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C. R. Biologies 332 (2009) 241­253
http://france.elsevier.com/direct/CRASS3/
Evolution / Évolution
"Changing by doubling", the impact of Whole Genome Duplications
in the evolution of eukaryotes
Olivier Jaillon a,b,c,
, Jean-Marc Aury a,b,c
, Patrick Wincker a,b,c
a Genoscope (CEA), 2, rue Gaston-Crémieux, CP 5706, 91057 Evry, France
b CNRS, UMR 8030, 2, rue Gaston-Crémieux, CP 5706, 91057 Evry, France
c Université d'Evry, 91057 Evry, France
Accepted after revision 21 July 2008
Available online 29 November 2008
Presented by Claude Combes
Abstract
Species are usually defined by reproductive isolation and are characterized by their gene repertoire. These two aspects are
consequences of events fixed during evolution, including whole genome duplications and other polyploidizations. Thanks to the
recent progress in genome sequencing, new light has been shed on these events. In this review, we will summarize these findings and
discuss the methodology involved. Evolutionary traces of such events have been evidenced in various lineages in plants, animals,
fungi and protozoa. Comparative analysis of synteny is a powerful approach to unveil evolutionary footprints of these events.
According to expectations, these events would facilitate speciation since some of them are thought to be at the base of major
radiations such as teleostei or eudicotyledons. After an initial amplification, the gene repertoire would be shaped by constraints
such as expression level and functional interactions that would tend to maintain only a tiny fraction of the duplicates over the long
term. Functional innovation from duplication may be a secondary effect, enabled by these duplicate retention mechanisms. To cite
this article: O. Jaillon et al., C. R. Biologies 332 (2009).
 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Résumé
 Changement par duplication , l'impact de la duplication totale de génome dans l'évolution des eucaryotes. Les espces
sont souvent définies selon leur isolement reproductif et caractérisées par leur répertoire de gnes. Ces deux traits résultent de
fixations, au cours de l'évolution, d'évnements parmi lesquels les duplications totales de génomes et autres polyplodisations.
Grâce aux séquences de génomes, des éclairages sur ces évnements sont apparus récemment. Nous les résumons ici, ainsi que
les aspects méthodologiques. Des empreintes évolutives de tels évnements ont été mises en évidence dans diverses lignées, parmi
les plantes, animaux, champignons et protozoaires. L'analyse comparée de synténie s'y révle une approche puissante. Comme
attendu, la spéciation serait facilitée ; il est accepté que certains de ces évnements seraient  la base de grandes radiations comme
les téléostéens ou les eudicotylédones. Le répertoire de gnes, aprs une premire amplification, serait façonné par des contraintes,
comme le niveau d'expression et les interactions fonctionnelles, qui tendraient  ne maintenir  long terme seulement qu'une
minuscule fraction des gnes en deux copies. L'innovation fonctionnelle  partir de duplicata serait un effet secondaire, permis par
ces mécanismes de rétention. Pour citer cet article : O. Jaillon et al., C. R. Biologies 332 (2009).
 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
* Corresponding author at: Genoscope (CEA), 2, rue Gaston-Crémieux, CP 5706, 91057 Evry, France.
E-mail address: ojaillon@genoscope.cns.fr (O. Jaillon).
1631-0691/$ ­ see front matter  2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.crvi.2008.07.007
Author's personal copy
242 O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253
Keywords: Whole Genome Duplication; Polyploidization; Comparative genomic; Dosage imbalance; Speciation; Neofunctionalization;
Subfunctionalization
Mots-clés : Duplication totale de génome ; Polyplodisation ; Génomique comparée ; Déséquilibre de dosage ; Spéciation ; Néofonctionalisation ;
Subfonctionalisation
1. Introduction
Most biologists are familiar with the interpretation
of sequence alignments between different species. Substitutions,
insertions and deletions that occurred since
the last common ancestor are commonly noted and analyzed.
Manipulating these evolutionary concepts is so
habitual that it has probably become an unconscious
process. However, capacities or rather deficiencies of
the tools that generate alignments probably orient and
bias our thought. Popular programs such as blast [1]
or blat [2] compare nucleic or protein sequences and
provide a measurement of local similarity through a
symmetrical result often represented as pairwise alignments,
but do not furnish a direct indication of duplications
inside a single genome. However, nearly forty
years ago in a landmark publication, Susumo Ohno proposed
that gene duplications represent a major force
in evolution. His basic premise is that by doubling the
number of the genes, WGD (Whole Genome Duplications)
would facilitate the emergence of new functions,
and also promote radiations [3]. Progress in cytogenetic
studies, followed by the recent explosion of the
number of sequenced genomes has provided the opportunity
to investigate the relics of such ancestral events.
