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Evolutionary Patterns of Recently Emerged Animal
Duplogs
Kiyoshi Ezawa1,2,4, Kazuho Ikeo2,3, Takashi Gojobori2,3, and Naruya Saitou1,*
1

Division of Population Genetics, National Institute of Genetics, Mishima, Japan

2

Human Genome Network Project, National Institute of Genetics, Mishima, Japan

3

DNA Data Analysis Laboratory, Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Japan

4

Present address: School of Advanced Sciences, Graduate University of Advanced Studies, Hayama, Japan*Corresponding author: E-mail: saitounr@lab.nig.ac.jp.
Accepted: 19 July 2011

Abstract
Duplogs, or intraspecies paralogs, constitute the important portion of eukaryote genomes and serve as a major source offunctional innovation. We conducted detailed analyses of recently emerged animal duplogs. Genome data of threevertebrate species (Homo sapiens, Mus musculus, and Danio rerio), Caenorhabditis elegans, and two Drosophila species(Drosophila melanogaster and D. pseudoobscura) were used. Duplication events were divided into six age-groups according
to the synonymous distance (dS) up to 0.6. Duplogs were classified into four equal-sized classes on physical distances andinto three classes on relative orientations. We observed the following shared characteristics among intrachromosomal
multiexon duplogs: 1) inverted duplogs account for 20–50%, and about a half of the physically most distant 25%; 2) except
for C. elegans, the composition of physical distances, that of relative orientations, and the proportion of inverted duplogs ineach physical distance category are more or less uniform; 3) except for C. elegans, the characteristics of the youngest (dS ,0.01) duplogs are similar to the overall characteristics of the entire set. These results suggest that intrachromosomal duplogs
with fairly long physical distances were generated at once, rather than resulting from tandem duplications and subsequent
genomic rearrangements. This is different from the three well-known modes of gene duplication: tandem duplication,
retrotransposition, and genome duplication. We termed this new mode as ‘‘drift’’ duplication. The drift duplication has been
producing duplicate copies at paces comparable with tandem duplications since the common ancestor of vertebrates, and it
may have already operated in the common ancestor of bilateral animals.
Key words: duplog, paralog, gene duplication, physical distance, transcriptional orientation, animals, genome-wide
analysis, cross-sectional analysis.

Introduction
Gene duplication has long been one of the major subjects ofevolutionary studies because it is considered as one of the
major sources of genomic innovations (e.g., Haldane 1932;
Muller 1935; Nei 1969; Ohno 1970; Lynch 2007). Genome
sequence data revealed that eukaryotic genomes are fairly
rich in duplicated genes (e.g., Lynch and Conery 2000;
Rubin et al. 2000; Wapinski et al. 2007), and thus supporting the above concept. Fitch (1970) proposed to call duplicated genes as paralogs. Paralogous genes may exist either
in the same species or in different species. Because duplicated gene pairs existing in one species are the main focusof this study, we would like to propose to call them ‘‘duplogs,’’ as a subclass of paralogous sequences. Duplog is

a synonym of ‘‘intraspecies paralogs’’ but is much shorterand easy to use. Wolfe (2000) proposed to call duplicatedgenes created through genome duplications as ‘‘ohnologs’’
after Susumu Ohno. All ohnologs in one species genome are
duplogs. Duplogs are somewhat related to ‘‘inparalogs,’’
paralogs in a given lineage that all evolved by gene duplications that happened after the radiation (speciation) eventthat separated the given lineage from the other lineage
under consideration (Sonnhammer and Koonin 2002).
Duplogs are, however, simply any kind of paralogs foundin one species genome.
In evolutionary genomics, three duplication mechanisms
have been well known to create duplogs of different positional relationships: 1) tandem duplication mostly creates

The Author(s) 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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FIG. 1.—Six species used and the time frame of this study. The
figure shows the six animal species used in this study (in boldface),
which have finished or nearly finished genome assemblies, and someclosely related outgroup species. Thick gray lines along the subject
species lineages roughly span the time intervals equivalent to the neutral
nucleotide divergence of 0.6 between duplogs.

