 1.00  105
ENSMUSP00000075814 Solute carrier family 22, member 21 1.22  104
ENSMUSP00000036682 WD repeat domain 20b 5.26  103
NOTE.Selection in the Pre-SD occurred in the ancestral rodent gene of the given mouse ID, whereas selection in the Post-SD set occurred in

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the branch corresponding to the given mouse or rat IDs.
*P value for positive selection using branch site test.

was the daughter. For the Pre-SD set, nine families (35%) Table 4. Tissue-Expression Divergence of Duplicated Genes.
could be similarly classified, and again, in all but one case,
dT
the postduplication branch corresponding to the daughter
was significantly faster evolving. Of these, three of the families Pre-SD Post-SD
had a postduplication branch that tested significant for Hs-F Hs-S F-S Hs-F Hs-S F-S
positive selection, which was the daughter branch in two Mean 0.79 0.36 0.85 0.60 0.24 0.61
cases. The presence of positively selected sites in a protein Median 0.82 0.23 0.92 0.80 0.00 0.83
suggests that it has acquired a novel or modified function. Standard deviation 0.20 0.31 0.23 0.39 0.32 0.41
Thus, the data support an important role for neofunctiona- NOTE.dT, tissue-expression divergence; Hs, Homo sapiens; F, fast mouse duplicate; S,
lization in the evolution of recent rodent gene duplicates. slow mouse duplicate.

Tissue-Expression Divergence Patterns
We investigated tissue-expression divergence (dT, see
Materials and Methods) of fast and slow evolving mouse
duplicates in comparison with the nonduplicated ortholo-
gous human gene. We employed 23 families from the
Post-SD set and 12 families from the Pre-SD set for which
RNAseq-based expression data were available from a recently
published mouse and human tissue panel (Brawand et al.
2011). We found that tissue-expression divergence (dT,
see Materials and Methods) between the faster evolving
copy and the human ortholog was significantly higher than
tissue-expression divergence between the slower evolving
copy and the human ortholog (P = 0.002 and P = 0.003
for the Pre-SD and Post-SD sets, respectively, Wilcoxons
test, table 4).
Tissue divergence remained significant when considering
just the 14 families from the Post-SD set for which we had
both expression data and classification into parent and
daughter, with dT between Homo sapiens (Hs) and the
parent copy equal to 0.138, and dT between Hs and the
daughter copy equal to 0.610 (P = 0.003, Wilcoxons test).
In general, daughter copies were observed to be expressed
in fewer tissues (2.2 on average) than parent copies (4 on
average). Tissue-divergence values between human and
mouse were not significantly different when comparing retro-
gene containing families to nonretrogene containing families
nor for families located on the same or different chromo-
somes (P = 0.349 and 0.658, respectively, Wilcoxons test).
In the five families for which we had expression data and
parentdaughter categorization in the Pre-SD set, we ob-
served that the daughter paralog always showed a restricted
expression profile, that was a subset of that of the parent
paralog, and typically was only expressed in one of the six
tissues examined. Five of 23 genes in the Post-SD set had a
clear signal of neofunctionalization in the expression data,
that is, they were expressed in at least one tissue in which
neither their duplicate copy nor the human ortholog was
expressed.
Indel Analysis
We investigated the distribution of coding sequence indels in
postduplication branches using a previously described proto-
col for identifying indels from alignments (Laurie et al. 2012).
We observed a total of 73 bona fide indels affecting 42 (27%)
postduplication branches in the Pre-SD set and 33 indels
affecting 22 (22%) postduplication branches in the Post-SD
set. Overall, we observed an excess of deletions relative to
insertions at a ratio of approximately 1.5:1, and although
approximately half the occurrences of each were of one
amino acid in length, there was a slight tendency toward
insertions being longer than deletions (see supplementary

1836
Neofunctionalization of Gene Duplicates . doi:10.1093/molbev/mst083
data, Supplementary Material online). We found a strong
tendency in both data sets for indels to be found on the
faster evolving branch following duplication; 35 of 47 cases
in the Pre-SD set (P < 0.01, binomial test), and 30 of 33
cases in the Post-SD set (P < 105).