Several ancient polyploidization events have now been
uncovered in Eukaryotes, and some of these are ancestral
to many lineages. Even though the fraction of
sequenced species remains marginal, repetitive findings
tend to confirm a relatively high frequency of polyploidization
during Eukaryote radiation. At present we
have evidence for the existence of a panel of events from
different ages and in different lineages. These findings
have made Ohno's theory quite popular and duplicated
genes from polyploidization have been called ohnologs
by some authors [4­6]. In prokaryotes, however, despite
a very large amount of genomic data available, the fraction
of characterized genomes is probably lower, and no
WGD event have been described to date. We can postulate
many reasons for this, based on the major structural
differences of DNA between prokaryotes and eukaryotes.
Each polyploidization is characterized by an immediate
amplification of the number of chromosomes.
One of the possibilities is that the circularity and often
single copy of DNA may be under constraint and could
represent a major limitation.
Although the theme of gene duplications in evolution
is usually attributed to Ohno, other authors had pointed
it out earlier since the beginning of the 20th century (see
[7] for a historical review). For example, in 1932 Haldane
proposed the possible advantage for duplications
to produce redundant copies that could lessen the risk
due to deleterious mutations [8].
Several WGD have been characterized in at least
three of the five supergroups of eukaryotes according
to the cladistic by Keeling [9], Chromalveolates, Plantae
and Unikonts (Figs. 1 and 2). Ever since such events
have been described, and because each event is specific,
new theories have been proposed to refine the original
model of Ohno. These theories often concern the functional
fate of duplicated genes (ohnologs). Beside their
fundamental interest, these models have probably been
motivated by a challenging conceptual problem. Suppose
a cyclist in the "Tour de France" has two copies of
all the components of his bicycle. To win, he can build
one new bicycle; at least the same if is ahead in the race,
or better, a more efficient one, but not two bicycles. He
has the option of keeping a component in duplicate as a
backup, or removing it, or innovating by using a component
copy for a novel function, or a combination of
these options.
Readers can find previous specialized reviews in
various aspects of polyploidizations [7,10­12]. Here
we describe methodological and evolutionary insights
about ancient polyploidization events that have come
from recent programs of whole genome sequencing.
2. Revealing ancient events
Because ancient polyploidizations (PLZs) are de
facto ancestral events, DNA from living species cannot
provide "formal" proof of their existence in the past.
Revealing an ancestral event always means providing
a list of arguments that are consistent with one's hypothesis.
Providing proof would require analysis of the
genetic material of a fossil. This cannot be achieved today.
Several methods have been used to demonstrate or
confirm ancestral PLZ. Most of the time, ohnologs surviving
from ancient PLZ represent a tiny fraction of the
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O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253 243
Fig. 1. Comparative representation of chromosomal topology of paralogous genes in modern species. Trees indicate the relative date of polyploidization
events in the lineages shown. Each circle represents chromosomes of one species, and each line connects two paralogous genes. For
each species, a core set of paralogous genes was identified by an all-against-all comparison of the proteome of each species against itself using the
Smith and Waterman algorithm [78]. Two genes, A and B were considered paralogs if B is the best match for gene A and if A is the best match of B
(Best Reciprocal Hit). Paralogous genes which are found on the same chromosome (in most of the cases they arise from segmental duplications)
were not drawn. Circular representations were produced using Circos (http://mkweb.bcgsc.ca/circos).
whole set of paralogs which is mainly composed of the
results of numerous small local duplications. The rationales
of the methods are different but their goals consist
of differentiating relics of a large-scale event from this
background noise.
Rejecting a hypothesis of PLZ is probably more difficult.
Under the hypothesis of a PLZ, we expect that
a small fraction of the genes can be maintained as duplicates
(most would be lost), and the genome would
return to a diploid state. We also expect intra- and interchromosome
rearrangements. So, an apparent absence
of any trace of PLZ in a genome cannot be completely
sufficient for the rejection of the hypothesis of this event
in the past. However, this problem is solvable indirectly
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244 O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253
Fig. 2. Representation of the successive duplications of the Paramecium
genome. The exterior circle displays all chromosomes, and the
two interior circles show the reconstructed sequences obtained by
fusion of the paired sequences from each previous step. Paralogous
genes connected by red lines are computed according to the BRH procedure
as in Fig. 1. Blue lines link pairs of genes with a non-BRH
match that were added on the basis of syntenic position. The position
of an ancestral block is unrelated to the position of its constituents in
the previous circle. See Ref. [46] for other details. (For interpretation
of the references to color in this figure legend, the reader is referred to
the web version of this article.)
via a related species where a PLZ could be demonstrated
and dated previous to the radiation.