physically close head-to-tail duplogs, 2) retrotransposition
mostly creates unlinked or very distant intronless duplogs(reviewed, e.g., in Babushok et al. 2007), and 3) whole genome duplication (WGD) doubles the gene complement atleast immediately after that (Ohno 1970). It is now believedthat the common ancestor of vertebrates underwent tworounds of WGD (fig. 1; see also Dehal and Boore 2005)
and that the common ancestor of teleost fish experienced
another round of fish-specific WGD (fig. 1; see also Jallion
et al. 2004; Woods et al. 2005). At the same time, we also
know that the WGD events account for only a fraction of the
duplicate genes of extant vertebrates.
Studying the long-term evolution of human and mouse
duplogs, Friedman and Hughes (2003, 2004) found negative
correlations between the proportion of linked duplog pairs
and the sequence divergence, and they concluded that duplicate genes have been generated mainly via tandem duplication and have been physically separated via genome
rearrangements. Since then, genome-wide studies haveshown that tandem duplicate genes account for a considerable fraction (18–34%) of all duplogs in vertebrate genomes(Shoja and Zhang 2006; Pan and Zhang 2008), but the remaining fraction was not characterized except retrotransposed genes (Pan and Zhang 2007). Shoja and Zhang(2006) also found that head-to-tail duplog pairs in their
set of ‘‘tandemly arrayed genes’’ tend to have smaller physical distances than inverted pairs. Combined with the arguments by Friedman and Hughes (2003, 2004), this may
indicate that inverted and physically relatively distant duplogs have resulted from chromosomal rearrangements.
To confirm that this is the case, however, detailed analyses
on recently created duplogs are indispensable.

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Recent duplications with 90% or more nucleotide identity,
excluding retrotranspositions, have been actively studied as
segmental duplications (SDs) since the advent of the humangenome assemblies (International Human Genome Sequencing Consortium 2001; Bailey et al. 2002; Bailey and Eichler
2006). It was revealed that the human genome is abundant
in interspersed SDs (Bailey et al. 2002, 2003; Bailey and Eichler
2006). A considerable fraction of interspersed SDs was explained by direct creations via mechanisms other than tandem
duplications, possibly mediated by interspersed elements(Bailey et al. 2003). The abundance of SDs was often considered as specific to human or hominoids (Bailey and Eichler
2006; Marques-Bonet et al. 2009) because early analyses
based on the whole genome shotgun (WGS) assemblies ofmammalian genomes, such as the mouse genome, showeda paucity of SDs (e.g., Cheung et al. 2003). Recently, more
careful analyses using the finished assembly of the mouse genome (She et al. 2008; Church et al. 2009) concluded that the
SD content in the mouse genome is ca. 5%, which is comparable with that of the human genome, and that the mouse
SDs are richer in tandem duplications. In these studies on primate and rodent SDs, however, tandem and interspersed SDs
were roughly distinguished based solely on physical distances(with low resolutions of at best 1 Mb) taking no account ofrelative orientations. Besides, they did not examine the dependence of relative positions on the duplication age, leaving it
unclear whether the interspersed SDs were indeed created directly or resulted from chromosomal rearrangements. As forrecent duplogs in nonmammalian vertebrates, there are virtually no studies so far on the evolution of positional relationships. Regarding invertebrates, Katju and Lynch (2003)
studied quite recent (synonymous distance [dS] , 0.1) duplogs in Caenorhabditis elegans. They found that inverted
pairs account for a majority of C. elegans duplog pairs physically close to each other and suggested a possible duplication
mechanism different from tandem duplication via unequalcrossing-over. But a question remains as to whether sucha mechanism is shared by other species or not.
To step up our understanding on duplog evolution, it isundoubtedly necessary to clarify whether physically relatively distant and/or inverted duplogs were created by
one-step mechanisms or by tandem duplications and subsequent genomic rearrangements, and whether such mechanisms are shared across animals or specific to lineages. For
this purpose, it is crucial to study the evolution of physicalrelationships between recently created duplogs. Theoretically, this could be achieved by comparing the chromosomal
positions of orthologs of duplicate genes in closely related
species. The problem is that species with finished-quality genome assemblies are still sparse in animal phylogeny (boldface species in fig. 1). It is now well appreciated that thedraft genome assemblies based on the WGS technique ispoor at locating duplicated DNA sequences, especially recently diverged ones (She et al. 2004; Church et al.