We examined whether there was any association between
signs of positive selection and the presence of indels following
duplication. Of the 20 branches in the Pre-SD set that tested
positive in the branch-site test, 15 had indels, whereas of the
remaining 136 branches, only 27 had indels, indicating a sig-
nificant relationship between positive selection and indels in
this data set (P < 105, Fishers exact test). A similar result was
found in the Post-SD set where 10 of the 15 branches that had
evidence of selection contained an indel event, compared
with 8 of the remaining 83 branches (P < 105, Fishers
exact test).
Discussion
In this study, we evaluate the dynamics of the evolutionary
rate of recent rodent duplicates, considering the ratio of
nonsynonymous to synonymous substitution rates (dN/dS)
before and after duplication, and after the speciation event
separating mouse and rat. Using dN/dS instead of other rate
measures, such as dN or the number of amino acid substitu-
tions, has the advantage that we can compare events that
occurred at different times. As both accumulation of changes
in pseudogenes and putative changes in badly predicted
genes will lead to an apparent acceleration in evolutionary
rate of the gene concerned and may mimic signals of positive
selection, here we have prioritized findings deriving from
functional data, rather than just predicted genes, despite
this resulting in a substantial decrease in the number of
families available for analysis. Our findings in terms of evolu-
tionary rate can be summarized into two main points:
acceleration after gene duplication is widespread but limited
to just one copy, and evolutionary rate of the accelerated
copy shows time dependence.
Acceleration in the evolutionary rate of paralogs compared
with orthologs after gene duplication has been widely
reported (Lynch and Conery 2000; Van de Peer et al. 2001;
Kondrashov et al. 2002; Nembaware et al. 2002; Jordan et al.
2004; Scannell and Wolfe 2008; Panchin et al. 2010). However,
when we consider the duplicates individually, we find that
they are evolving asymmetrically and, more remarkably, that
it is just one of the two copies that shows acceleration, the
other copy maintaining the constraints of the ancestral gene.
A previous study suggested that asymmetry in evolutionary
rates was a general trend in recent human duplicates (Zhang
et al. 2003). Focusing on yeast duplicates after a WGD event,
Scannell and Wolfe (2008) found a burst of protein sequence
evolution in both initially fast (5.4 times accelerated) and
initially slow duplicate clades (2.2 times accelerated) and
thus proposed that a form of subfunctionalization leads
to the preservation of gene duplicates (Scannell and Wolfe
2008). A further study failed to find significant asymmetry
between recent pairs of duplicates, though they were
observed to evolve faster than single-copy orthologs
(Kondrashov et al. 2002). However, asymmetry may have
MBE
been underestimated in this study because they discarded
those triplets in which the outgroup was closer to a certain
paralog than the other paralog.
In this study, we have observed that acceleration of evo-
lutionary rate is only apparent in one of the two duplicates.
Here, we have estimated the evolutionary rate of the predu-
plication branch by using two outgroup species instead of
one, in contrast to most previous studies. Analysis of the
genomic location of duplicated copies shows that the
parent copy evolves in a highly constrained manner, most
likely preserving the ancestral function, whereas the daughter
copy diverges away. This implies that the two copies are not
equal at birth, not only for copies relocated to a different
chromosome or retrogenes (Cusack and Wolfe 2007) but
also for copies located proximally to each other. These obser-
vations strongly support the neofunctionalization model in
this data set. To our knowledge, acceleration being limited to
Downloaded from http://mbe.oxfordjournals.org/ at Bose Institute on February 24, 2014
just one copy has only previously been reported in a study of
yeast duplicates that originated through WGD. In that case,
only 17% of the gene pairs showed accelerated evolution after
the duplication (Kellis et al. 2004), whereas here the fraction
was higher (65.38% and 20.41% for the Pre-SD and Post-SD
sets, respectively).
We have observed that evolutionary rate is not constant
through time. The initial dN/dS increase, as measured in the
Post-SD set (where duplicates are younger than 16 My), is
about 5-fold. However, after the initial acceleration, the rate
gradually decreases. The less pronounced dN/dS increase after
duplication in the Pre-SD set, which contains duplicates that
have evolved for a much longer time, as well as the decrease of
the rate after speciation, provide strong evidence of this
slowdown.