Lack of data is probably the main source of recurrent
debates between experts. Ohno proposed that three
rounds of WGD occurred at some phylogenetic positions
in the evolution of vertebrates. The third round
(called 3R) which is thought to be at the origin of
the teleost fish lineage had been widely discussed before
relatively recent efforts in genome sequencing of
several vertebrate lineages. Indeed, this hypothesis was
supported by several observations of gene families in
which the number of members doubles at some major
vertebrate radiations. The example the most frequently
described is the family of Hox genes which is present
in one copy in invertebrates, in four copies in mammals
and in more than four in teleost fishes [13,14]. In
teleosts, copies of genes may be lost or maintained depending
on lineages. However, because these observations
were based on partial data which may not be representative
of a genome wide scale, the question remained
controversial [15]. Thanks to the availability of both the
nuclear DNA sequence of a teleost fish Tetraodon nigroviridis
and of the human sequence which was used
as an outgroup of the teleost lineage, convergent results
in agreement with 3R were obtained at large scale and
(a)
(b)
Fig. 3. Examples of topology of phylogenetic trees to date a duplication
event related to a radiation. Trees could be constructed using
one gene which exists in two species (A and B) in different number
of copies, one copy in A and two copies in B (B and B). When duplication
occurs after the split between A and B (tree a), a topology
where B and B are closer to each other than to A is expected. In
this case, species A could considered as an outgroup. When duplication
occurs before the split (tree b), if A has been lost, B can be
closer to A than to its paralogs B.
from two independent methods resulted in the resolution
of this issue [16].
2.1. Using phylogenetic analysis
Phylogeny treats each gene family independently and
reveals whether a putative duplication is anterior or
posterior to a radiation. For each family of genes, the
method requires only the sequences of 3 genes, 2 paralogous
from one species and one from another species.
Two paralogous genes that duplicated after the split
between the two species should be branched closely.
The paralogous distance is shorter than orthologous distance.
In a situation where the duplication is older than
the split, then the orthologous distance can be shorter
than the paralogous distance (Fig. 3). The percentage of
gene families respecting the first or the second topology
provides an argument either in favor of, or against
ancestral large scale duplication.
This approach can be useful to test the hypothesis
of a WGD with either a complete set of proteins [17]
or a partial proteome of a species [15], and also when
methods based on synteny are deficient (either due to
a lack of data, or due to highly rearranged genomes).
In a context where a WGD is admitted in a lineage,
this method also permits dating the duplication of each
pair of paralogous genes, either before or after the WGD
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O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253 245
[18]. After a WGD, a fraction of ohnologs possesses a
similar rate of mutation in the two copies (symmetric
evolution). However, in the other fraction, the evolution
rate is significantly different between the two copies
(asymmetric evolution). It has been demonstrated than
phylogenetic trees reflect correctly the first type of situation,
but tend to be misleading in the latter situation. An
artifact named as "Long-Branch Attraction", causes the
dating of duplication too early, before the split with an
outgroup species lacking the duplication [19]. Finally,
this approach may be relevant for dating several PLZ in
different lineages relative to each other (see below).
2.2. Using distributions of neutral substitution rates of
genes
Since copies of genes resulting from a WGD have
the same age, it is thus tempting to use this property.
The rate of synonymous substitution between two paralogs
can be used as a proxy to a relative date for their
duplication. A significant fraction of paralogs with a
similar rate of synonymous substitution would be an
argument for a large-scale duplication. Because more
than one substitution could occur at the same site, and
therefore cannot be measured directly, different methods
exist for providing estimations. The distribution of
Ks (fraction of synonymous substitutions per synonymous
site) between paralogs was used at large scale initially
to evaluate the extent of duplication events in Arabidopsis
[10,20­23]. Theoretically, the distribution of
Ks follows a decreasing exponential curve, from low Ks
values corresponding to recent duplications, to higher
Ks values at the flat tail. The decay rate of exponential
decrease depends on the rate of progressive losses
of duplicated genes. The presence of a peak at low Ks
values would be due to many and recent local duplications.
Other peaks would correspond to bursts of gene
duplications. In Arabidopsis, the shape of Ks distribution
led to the conclusion that one recent event occurred
and masked at least one earlier event. However, other
authors concluded that the distribution is in agreement
with 3 successive rounds of WGD along with the two
earlier major radiations of the angiosperm lineage [24].
But some of these conclusions are in contradiction with
more recent synteny data between various genomes of
angiosperm [25]. This caveat from Ks analysis is due to
some major limitations of this method. Old events are
hardly distinguishable due to saturation of substitutions
on synonymous sites. Mutation rates are possibly not
constant over a long evolutionary time-scale and in different
lineages. Inferring the level of polyploidy seems
hazardous by this means. Also, sub-populations of ancient
paralogous genes that underwent gene conversions
would be characterized by low Ks values that would be
interpreted as signals of recent duplication.
2.3. Using synteny conservation with other species
Genomes descending from a common ancestor accumulate
inter and intra-chromosomal rearrangements.
Depending on both the time separating two species, and
on the rate of genome shuffling, genomic regions conserving
ancestral gene content are more or less short
and numerous. By counting the number of events such
as inversions and translocations that occurred since the
last common ancestor, a genomic distance can be computed
[26]. A WGD is comparable in the sense that
these events occur in paralogous chromosomes instead
of orthologous chromosomes. A linear conservation of
gene order between two genomes is usually represented
and can be noticed as clear lines in representations such
as dot plot figures. However, a WGD exclusive to one
of the two species may lead to fragmentation and blur
lines because orthologous genes are projected on two
distinct chromosomes (Fig. 4a). When duplicated genes
are maintained, each pair can be connected to a single
ortholog from an outgroup species, and we could obtain
a significant number of relations of type 1-2, the partial
signature of a WGD. But when loss of duplicates is massive
after a WGD and is randomly distributed between
each paralogous chromosome, we expect essentially relations
of type 1-1 between paralogous genes.