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Evolutionary Patterns of Animal Duplogs

FIG. 2.—Cross-sectional analyses based on duplog pairs and those based on duplication events. For the illustration purpose, we showed themethods applied to a fictitious gene family, whose phylogenetic tree is given on the panel A. We distributed gene pairs into ‘‘age-groups’’ according totheir duplication dates and then took statistics of physical properties on each age-group, as shown in panel B. Then, weighted statistics are assigned sothat the weight factors for each duplication event add up to one, as shown in panel C. For details on the assignment of weight factors, see
supplementary materials and methods (Supplementary Material online). After that, the statistics for different age-groups are juxtaposed for comparison,as shown in panel D.

2009). At present, therefore, such ‘‘longitudinal’’ analyses
cannot be used to track the evolution of duplog positions.
Nevertheless, ‘‘cross-sectional’’ analyses may be used to infer the mechanisms forming relatively physically distant duplog pairs in the species with finished genome assemblies.
A cross-sectional analysis examines the distributions ofphysical relationships between duplogs at different ages
(fig. 2). If relatively distant duplogs resulted from tandem
duplications and subsequent rearrangements, they will account for only a small portion of the youngest age-groupand their proportion will increase with age (fig. 2D). If, incontrast, most of them were created de novo via one-stepmechanisms, their proportion will be more or less uniform
across ages, and the youngest age-group will containa similar proportion of distant duplogs as the whole setof duplogs does.
Motivated by the above consideration, we conductedcross-sectional analyses. Because this study requires highquality genome assemblies, we restricted our analyses to
six animal species with finished or nearly finished genome
assemblies (fig. 1): human (Homo sapiens), mouse (Musmusculus), zebrafish (Danio rerio), nematode worm (C. elegans), and two fruit flies (Drosophila melanogaster andD. pseudoobscura). Our cross-sectional studies revealed several trends shared by most of the species studied, which
point to the one-step creation of randomly oriented duplogs

at relatively large physical distances, as well as behaviors
specific to one species, especially C. elegans.

Materials and Methods
Selection of Subject Animal Genomes
As of 12 May 2011, there are 128 animal species whosegenome sequences are assembled (see National Center for
Biotechnology Information Genome Project Statistics at
http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html).
Unfortunately, most of them are draft assemblies based on
the WGS sequencing. Recent studies showed that WGS assemblies grossly underrepresent duplicated regions of the genomes, especially recent duplications (She et al. 2004;
Church et al. 2009).
Because this study critically depends on the exact compositions and positions of duplogs, we restricted our analyses to theanimals that have high-quality genome assemblies. So far, only
four animal species have genome assemblies of finished qualities based on clone-by-clone sequencing: human (H. sapiens;
International Human Genome Sequencing Consortium 2004),
mouse (M. musculus; Church et al. 2009), nematode worm
(C. elegans; The C. elegans Sequencing Consortium 1998;
Hillier et al. 2005), and fruit fly (D. melanogaster; Celniker
et al. 2002; Ashburner and Bergman 2005). In addition to these

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four animals, we also included zebrafish (D. rerio) and anotherfruit fly (D. pseudoobscura) in our subject species.
Zebrafish was added with the hope that we could uncover
duplication mechanisms operated in the genome of the vertebrate ancestor. Although it is still a draft assembly, the zebrafish genome sequence (the Sanger Institute genome build
version Zv7) is built by tiling bacterial artificial chromosome
clone sequences and by supplementing it with the WGS assembly (Danio rerio Sequencing Project 2007). We thus considered the zebrafish genome as more suitable for the study ofrecent duplogs than the genome of any other fish such as medaka (Kasahara et al. 2007) or tetraodon (Jaillon et al. 2004),
whose genome assemblies are based mostly on WGS. After
preliminary analyses, we found that the number of recent duplication events in the D. melanogaster genome is about anorder of magnitude smaller than in other animals. So we tried
to enhance the statistics on fruit fly duplication events by analyzing another species. Drosophila pseudoobscura was chosen because its genome assembly is more than just a WGS
assembly, as it is augmented by separate sequences of repeat
regions (Richards et al. 2005).