Gene conversion is a recombination process that homog-
enizes gene pairs and which is believed to be especially im-
portant when the copies are nearly identical (Nagylaki and
Petes 1982; Innan 2002; Casola et al. 2012). The incidence of
this process in our data set is probably very small, as most
gene pairs separated many millions of years ago and show
very distinct evolutionary rates. Gene conversion may lead to
an underestimation of the age of gene duplicates. Although
we observe more duplicates classified as younger than as
older in the Post-SD set, this is not the case for the Pre-SD
set. This seems to suggest that the youngest duplicates are
more prone to be lost over time.
We divided the Pre-SD and Post-SD sets into older and
younger duplicates to gain further insights into the evolution-
ary rate changes through time. After this partitioning, we
concluded that a period of approximately 40.5 My may be
enough to counteract the spike in evolutionary rate following
duplication. This is in contrast to observations following
WGD in yeast where acceleration appears to have been main-
tained for over 100 My (Scannell and Wolfe 2008). Assuming
that synonymous substitutions per synonymous site and mil-
lion years in rodents is approximately 0.007 dS /My (Toll-Riera
et al. 2011), a genetic distance of about 0.28 dS (0.007  40.5)
appears to be sufficient to erase any duplication-associated
protein evolution acceleration.
1837
Pegueroles et al. . doi:10.1093/molbev/mst083
Previous studies focusing on C. elegans and yeast (Davis
and Petrov 2004; Scannell and Wolfe 2008) found that evo-
lutionarily constrained genes tend to duplicate more
frequently than less-constrained genes. However, our set of
152 recently duplicated gene families was composed of sig-
nificantly accelerated genes when comparing dN/dS of the
nonduplicated rodent to an exhaustive set of 1:1 ortholog
genes from Toll-Riera et al. (2011) (P = 0.004, Wilcoxons
test after removing families with dS > 2 and < 0.01).
Subsequent examination of gene function showed that the
increased rates were mainly due to 25 families defined as
receptors, as there were no significant differences when we
excluded them from the comparison (P = 0.071, Wilcoxons
test).
In this data set, we found that a substantial fraction of
duplicates exhibited signals of positive selection, suggesting
that some of the changes contributing to asymmetry are
adaptive. Conant and Wagner (2003) suggested that asym-
metric divergence is mainly caused by relaxed selective con-
straints, though in some cases it may be the result of positive
selection, whereas Han et al. (2009) also found that the fre-
quency of duplicates undergoing positive selection is larger
than that of single-copy orthologs and that daughter copies
transposed to new chromosomal locations are significantly
more likely to have undergone positive selection. The obser-
vation that gene duplicate retention is higher in mouse than
in human has also been taken as evidence that positive
selection plays a key role in duplicate retention, as selection
is more effective in large populations (Shiu et al. 2006). Taken
together with our findings, these observations suggest that
positive selection may play a major role in the maintenance of
new gene duplicates in the genome.
The observed association between fast evolving branches
and the presence of coding sequence indels is in accordance
with previous work in which we observed a positive correla-
tion between indels and dN/dS in one-to-one orthologs in
rodents (Laurie et al. 2012). The finding here that this associ-
ation also extends to proteins for which there is evidence of
positive selection is interesting in light of a recent finding
suggesting that indels themselves may increase the likelihood
of mutation in the surrounding sequence when segregating in
the heterozygous state (Tian et al. 2008). Positive selection
can only act upon mutations that have already occurred, and
thus it would be interesting to know whether the observed
indels predate sequence mutations that were subsequently
positively selected in these young duplicates. Unfortunately,
there are no available models to test whether indel events
themselves may be selected for.
Taking into account the important amino acid substitu-
tion rate differences between duplicated copies, one might
expect diverse gene expression patterns as well. We observe
that, in general, constrained copies retain a similar pattern to
that of their single-copy ortholog, whereas accelerated dupli-
cates present a significantly different expression pattern com-
pared with the constrained form. Again, these findings
support the idea that slow evolving copies preserve the orig-
inal function, whereas fast evolving copies gain new functions,
in agreement with the ortholog conjecture hypothesis,
1838
MBE
which states that function is more conserved between ortho-
logs than paralogs but that it can change rapidly after gene
duplication, as recently validated using experimental data
(Altenhoff et al. 2012). Tissue-divergence patterns seem to
be related to the genomic location of duplicates, because
patterns when comparing fast- and slow evolving copies
were distinct for duplicates located on different chromo-
somes but not when considering only retrogenes. It is
worth noting that significant differences in tissue-divergence
patterns were detected even though it is likely that the true
values are being underestimated, firstly due to the limited
number of tissues for which data was available, and secondly
because we only tested tissue-related differences.