So when the number of duplicate losses is significantly
higher than the number of chromosome rearrangements,
relations of type 1-2 are computable
not between genes, but rather between larger genomic
segments. For example, two duplicated genomic segments
descending from a single region with 6 genes
[a,b,c,d,e,f ] before the duplication, and maintaining
3 genes each after the duplication, [a,c,e] and
[b,d,f ], can both be connected to a single segment
[a,b,c,d,e,f ] that would exist in a related but nonduplicated
genome (Fig. 4b). This kind of double
conserved synteny (DCS) was initially described at a
genome-wide scale to demonstrate an ancestral WGD
in the yeast Saccharomyces cerevisiae by comparison
with the non-duplicated Kluyveromyces waltii [27]. This
was the first analysis using genome wide comparison
between one species that undergone a WGD and an
outgroup species. A posteriori it could seem surprising
that this WGD was controversial, because 81% of the
5714 genes of S. cerevisiae are involved in DCS blocks.
Only small genomic regions resulting from rearrangements
and containing 3 genes on average do not show
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246 O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253
clear DCS patterns. But before the availability of any
external non-duplicated genome sequences, only 457
gene pairs were characterized which could result from
distinct local duplications. Similarly in vertebrates, to
highlight the third round of whole genome duplication
in the teleost fish lineage (3R), 6684 orthologous relations
were computed between the genome sequence
of Tetraodon nigroviridis and of Homo sapiens [16].
Analysis of the topology in the chromosomes of these
(a)
(b)
relations revealed that 75% of orthologs are involved in
DCS. Typically, along a single region of a human chromosome,
series of genes are orthologs with Tetraodon
genes located alternatively on two chromosomes. By
comparison, 748 pairs of Tetraodon paralogous genes
are maintained from the WGD (Fig. 1). So, at least in
yeast and in teleost, the sequence of a non-duplicated
genome provides 9­10 times more markers for revealing
ancient WGDs.
Using synteny conservation to uncover ancestral
WGD or PLZ is efficient using a non-duplicated genome
as a reference. Conversely, comparing one genome
known to be duplicated to another evolutionary related
genome permits to test whether the event predates or
not the split. An event predating the split would allow
time for paralogous chromosomes to diverge sufficiently
from each other before speciation that would
lead to relations of type 1-1 after speciation. This rationale
has been used to decipher the chronology of
PLZ events in flowering plants. Large genome duplications
and other polyploidizations seem to be more
common, and better tolerated in plants than in animals
and perhaps in protozoa [28,29]. Since the publication
of the complete sequence of Arabidopsis thaliana,
several studies led scientists to postulate that at least
one WGD occurred in its evolution [22,30­32], and
an old WGD would be common to many dicotyledons.
Fig. 4. (a) Visualizing PLZ using macrosynteny grids. Four arbitrary
evolutionary times are represented during which two distinct
genomes diverge from a common ancestor through one WGD in one
lineage. Columns and rows inside grids represent chromosomes and
dots orthologous markers (genes, genomic regions...). The first grid
corresponds to one genome A compared against itself leading to a
clear diagonal crossing entirely the grid through every chromosome.
In step 1, one WGD leads to a new lineage due to speciation (see text)
and emergence of species B. At this step, by comparing A and B two
diagonals would be represented if chromosomes are sorted. Genes and
genomic regions in A and B, could be connected in relations of type
1:2. In steps 2 and 3, some duplicated genes in B are progressively
pseudogenized or lost. More and more genes attain a relation of type
1:1 between A and B. Intra- and inter-chromosomal rearrangements
contribute to obscure synteny. They affect 2 columns if they occur
in A, but only one in B. Then, pairs of duplicated chromosomes in B,
conserve similar profiles. For example, B3 and B6 correspond ancestrally
to A3, but have orthologs in every chromosomes on A in the last
grid, and these 2 columns (B3 and B6) must be visualized and compared
entirely. So two duplicated chromosomes, or genomic regions
in B, could have no gene maintained as a duplicate but could be seen
as paralogous because they conserve similar profiles on A. (b) Representation
of Double Conserved Synteny (DCS). Grey boxes indicate
genes along one chromosomal region in a non-duplicated genome
(A1), compared to two paralogous regions in a duplicated genome (B1
and B4). Because of many gene losses that occur after a WGD, a few
genes remain in 2 copies in B. The ancestral order of the genes is conserved
but B1 and B4 share very few genes.
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O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253 247
Two other genome sequences of dicotyledonous plants
are now available, Populus trichocarpa [33] and the
grapevine Vitis vinifera [25]. The synteny analysis of the
three possible pairwise comparisons makes it possible
to define the number of polyploidization events that are
unique to a lineage, or shared. The genome sequence of
the grapevine revealed that this plant derives from one
or more events that led to a hexaploid nuclear content.