Peptide and cDNA Sequence Information Used inThis Study
We downloaded files of the gene transcript (cDNA) sequences and the peptide sequences predicted in thehuman (H. sapiens), mouse (M. musculus), and zebrafish(D. rerio) genomes from the FTP site of the Ensembl database (Hubbard et al. 2009) version 52. The cDNA and peptide sequence files for two fruit fly species (D. melanogasterand D. pseudoobscura) were downloaded from the October
2008 version of FlyBase (Tweedle et al. 2009). Those fornematode worm C. elegans were downloaded from WormBase (Harris et al. 2010) version WS200.
We only used cDNA sequences with peptide counterparts, excluding cDNA products of the mitochondrial genes.
The genomic map of exons, exon–transcript relationship,transcript–gene relationship, and translation starts and endsof the gene transcripts (cDNAs) were extracted from themysql dumps provided at the Ensembl FTP site for vertebrates. The corresponding information for fruit flies and thatfor C. elegans were extracted from the genome feature tables in the GFF format provided at the FTP sites of FlyBase
and WormBase, respectively.

Duplogs from the Six Animal Species
We conducted a series of screenings to retrieve pairs of duplogs, or intraspecies duplicated sequences, that have duplicated relatively recently in the genomes of the six animal
species. We conducted BlastP (Altschul et al. 1990) homology
searches against the set of translated cDNA sequences fromthe six species and their respective outgroup species that aresupposed to have diverged from the subject species much

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earlier than the period we studied. Five vertebrate species(chicken, Xenopus tropicalis, zebrafish, Tetraodon nigroviridis, medaka) were used as outgroup for human and mouse,and eight outgroup species (human, mouse, chicken, X. tropicalis, T. nigroviridis, fugu, medaka, and stickleback) wereused for zebrafish. Caenorhabditis japonica and Pristionchus
pacificus were two outgroup species used for C. elegans,
while five Drosophila species (D. persimilis, D. willistoni,
D. virilis, D. mojavensis, and D. grimshawi) as well as its counterpart species were used as outgroup for D. melanogasterand D. pseudoobscura.
We then screened the resulting homologs using the numerical cutoff of 35% peptide identity, as well as a ‘‘natural
cutoff’’ determined from the best outgroup homologs if
available (Ezawa et al. 2006). Average dSs between thequeries and the orthologs from the outgroup species aremuch larger than 0.6 (and typically larger than 2). Therefore,
the introduction of such a natural cutoff will never missa substantial fraction of duplogs with dS , 0.6. The surviving duplogs were aligned at the peptide level with the query
sequence via Smith and Waterman’s (1981) algorithm,
which is implemented in the ‘‘ssearch’’ program of the FASTA
package (available at http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml). The resulting pairwise alignments were transformed into their cDNA counterparts.
We then masked the CpG dinucleotides for human andmouse because these sites are known to be hypermutablein mammals (Ehrlich and Wang 1981). For zebrafish,
C. elegans, D. melanogaster, and D. pseudoobscura, wemasked the repeat regions to avoid the misassignment of
duplogs due to repeat sequences. We discarded the alignments containing less than 150 unmasked nucleotide sites
excluding gapped sites. Next, we estimated the counts ofsynonymous sites and dS between the query and intraspecies paralogs via the ‘‘yn00’’ program of the PAML package(Yang 1997). Finally, we only kept duplog pairs whose dSs
are less than the threshold value of 0.6. Among these recently generated duplog pairs, we only kept for our analyses
those pairs 1) that have 100 or more synonymous sites and2) that consist only of genes mapped onto chromosomes.

Cross-Sectional Analyses Based on Duplog Gene
Pairs
Our main results were obtained through event-based crosssectional analyses explained in the next subsection. In thissubsection, however, we will explain pair-based analyses, because they provide a foundation of the event-based analyses.
First, to get a broad sense of the age dependence, we subdivided the evolutionary period from the upper bound of dS
5 0.6 to the present (dS 5 0) into three time intervals using
the boundaries at dS 5 0.2 and 0.4. After confirming that
duplication events in the youngest interval of 0  dS , 0.2
are much more abundant than those in the other two intervals (0.2  dS , 0.4 and 0.4  dS , 0.6) for most of the