Previous studies have failed to find a correlation between
tissue-divergence expression and the evolutionary rate of
coding sequence (Wagner 2000; Conant and Wagner 2003)
or found only a weak correlation, for example, only significant
Downloaded from http://mbe.oxfordjournals.org/ at Bose Institute on February 24, 2014
for dN < 0.2 (Makova and Li 2003). These findings have led to
the suggestion that expression divergence may be mainly
related to changes in the promoter region, because evolution
of coding sequence appears to be only weakly coupled to
gene expression (Wagner 2000). Though the analysis of pro-
moter regions remains a difficult task, a modest but signifi-
cant correlation between the 2 kb region upstream of the
transcription start site and expression divergence has been
observed (Farre and Alba` 2010), suggesting a role of promoter
regions in modifying expression patterns of duplicated genes.
However, in both the Pre-SD and the Post-SD sets, we have
detected a relationship between tissue-expression divergence
and evolutionary rate, because in general faster evolving
daughter copies show a more divergent tissue-expression pat-
tern. Thus, we suggest that the evolution of gene expression
in gene duplicates may be driven by changes in both coding
and regulatory regions.
In summary, by using a collection of rodent duplicated
gene pairs of different age, we have been able to unravel
important inherent features of the gene duplication process.
We have discovered that evolutionary rate acceleration after
duplication is widespread but restricted to one of the copies,
which increases its rate about 5-fold on average, whereas
the other copy maintains the ancestral constraints. The
copy-specific acceleration is associated with positive selection
and functional diversification, in agreement with the neo-
functionalization model. The acceleration period has a limited
duration, and the rate gradually decreases until it returns to
its initial values. An interesting possibility is that the acquisi-
tion of a novel function by one of the copies is facilitated by
the existence of side activities in the ancestral gene that
become more important in the newly generated copy
(Bergthorsson et al. 2007; Nasvall et al. 2012). Optimization
of the secondary function would apparently require less than
40.5 My of rodent evolution.
Materials and Methods
Data Retrieval and Sequence Alignments
We obtained an exhaustive list of paralagous mouse and
rat protein coding genes that arose as a result of a single
Neofunctionalization of Gene Duplicates . doi:10.1093/molbev/mst083
duplication event following divergence from the primate
lineage, together with their corresponding human ortholog,
using Biomart at Ensembl (r65) (Flicek et al. 2012). To
ensure a high-quality data set, we applied the following
initial filtering criteria to all pairs of duplicates: We dis-
carded pairs where the percentage of identity between
copies was lower than 50%, pairs with overlapping location
(which are likely to represent distinct splice forms rather
than duplicated genes), and genes lacking experimental
evidence of expression. We also removed one family
containing selenocysteines as these are interpreted as
stop codons by CodeML. Genomic location and the per-
centage of identity between copies were obtained from
Biomart, and experimental evidence of expression from
the University of CaliforniaSan Cruz (UCSC) Table
Browser (considering both mRNA and EST tracks), using
the mm9 and rn4 databases (Karolchik et al. 2004).
Nonduplicated orthologs from cow, dog, horse, or cat
were used as outgroups. We used an in-house Perl script
to select sets of transcripts with the most similar length
across species, for use as the representative protein.
Protein families were split into two groups according
to whether duplication occurred before (prespeciation
duplication set, Pre-SD) or after (postspeciation duplication
set, Post-SD) the mouse-rat speciation event. The
speciation event provides a natural landmark in time
allowing us to compare dN/dS before and after gene
duplication, and during different time intervals in the
evolutionary history of these two groups of genes.
Concerted evolution is a process involved in the mainte-
nance of sequence similarity between gene copies through
different recombination mechanisms, such as gene conver-
sion (Ohta 2010). If concerted evolution occurred in
families where both mouse and rat genes were duplicated
before the speciation event, the observed tree might be
inconsistent with the real tree (Teshima and Innan 2004).