The current diploid state results from consecutive rearrangements
that affected the original three components.
Surprisingly, this event is dated earlier than the radiation
between these three dicotyledonous species. Indeed
single genomic regions of Arabidopsis and of the poplar
are never syntenic with three other counterparts in grape
but with only one. Conversely, an independent recent
WGD in poplar [33] is clearly confirmed here by relations
of type 1-2 with the grape genome entirely. The
patterns of conservation of the grape genome with Arabidopsis
are more fractioned but a clear correspondence
of type 1-4 is established for many genomic regions.
This result indicates that at least 2 WGDs occurred in
the evolution of Arabidopsis after the formation of the
paleo-hexaploid ancestor, and after the split with the
grape.
Although the paleo-hexaploid ancestor was apparently
common to many dicotyledons, it does not appear
to be shared by rice Oryza sativa which is the only
monocotyledon completely sequenced to date. In this
case, constituents of grapevine triplets are orthologous
to the same regions in rice.
Overall, these comparisons of plant genomes have
pointed out that the type of polyploidization that may
be at the origin of the dicotyledonous plant radiation is
the formation of a hexaploid.
Because several aspects can be integrated at the same
time, using synteny conservation to analyze WGDs and
other polyploidizations is highly efficient. The orthologous
relationships, thanks to the knowledge of their
location on chromosomes, are used as markers of the
dynamics of the genomes. But manipulating a large
amount of data requires the availability of almost complete
genome sequences with a significant level of anchorage
on chromosomes.
3. Inferring ancestral genome organization
predating WGD
A direct consequence of the analysis of WGD by
synteny conservation between two species is the ability
to infer more or less precisely an ancestral organization
of the chromosomes. The principle commonly used is
essentially based on parsimony. Genes which are topologically
conserved between 2 species, i.e. a weak genomic
distance, were also co-located on the sequence
of the last common ancestor. Then, it is possible to infer
the gene composition of ancestral linkage group,
by clustering groups of genes which are conserved on
identical chromosomes. Again, following a parsimony
principle, with a third genome as an outgroup it is possible
to cluster ancestral regions into large parts of ancestral
chromosomes, as well as inferring some events
that occurred in specific lineages such as translocations,
chromosomal fusions or splits. The term paleogenomics
has been proposed for this new discipline [34,35]. Notably,
several efforts are concentrated on deciphering the
sequences of ancestral mammals at different nodes that
would help in understanding our recent evolution [36­
40]. In that context, deciphering a pre-WGD situation
is a special case in which only two species are needed.
A non-duplicated genome must serve as outgroup, and
is compared to the two components of the duplicated
genome that are treated individually even if they are
not independent. In the case of the WGD at the base
of the teleost fish lineage, a protokaryotype of an ancestral
vertebrate predating the WGD was inferred, firstly
by comparing near complete sequences of Tetraodon
with human as outgroup. Blocks of DCS (double conserved
synteny) used to reveal the WGD were clustered
into 12 types according to the chromosomes they connected
between human and Tetraodon. Each of these 12
types of DCS, named A to L, would contain genes located
ancestrally in the same linkage group [16]. The
availability of a bird's sequence, another outgroup from
teleost WGD, could refine this scenario. In fact, computing
DCS between Tetraodon and the sequence of
the chicken Gallus gallus [41] leads to the same 12
types of DCS (unpublished). However, some regions in
Tetraodon that were too scrambled using the human outgroup,
can be included in DCS. Due to the potentially
high level of scrambling in gene order between ancestrally
duplicated chromosomes, a statistical estimation
of the accuracy of paralogous regions that are detected
is important [11,17]. The accuracy of this kind of results
depends greatly on the quality of the sequence assembly
of living species, especially the fraction anchored
on chromosomes. More than 90% of the chromosomes
of the teleost fish medaka, Oryzias latipes, are now covered
by its sequence assembly, contrasting with 61% for
the Tetraodon. Blocks of DCS were also computed between
Medaka and human with a similar strategy, and
also by using the sequence of Tetraodon and the zebrafish
[42]. These authors proposed a more precise scenario:
rapidly after the WGD, the last common ancestor
of these three fishes had 24 chromosomes that resulted
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248 O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253
from 8 major rearrangements. But the last common ancestor
prior to WGD had probably 13 chromosomes A
to M. Another group, by using partial data available but
on more species, proposed instead a situation with 11
chromosomes [43]. Earlier studies based also on synteny
analysis but lacking complete genome sequences
suggested an ancestral karyotype of the vertebrate lineage
with 12 or 13 chromosomes [44,45].
The genome sequence of the ciliate Paramecium
tetraurelia provides strong evidence for a highly conserved
WGD. A majority of the genes, around 68% are
present in 2 copies. Moreover, the location of the genes
is so preserved that it is possible to infer almost exactly
their ancestral order without the need of another
genome. A two-step procedure has been developed to
find traces of ancestral WGDs and to infer the ancestral
order of genes. Recursively, this method has been applied
three times revealing at least three WGDs, which
occurred successively, but at separate time during the
evolution of Paramecium [46] (Fig. 2). In terms of protein
conservation the age of the third WGD can be estimated
at an ancient time point in the evolution of the
ciliate clade. This is a unique situation so far, in which it
has been possible to access a very ancient genomic organization
without an external non-duplicated genome.