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species, we further subdivided this youngest class with theboundaries dSs 5 0.01, 0.03, and 0.1 to get a finer view of
the age dependence. Especially, dS 5 0.01 was chosen as itis the smallest measurable distance under our condition of
100 or more synonymous sites. Then, we classified each duplog pair into an age-group according to the interval the dS
falls into (fig. 2B). The age-groups were labeled as follows:
C1, 0  dS , 0.01; C2, 0.01  dS , 0.03; C3, 0.03  dS ,0.1; C4, 0.1  dS , 0.2; C5, 0.2  dS , 0.4; and C6, 0.4 dS , 0.6.
After that, we obtained statistics on the physical proximity and relative transcriptional orientations for each agegroup. The ‘‘physical proximity’’ of a gene pair is a combination of the linkage and the physical distance. The pair iscalled ‘‘linked’’ when the genes are on the same chromosome and ‘‘unlinked’’ when they are on different chromosomes. The physical distance of a pair is defined by thelength (in base pairs) of the sequence between the coding
regions of the member genes. For each species, threeboundaries were chosen to divide the entire set of linked
duplog pairs from each species into four subsets of almost
equal sizes. Then, to facilitate the comparison, we arranged
the distributions for different age-groups in order of age(fig. 2D).

Cross-Sectional Analyses Based on Duplication
Events
The cross-sectional analyses based on duplog gene pairs
could potentially result in biased distributions dependingon the histories and physical properties of large families.
To alleviate such bias, we conducted cross-sectional analyses
based on duplication events, by counting each duplication
event, instead of each duplog pair, as a unit. We first merged
duplog pairs that share the member genes into clusters (orfamilies) of duplogs by using a single-linkage algorithm.
Each of the resulting clusters should consist of duplogs thatoriginated from a common ancestor gene since the timemeasured by dS 5 0.6. The dS has been used to approximate the nucleotide substitution rate under neutral evolution (e.g., Lynch and Conery 2000) because synonymous
substitutions by definition do not change amino acids
and therefore are under weak, if any, selective pressure.
Then, we constructed a rooted phylogenetic tree for eachcluster via UPGMA (Sneath and Sokal 1973). Because thetree is rooted, we can identify the duplication event each
duplog pair diverged from. By collecting the duplog pairs
diverged from each duplication event, and by assigning
an appropriate weight factor to each of the duplog pairs,
we converted statistics on duplog pairs into those on duplication events (fig. 2C). The weight factors must add up tounity across the duplog pairs diverged from each duplication
event, for we count each duplication event as a unit. How weassigned a weight factor to each gene pair is described in thesupplementary materials and methods (Supplementary

Material online). Then, we subdivided the duplication events
into six age-groups using the same set of boundaries for dS
values as in the above subsection. Finally, we added together
the weight factors for duplog pairs belonging to each agegroup and each class of physical properties (fig. 2D). Note
that, in the event-based analyses, the three boundaries forthe physical distance were chosen to divide the entire setof linked duplication events into four subsets of almost equal
sizes. Note also that, in our method, the number of duplication events is defined as the number of duplog copies generated during the time period in question.

Results
Statistics on the Six Animal Genomes
Basic statistics on the genomes of six animals used in thisstudy are summarized in supplementary table S1 (Supplementary Material online). Broadly speaking, the genomes of invertebrates (C. elegans, D. melanogaster, and D. pseudoobscura)are an order of magnitude smaller than those of vertebrates(human, mouse, and zebrafish), whereas the numbers ofgenes are almost the same between invertebrates and vertebrates, with the former more than a half of the latter. So, we
expect that the average physical distance between neighboring duplog genes should be an order of magnitude smaller in
the invertebrates than in the vertebrates. This turned out to beroughly true, as we can see from the three boundaries of theinter-duplog physical distance at 25%, 50%, and 75% of all
linked duplication events for each species (supplementarytable S1, Supplementary Material online). For example,
the median physical distances between linked vertebrate
duplogs are 155, 95, and 78 kb for human, mouse, andzebrafish, respectively, whereas those between linked
invertebrate duplogs are 7.4, 7.1, and 4.0 kb for C. elegans,
D. melanogaster, and D. pseudoobscura, respectively.