To minimize this bias, we constructed phylogenetic trees
using ML methodology as implemented in the Proml pro-
gram of the Phylip package (Felsenstein 2005), and we just
kept those families where the topology was the same as
that obtained from the Ensembl Compara pipeline (Vilella
et al. 2009) and discarded those with noncoinciding topol-
ogies. A total of 117 families remained for the Post-SD set
(in 15 of them both mouse and rat genes were duplicated)
and 35 families for the Pre-SD set (supplementary data,
Supplementary Material online). The 117 Post-SD families
contained a total of 132 duplicated gene pairs (264 indi-
vidual duplicated genes) as in a small subset of them
the gene had duplicated in both the mouse and rat
branches. The 35 Pre-SD families contained 70 duplicated
gene pairs represented in both mouse and rat, for a total
of 140 individual duplicated genes. For each protein family,
we performed a multiple protein sequence alignment
with the program Prank + F (Loytynoja and Goldman
2008) using the previously obtained ML trees as a guide
tree. Corresponding nucleotide sequence alignments were
generated from the Prank alignments using PAL2NAL
(Suyama et al. 2006).
MBE
Estimation of Evolutionary Rates and Positive
Selection Tests
The number of nonsynonymous substitutions per nonsy-
nonymous site (dN), synonymous substitutions per synon-
ymous site (dS), and the corresponding dN/dS ratio were
estimated using the ML method implemented in the
CodeML program of PAML version 4.4 (Yang 2007).
We used the free-ratio branch model, which allows the
calculation of a distinct ratio for each branch, and equilib-
rium codon frequencies as free parameters (Codonfreq = 3).
To determine the degree of acceleration and the percent-
age of asymmetry between duplicates, we compared
the evolutionary rates between the preduplication and
postduplication branches, and between the two paralogs
using Fishers exact test.
We tested for positive selection with the branch-site test
(Test 2 implemented in CodeML) (Zhang et al. 2005). This
Downloaded from http://mbe.oxfordjournals.org/ at Bose Institute on February 24, 2014
test compares the null model where codon-based dN/dS for
all branches can only be 1, with the alternative model where
the labeled foreground branch may include codons evolving
at dN/dS > 1. The likelihood ratio test was used to compare
the two models. It was calculated as 2*(L1  L0), where L1 is
the ML value of the alternative hypothesis and L0 the ML
value of the null hypothesis. A 2 distribution with 1
degree of freedom was used to calculate the P value, which
was adjusted for multiple testing using the false discovery rate
method of Benjamini and Hochberg (1995) within the mult-
test package of R.
Data Filtering
Additional filters were applied to our data sets to ensure a
high-quality data set for evolutionary rate calculations, as
previously described elsewhere (Toll-Riera et al. 2011). We
removed families with very short alignments, that is, less
than 100 triplets excluding gaps, families with dS > 2 be-
cause such high values may indicate incorrect ortholog
identification (1 family), and families with dS < 0.01 be-
cause very low dS values may lead to erroneously high
estimates of the dN/dS ratio (82 families). Two further
families were removed from the Pre-SD set due to the
Ensembl gene prediction for one of the rat orthologs in
each containing many dubiously short introns. For the
Post-SD set, we analyzed separately those gene duplicates
that occurred independently in the mouse and the rat
lineages in a certain protein family. Comparison of rates
in the different branches of the same family was only per-
formed for families in which all branches had passed the
filters, that is, 49 families for the Post-SD set and 26 families
for the Pre-SD set. The Pre-SD set contained 52 mouse
duplicated genes, 52 rat duplicated genes, and 26 single-
copy human orthologs, whereas the Post-SD set comprised
49 protein families, formed from 74 mouse duplicated
genes, 24 rat duplicated genes, and 49 single-copy human
orthologs. In five gene families of the Post-SD set, the gene
duplication occurred after the speciation event, according
to Compara prediction and the ML tree constructed using
ProML (see earlier).
1839
Pegueroles et al. . doi:10.1093/molbev/mst083
Duplicate Classification
The genomic location of duplicated genes and number of
exons in each were obtained from Biomart (Haider et al.