4. Consequences of WGD
4.1. Structural modifications due to WGD
4.1.1. Speciation
PLZ can lead to speciation at two distinct stages:
­ Fixation of the polyploid organism;
­ Emergence of numerous species.
Starting from a diploid species, a WGD creates
a tetraploid genome. A cross between diploid and
tetraploid would create triploid having a high probability
of sterility (odd number of chromosomes leading
to problems during segregation). Thus, tetraploid
species are reproductively isolated. Coyne and Orr say
that "The discovery of polyploidy speciation represented
the first major triumph in the genetics of speciation"
and underlines the note of Haldane that speciation by
polyploidy represents "the most important correction
which must be made to Darwin's theory of the origin of
species" [47].
By doubling the chromosomes and thus the genes,
PLZ can raise some constraints that affect the structure
of the genome. Notably, chromosomal exchanges between
paralogous arms can be facilitated due to high
similarity. Thus, ancestral structure would be modified
when local rearrangements or genes losses occurred in
the meantime inside only one paralogous arm. Overall,
rearrangements and gene losses tend to decrease
similarity and colinearity between paralogous chromosomes.
As time goes by, reproductive isolation may become
firmly established, in favor of the emergence of
new species. But the principal factor of speciation is
probably the consequence of reciprocal gene loss (RGL)
which occurs when one copy of an essential gene is lost
independently in two sister groups that descend from the
same WGD. In this model, the two sisters lose the same
copy in half the cases, and lose the reciprocal copy in
the other half. Thus, a double null homozygote, lethal,
would be produced in 1/16 of F2 hybrids, and naturally
the reduction of hybrid fertility is proportional to the
number of gene silencing [23,48]. This passive mechanism
would contribute to speciation in agreement with
the Bateson­Dobzhansky­Muller model.
Analyses of RGL have been performed by comparing
different species descended from a WGD in yeast
and in teleost fishes. Two different studies that compared
zebrafish with Tetraodon or with medaka respectively
showed evidence of RGL after the two speciations.
The rate of ancestral genes that underwent RGL
would be about 8% [44,49]. In yeast, a similar rate
has been measured between S. cerevisiae and S. castelli
(6%) and between C. glabrata and S. castelli (7%),
but a lower rate was observed between C. glabrata and
S. cerevisiae (4%). Some of essential genes of S. cerevisiae
correspond to half of RGL situations with S. castelli,
enabling estimation of the reduction of viability for
hypothetical hybrid spores to be 6 × 10-9 [50].
The level of chromosomal rearrangements may also
play a role in speciation. In yeast, some species of the
Saccharomyces genus can be crossed but produce sterile
hybrids. But in some cases at least, sterility seems to
be due to differences in chromosome organization rather
than in gene content. After modifying the order of genes
of one yeast species in order to obtain colinearity with
another species, hybrid spores are viable [51]. However
macrosyntenic rearrangements do not seem to be a prerequisite
for speciation in yeast [52].
During mitosis, the mismatch repair system prevents
recombination between dispersed repeated sequences
and therefore contributes to a reduction in the risk of
lethal rearrangements and deletions. But it has been
shown in yeast that the same mechanism acts as a postzygotic
barrier [53]. Crosses between different strains of
Saccharomyces cerevisiae, or of Saccharomyces paradoxus
which are supposed to be species which diverged
a long-time ago are partially sterile. However, disrup-
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O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253 249
tion of the mismatch repair system reduces reproductive
isolation. These probable roles of this system in speciation,
but also in gene conversion during meiosis could
act as well after WGD to complement RGL.
The hypothesis that major evolutionary lineages
emerge from PLZ events is becoming increasingly accepted.
In plants, 235 000 angiosperm species could
be descended from one or several successive common
PLZ [25,28], and cereals would share an ancient
PLZ [54]. The diversification of the 12 000 species
of homosporous pteridophytes with high chromosome
numbers may be related to an ancient PLZ [48,55,56].
Among protozoa, at least two of the three WGDs of
Paramecium can be placed at the base of a radiation
[46]. Similar co-occurrences have been discussed in
yeast [27,57­59]. Among vertebrates, the Euteleostei
group derives from the 3R WGD in vertebrates and represents
in terms of number of species (24 000) and of
variety of morphological adaptations, the largest phylum
[60]. Also, in parallel, several lines of evidence indicate
that the 3R WGD is not present in non-euteleostei
fishes [61,62]. The hypothesis of two rounds of WGD
at the base of the vertebrates is more and more supported
by genomic data [63]. The final statement about
this question came from the genome sequence of Amphioxus,
Branchiostoma floridae. A pattern of genomewide
quadruple conserved synteny with vertebrates has
been shown [64] thus confirming the intuition of Ohno
"It is our contention that the ancestors or reptiles, birds,
and mammals have experienced at least one tetraploid
evolution either at the stage of fish or at the stage of amphibians"
[3]. An overview of the relics of major events
in yeast, plant and vertebrate evolution is displayed
in Fig. 1 with comparative examples from duplicated
genomes and outgroups in each lineage.