Sets of Duplogs in the Six Animal Genomes
For each of the six animal genomes, with homology searches
via Blast and a series of screening described in the Materials
and Methods, we gathered a set of duplogs, or intraspecies
paralogs, whose dSs are less than the threshold of 0.6
synonymous substitutions per synonymous site. We are interested in recent duplication events because only theshort-term age dependence of duplog positions can unravel the mechanisms that form relatively physically distant
duplogs, as explained in the Introduction. The thresholdvalue of dS 5 0.6 was chosen to avoid the historical correlation (or redundancy) between the human and mouse
duplogs (fig. 1). The overall statistics on the set of duplog
pairs is given in table 1. Broadly speaking, vertebrate genomes have an order of magnitude more duplog pairs thaninvertebrate genomes.
Figure 1 displays the time intervals used in this study as
thick gray lines from the exterior nodes on the species

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Table 1
Overall Counts of Duplogs and Duplication Events
Duplog Pair Typea
Multiexona
No. duplog pairs
Human
Mouse
Zebrafishb
Caenorhabditis elegans
Drosophila MelanogasterD. pseudoobscura
No. duplication events
Human
Mouse
Zebrafishb
C. elegans
D. melanogasterD. pseudoobscura
Percentages of linked duplog
generations among duplication eventsc
Human
Mouse
Zebrafishb
C. elegans
D. melanogasterD. pseudoobscura

Single Exona

Mixeda823
2,582
1,411 (187)
39
19
96

Total

4,524
5,048
21,258 (3,071)
1,950
213
408

789
5,880
1,063 (277)
412
763
162

6,136
13,51023,732 (3,535)
2,401
995
666

1,215.3 [66.8]
1,467.0 [46.0]
2,448.8 (871.1)[86.5 (83.4)]
1,340.0 [92.0]
115.5 [50.7]
324.5 [66.1]

263.2 [14.5]
1,084.7 [34.0]
188.3 (105.2)[6.7 (10.1)]
103.2 [7.1]98.1 [43.0]
106.5 [21.7]

340.8 [18.7]
635.0 [19.9]
194.0 (67.7)[6.9 (6.5)]
13.8 [1.0]14.3 [6.3]60.0 [12.2]

1,819.2
3,186.6
2,831.0 (1,044.0)

78.7
67.9
67.9 (74.5)
82.3
97.4
69.0

67.9
76.8
73.2 (93.3)
71.9
99.0
66.2

28.3
25.4
70.3 (65.1)
42.8
62.9
52.0

67.7
62.5
68.4 (75.7)
81.2
95.9
66.3

1,457.0
228.0
491.0

NOTE.—We counted only those gene pairs mapped on chromosomes. The ‘‘number of duplication events’’ actually means the summation of weight factors from the gene pairs
belonging to respective subsets. For details on the weight factors, see supplementary materials and methods (Supplementary Material online). The numbers therefore can be fractional
and were rounded off to the nearest tenth. Numbers in brackets in ‘‘no. duplication events’’ denote the percentages of duplication events of each type (column) accounting for eachspecies (row).
aTypes of contributing gene pairs. ‘‘Multiexon’’ denotes a subset of gene pairs each consisting only of multiexon genes. ‘‘Single exon’’ represents a subset of pairs consisting onlyof single-exon genes. And ‘‘mixed’’ is designated for a subset of pairs each consisting of a single-exon and multiexon genes.
b
Numbers in parentheses in this row are for a set of ‘‘stable’’ zebrafish duplogs that are consistently annotated on both the Zv7 and Zv8 assemblies (for details, see
supplementary materials and methods, Supplementary Material online).
c‘‘Linked’’ denotes a set of gene pairs each of which consists of genes on the same chromosome.

phylogeny. Time intervals corresponding to 0  dS , 0.6
considerably vary among the species because of the variation of synonymous substitution rate, measured per year,
among the species. Broadly speaking, the neutral substitution rate is an order of magnitude higher in the invertebrates
than in the vertebrates. Neutral substitution rate ( per site/year) was estimated to be (5.4 – 23)  10 8 for C. elegans
and (2.9 – 12)  10 8 for D. melanogaster (Cutter 2008),
and those for human and mouse were estimated as (0.8 –1.2)  10 9 and (2.5 – 5.0)  10 9, respectively (Yi et al.
2002). Consequently, the time intervals corresponding to
0  dS , 0.6 are an order of magnitude shorter for invertebrates than for vertebrates. As figure 1 indicates, the duplication events studied here are quite recent, hence theduplog pairs generated by the ancient WGD events (Ohno
1970; Jallion et al. 2004; Dehal and Boore 2005; Woods
et al. 2005) should be negligibly few, if any, in our set of
duplogs. We also know that animal genomes, especiallymammalian genomes, have been bombarded with retrotransposition, and intronless retrotransposed duplicates ofgenes are abundant in these genomes (e.g., Babushok