2009). We classified as possible retrogenes those genes
having a single exon when the corresponding paralog
and ortholog copies are composed of multiple exons
(Farre and Alba` 2010). Where possible, genes were classified
into parent and daughter copies by looking for conserved
synteny using the Galaxy server to access the UCSC
Genome Browser (Goecks et al. 2010) under the assump-
tion that the parent gene will have a longer extent of
conserved synteny than the daughter copy. The Extract
MAF blocks tool was used to obtain multiple alignments
for a given set of genomic intervals, and the resulting pre-
dicted parentdaughter relationships were manually com-
pared with Ensembl r65 to check for retention of gene
order. Duplicates closely located on the same chromosome
(<300 kb) were excluded from this analysis because synte-
nic relationships are less clear at such short distances.
Correspondence was found in all manually checked cases,
indicated by the observation that the order of the genes
surrounding the predicted parent copy and the ortholo-
gous human gene were similar. A total of 46 genes for the
Post-SD set and 24 genes for the Pre-SD set could be
classified into parent and daughter copies.
Gene Expression
Human and mouse gene expression data were obtained from
a recent large-scale comparative RNA-seq study that used the
Illumina Genome Analyzer IIx (Brawand et al. 2011). Data
were available for human and mouse for six tissues: brain,
cerebellum, heart, kidney, liver, and testis. Though the
number of replicates varied between tissues, for most tissues
there were data from one female (except testis and human
liver) and two males (with the exception of human cerebel-
lum [1 male] and brain [5 males]). Gene expression levels
were reported as the mean per-base read coverage, and the
authors consider values lower than 3 to be indicative of low
expression. We used this value as a cutoff to discriminate
between well-supported expression and background noise.
We extracted gene expression data for a total of 184 genes
(122 mouse and 62 human) from out data set, which corre-
spond to 25 complete (i.e., a single-copy human gene and
two mouse duplicates) Pre-SD and 33 complete Post-SD
families. Tissue-expression divergence (dT) was calculated
by subtracting the number of tissues where both duplicates
were expressed (i.e., the intersect) from the number of tissues
in which at least one of the duplicates was expressed (i.e., the
union), and dividing by the latter, as described in Farre
and Alba` (2010). Because of low expression of the human
copy or one of the two paralogs in all examined tissues,
tissue-expression divergence could not be estimated for
23 families (13 Pre-SD and 10 Post-SD). We performed calcu-
lations comparing the single-copy human gene to each of
the orthologous mouse duplicates and comparing the
two mouse paralogs to each other. We also identified puta-
tive cases of neofunctionalization, defined as cases where
1840
MBE
one paralog is expressed in a specific tissue, in which neither
the second paralog nor the single-copy ortholog are
expressed.
Indel Analysis
The position and size of all indel events within the coding
sequence of the proteins analyzed, as determined from the
multiple alignments, were identified using an in-house Perl
script as described elsewhere (Laurie et al. 2012). Indels were
discarded if they were found to be proximal to an Ensembl-
defined exon boundary, as such cases are more likely to
represent errors in exon definition within the gene-building
algorithm rather than true indel events.
GO Analysis
A GO (Ashburner et al. 2000) enrichment analysis was per-
formed for all 152 gene families using the FatiGO program of
Downloaded from http://mbe.oxfordjournals.org/ at Bose Institute on February 24, 2014
the Babelomics v4.3 integrative platform (Medina et al. 2010).
FatiGO tests for an enrichment in any of the three ontologies
(biological process, cellular component, or molecular func-
tion) from levels 3 to 9, calculating a two-tailed Fishers
exact test and adjusting for multiple comparisons
(Al-Shahrour et al. 2007).
Statistical Tests and Graphs
Wilcoxons, 2, Fishers exact, proportion tests, and Spearman
correlations were performed using the R statistical software
package (R Development Core Team 2010). Graphs were
also produced using R and in particular the ggplot2 package
(Wickham 2009). False discovery rate corrections for simple
multiple testing were calculated following the Benjamini
and Hochberg procedure (Benjamini and Hochberg 1995)
using the multtest package from http://bioconductor.org/
biocLite.R (Gilbert et al. 2009).
Supplementary Material
Supplementary data are available at Molecular Biology and
Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
This work was supported by Ministerio de Economa y com-
petitividad, Spanish Government (Plan Nacional BIO2009-
07120 and BFU2012-36820), Generalitat de Catalunya (FI pre-
doctoral fellowship to S.L.), and Fundacio ICREA to M.M.A.
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