4.1.2. Gene loss and pseudogenization
As we discussed previously, lack of data represents
a major limitation in finding the trace of an ancestral
WGD. The most well-known studied WGDs, in vertebrates
and in plants are old, and these organisms have
lost a large majority of their duplicated genes. From a
technical point of view, synteny breakage due to gene
loss complicates genome-wide comparative analysis between
species having different PLZ.
Reduction of ploidy of a duplicated genome (tetraploidy
to diploidy for example) is one consequence of
gene loss. Potential structural and functional biases in
the ways in which genes are lost are open questions.
Among the duplicated chromosomes from the most recent
WGD in Paramecium (Fig. 2), the size of the genomic
regions that are maintained as single copies, corresponds
to the lost sibling. The pattern is compatible
with a mechanism which acts at the gene level or at
least on a small scale. The range of decay state observed
indicates that pseudogenization is probably a progressive
process rather an abrupt phenomena immediately
after WGD. However, comparative analyses of yeast
genomes suggest a rapid phase of gene loss immediately
after WGD [50]. One might expect a random distribution
of gene deletions between duplicated chromosomes
as is the case in the sequences of various teleosts and in
Paramecium [16,44,46]. However, different species of
yeast tend to lose the same duplicate (orthologs rather
than paralogs) independently. This leads to the conclusion
that different pressures affect the two paralogs [65].
Nevertheless a topological bias exists in the Arabidopsis
sequence where Thomas et al. showed clusters of
genes preferentially retained in two copies [66]. Genes
that are lost or maintained in duplicate shape the emerging
species functionally. Moreover, duplicates are more
or less preferentially lost over the short term depending
on some functional biases (see below). Massive gene
loss is the more visible long term effect and seems to be
the most predictable fate that has been noticed in every
lineage concerned by PLZ.
4.1.3. Chromosomal rearrangements
Whole genome comparisons between species provide
clues about differences in the frequencies of rearrangements
in lineages and even in some chromosomes.
In the same manner, analysis of the chromosomal topology
of ohnologs highlights type of rearrangements that
occurred post-WGD. This kind of event seems to spare
some chromosomes more or less. In the poplar, chromosomes
PtVIII and PtX have remained stable since the
recent WGD, with no large inter-chromosomal translocation,
whereas PtI is a combination of 4 ancestral linkage
groups [33] (Fig. 1). Surprisingly, such differences
in structural evolution of chromosomes do not diminish
with time but persist. After 400 million years of
evolution since the WGD, paralogs of Tetraodon chromosome
Tn14 are almost exclusively located on Tn10,
which is, however, connected essentially to Tn1, Tn7,
Tn14 and Tn21 (Fig. 1 and Ref. [16]). In Paramecium,
the level of conservation at the proteic level between
paralogs of the recent WGD is comparable to that observed
between humans and mice. But whereas hundreds
of large blocks of synteny separate the two mammal
orthologous sequences [67], in Paramecium the rate
of rearrangements is so low that it would indicate a more
general constraint that affects the chromosome structure
[68]. Depending on the phylum, we observe different
patterns of conservation of the ancestral structure of the
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250 O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253
chromosomes during the evolutionary transition from
polyplody to diplody, probably as a result of forces of
different intensities (compaction, transposon activities,
population size, generation time, etc.). It has been suggested
that the rate of rearrangements would be accelerated
after WGD but this hypothesis cannot be rejected
or accepted at the moment due to the small amount of
data available [69].
4.2. Functional consequences at the gene level
One of the most fascinating theoretical consequence
of PLZ is the potential for functional innovation inherent
in ohnologs [3]. It seems evident that this fate
could only be achieved if both paralogs are maintained
(no gene lost). But maintaining genes in several
copies may have functional consequences which
affect the metabolism globally. Historically, Susumo
Ohno assumed that one copy could maintain the ancestral
function while its sibling could accumulate mutations
until eventually being selected with new function.
This scenario for novelty acquisition is known as
neofunctionalization. Conversely, under the subfunctionalization
model, distinct functions from the preduplicated
gene are distributed, with more or less overlap,
between the two sister genes. The most common
situation, nonfunctionalization concerns copies that
firstly become pseudogenes and are then lost. A formal
description of this model, Duplication-DegenerationComplementation
(DDC model) was presented by Force
and co-authors [70,71]. One surprise from the various
descriptions of PLZ is the paucity of functions created
by neofunctionalization that have been demonstrated
to date. However, beyond the rare cases in which neofunctionalization
has been functionally tested, some
insight about the real importance of this type of gene
evolution is provided by computational simulations.