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et al. 2007). To mitigate their influences, we divided duplog
pairs into three categories: ‘‘multiexon’’ pairs consisting
solely of genes with more than one exon, ‘‘single-exon’’pairs consisting solely of genes with one exon, and ‘‘mixed’’
pairs consisting of both single-exon and multiexon genes(table 1). Because most retrotransposed duplicates are expected to be in the sets of single-exon and mixed pairs,
we mainly used multiexon pairs to examine the patternsof duplication events due to mechanisms other than retrotransposition. In most cases, the multiexon duplog set behaved similarly to the whole duplog set. Because it ismultiexon duplogs that are essential to this study, we will
hereafter show the results on multiexon duplogs unless explicitly stated otherwise.
The total numbers of duplication events (with dS , 0.6)
are more or less similar among the studied animals except
two Drosophila species, which have experienced about anorder of magnitude fewer duplication events compared withmouse (table 1), consistent with previous studies (Hedger
and Ponting 2007; Zhou et al. 2008). Figure 3 shows thesizes of age-groups of all duplogs. We see that each of

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FIG. 3.—Sizes of age-groups of duplication events classified into contributions from multiexon, single-exon, and mixed pairs of duplogs. The red,white, and black bars stacked in each age-group represent the numbers of duplication events attributed to multiexon, single-exon, and mixed duplog
pairs, respectively. Here, a ‘‘mixed’’ duplog pair consists of two duplogs, one multiexon and the other single exon. Only those pairs mapped on
chromosomes are counted. Age-groups are defined by the time intervals measured in terms of the dS between duplogs. The age-groups used are as
follows: C1 (0  dS , 0.01), C2 (0.01  dS , 0.03), C3 (0.03  dS , 0.1), C4 (0.1  dS , 0.2), C5 (0.2  dS , 0.4), and C6 (0.4  dS , 0.6). The
panels A, B, C, D, E, and F show the graphs for human, mouse, zebrafish, Caenorhabditis elegans, Drosophila melanogaster, and D. pseudoobscura,
respectively. Caution must be exercised when comparing the age dependence of (observed) duplication events per unit time because the time interval(in dS) varies across the age-groups.

the age-groups consist of more than 100 duplication eventsin general, except those for fruit flies and the age-groupwith 0.01  dS , 0.03 for C. elegans. These sample sizes
are appropriate for statistical analyses for most of the cases.
Taking account of the approximately logarithmic scaling of
the time intervals for the age-groups, the number of observed duplication events per unit time seems negatively correlated with the age of the events in all species. This wascommonly observed in the previous analyses (e.g., Lynch
and Conery 2000; International Human Genome Sequencing Consortium 2004) and is probably due to the loss of(functional) duplicate copies over time. Especially, the youngest age-group is expected to contain a substantial numberof duplicates that are not fixed yet, as confirmed in recent
studies (e.g., She et al. 2008; Zhou et al. 2008). Regarding
the type of duplication events, multiexon duplogs accountfor 67%, 46%, 86%, 92%, 51%, and 66% of all duplogs inhuman, mouse, zebrafish, C. elegans, D. melanogaster, andD. pseudoobscura, respectively (table 1 and fig. 3). In mouseand D. melanogaster, single-exon duplogs give fairly large
contributions. Functional analyses revealed that G-protein-coupled receptors including olfactory receptors and his-tones give major contributions to mouse and D.
melanogaster duplogs, respectively.

Cross-Sectional Analyses
To figure out dominant mechanisms of physically relatively
distant and/or inverted duplog formation, we examined theevolutionary patterns of positional relationships between
duplogs, via cross-sectional analyses based on duplication
events (fig. 2; see Materials and Methods for details). We
first examined the composition of physical proximity, namely
the linkage and the physical distance between the duplogs(fig. 4 and supplementary fig. S2, Supplementary Material
online). In the set of all duplication events (supplementary
fig. S2, Supplementary Material online), the proportion of
unlinked events seems to show different age dependence
from species to species. For events resulting in multiexon duplog pairs (fig. 4), the proportion of unlinked events seems
to either remain almost constant (zebrafish, D. melanogaster) or gradually increase with the age for a while (human,
mouse, D. pseudoobscura), except C. elegans. The latter behavior appears consistent with the production of unlinked
duplogs from linked ones via genomic rearrangement

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FIG. 4.—Age dependence of the composition of physical proximities between multiexon duplogs. In each panel, the black dashed line shows theage (in dS) dependence of the proportion of unlinked duplication events out of all multiexon duplication events mapped on chromosomes. The solidlines colored blue, pink, orange, and light blue, respectively, show the age (in dS) dependence of the proportions of the first, second, third, and fourth
quartiles of the physical distance in the set of linked multiexon duplogs.