Indeed, two paralogs of proteins that evolved to neofunctionalization
may retain a trace of an asymmetrical
evolution. The signal which indicates an asymmetrical
rate of mutations is measurable, using the length of
branches in a phylogenetic tree, at least if an outgroup is
available. The fraction of paralogous proteins that have
experienced asymmetrical evolution in Paramecium,
plants, and yeast is significant and tends to increase with
time [46,72,73]. This tendency can also be observed in
teleost fishes despite the low number of unambiguous
ohnologs [18,74].
In the model of subfunctionalization, separation of
ancestral functions between two sister genes could be
either in space, in time or in functions. Numerous cases
are characterized by analysis of the level of gene expression
at different stages or in different tissues of an
organism. In the teleost zebrafish, for example, the engrailed
gene is present in two copies, eng1a and eng1b.
In vertebrate species outside the 3R lineage, such as
mouse and chicken, eng1 is a single gene and is expressed
both in the hindbrain and in spinal neurons
whereas in zebrafish, the expression of each copy is specific
to one area [71].
However, functional innovation would not be the
main cause for retention of ohnologs. Rather, most
ohnologs would be retained as a side effect of their
function. Several functional biases have been observed
among the ohnologs maintained. In particular, some
types of functions such as signaling molecules and transcription
factors are preferentially retained for a long
time after a WGD in plants, yeast and paramecium, but
are not enriched among local duplications [24,46,65,73,
75]. Several models exist to predict or to explain such
biases in function of genes maintained as duplicates after
a WGD and analysis of the genome sequence of
Paramecium confirmed two wide effects that had been
predicted (the models are nicely reviewed in [12]). First,
interactions, pathways, networks or complexes formed
by proteins would create constraints on their stochiometry
which is noticeable at the gene level. Indeed such
genes are preferentially co-retained at first, and co-lost
a long time after the WGD. Disrupting the equilibrium
of stochiometry would be then counter-selected, leading
to a rule of the "all or none" type. This effect previously
proposed as a "dosage imbalance effect" has been also
observed in yeast [76,77] and would explain the difference
in functional biases of the genes that are retained
in duplicate after PLZ or after small-scale duplications.
Second, highly expressed genes are also preferentially
retained in duplicate, both short and long periods after
the WGD. We can postulate that a high expression
level of some genes may be selected due to a gain of
fitness. But in certain cases, the expression level may
reach the upper limit of the transcription machinery efficiency.
Then, having another functional copy would
make it possible to raise this limit. Some authors have
also noticed a fraction of ohnologs with a very low evolutionary
rate, possibly resulting from gene conversions.
Here again, those genes are functionally biased.
5. Conclusion
In considering the evolution of species, certain types
of events are correlated with the emergence of large radiations.
Endosymbiosis, at the base of several major
lineage, is one of these. Their impact is genetic, they
provide a pool of potential functions but may also be
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O. Jaillon et al. / C. R. Biologies 332 (2009) 241­253 251
structural when they provide a supplementary compart-
mentalization.
Other events lead to increased genetic variability
such as sexuality and meiosis. In a diploid species, each
gene, or at least most of them are present in 2 allelic
forms. The possession of a double genome per individual
allows a genetic mixing of the population. The
repertoire of alleles that exists in a population at a given
time represents opportunities for adaptations and for the
emergence of new functions by mutations.
Whole Genome Duplication leads also to doubling
each gene of an individual, but over a long time and under
constraints such as gene dosage, and other types,
certain copies are lost or maintained. Some of the
retained copies tend to a specialization or to a new
function via sub- or neo-functionalization. Gradually,
the genome returns to diplody from a transitional
tetrapody status. All of these steps are relatively long,
and mainly due to differential gene losses, emergence of
a new species is facilitated. Each hybridization between
sub-populations having different losses of duplicates, is
a genetic combination that may lead to speciation and
may then be potentially innovative.
Whereas diplody and sexuality allows a genetic
mixing between each generation of a species and between
every individual, WGD makes the stoichiometry
of the genes more complex and bootstraps the genetic
pool favouring emergence of new species. A genetic
variability could then be exploited between individuals
but also between emerging lineages which are still inter-
fertile.
Half a century of fundamental hypotheses about the
impact of polyploidizations on evolution has begun to
be confronted with observations. Darwin's theory of
natural selection has never been seriously rejected but
has been refined to take into account new findings, such
as the neutral evolution theory for example. Similarly,
current authors support the vision of Susumo Ohno
about facilitation of emergence of new functions and of
species by duplications, but refine it with a supplementary
hypothesis about short-term regulation of the forces
that lead to these long-term results. It is not unexpected
that these questions can be addressed within a genomic
framework. Recent findings depend on comparisons between
complete DNA sequences of chromosomes from
model species. In the near future, we have the technical
possibility to be confronted with a range of complete sequences
from genomes with different separation times
since PLZ that would furnish clues about the regulation
of gene repertoire shaping over time. We must not forget
that the human species is a result of ancient PLZ
too. Future scientific plans must continue in the path of
obtaining excellent genetic maps and excellent quality
sequences. No major finding about nature could emerge
from data which is too partial.
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