(e.g., Friedman and Hughes 2003, 2004; Conceic
xão and
Aguadé 2008). In some species, for example, mouse and zebrafish, however, the unlinked multiexon events account fora considerable portion of the youngest age-group, suggesting their de novo generation via mechanisms other than retrotransposition. Within the linked multiexon duplication
events of each studied animal, the proportions of the thirdand fourth quartiles of the physical distance either remain
almost the same through 0  dS , 0.6 or gradually decrease
with age except for the fourth quartile in C. elegans (fig. 4;
for statistical test results, see supplementary table S2, Supplementary Material online). This indicates that most of
linked duplogs belonging to the most distant class (top
25% of events in the physical distance) were created de novo via mechanisms different from retrotransposition. Caenorhabditis elegans is unusual in that the proportion of the
fourth quartile (top 25% in the physical distance) is fairly
small (ca. 5%) in the youngest age-group.
Next, we examined relative orientations between linked
multiexon duplogs. Overall, the proportion of inverted events(tail-to-tail and head-to-head events) varies across species
from ca. 20% in D. melanogaster to over 40% in human(table 2). To get a clue about whether this is the nature of
duplication events themselves or due to the secondary

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changes, we examined the age dependence of the composition of relative orientations (fig. 5) within linked multiexon duplogs. The proportion of inverted events either remains nearly
constantthroughout 0  dS, 0.6 or graduallydecreasesasthe
age increases (fig. 5; for statistical test results, see supplementary table S2, Supplementary Material online). And the proportion of tail-to-tail events and that of head-to-head events areapproximately equal, aside from some fluctuations. This indicates that most of the inverted pairs in the genome are createdfrom the beginning via mechanisms other than retrotransposition and did not result from the inversion of tandemly duplicated pairs. Caenorhabditis elegans seems anomalous: The
proportion of inverted events in the age-group 0.01  dS ,0.03 (77%) is significantly larger than in the age-group dS, 0.01 (49%; P value 5 0.0056 in Fisher’s exact test).
The age dependences of the compositions of physical distance and of relative orientations observed above (figs. 4
and 5) seem to indicate one-step creation of duplogs withrelatively large physical distance and/or inverted orientation.
To further clarify the nature of such duplication mechanisms,
we need to examine the physical distance dependence ofthe relative orientation composition, as well as the age dependence of such dependence. For this purpose, we first examined how the composition of relative orientations

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Table 2Compositions of Relative Orientations
Relative Orientationa

Head-to-Taila

Tail-to-Taila

Head-to-Heada

Total

716.4 (58.2)
1,374.7 (69.0)
1,443.3 (74.6)
623.7 (78.9)
745.2 (63.0)
176.0 (80.5)
219.8 (67.5)

255.6 (20.8)
301.3 (15.1)
236.6 (12.2)
87.5 (11.1)
190.1 (16.1)
17.3 (7.9)
50.8 (15.6)

259.6 (21.1)
315.4 (15.8)
256.1 (13.2)
79.5 (10.1)
247.1 (20.9)
25.4 (11.6)
55.0 (16.9)

1,231.6
1,991.5
1,935.9790.7
1,182.4
218.7
325.6

539.5 (56.4)
694.4 (69.7)
1,220.5 (73.4)
496.8 (76.6)
709.7 (64.4)
93.0 (82.6)
150.8 (67.3)

203.6 (21.3)
145.0 (14.5)
222.6 (13.4)
78.7 (12.1)
170.6 (15.5)
10.5 (9.4)
38.5 (17.2)

213.3 (22.3)
157.4 (15.8)
218.7 (13.2)
73.1 (11.3)
222.1 (20.1)
9.0 (8.0)
34.6 (15.4)

956.4
996.8
1,661.8
648.6
1,102.4
112.5
224.0

b

All linked duplication events
Human
Mouse
Zebrafish
Zebrafish (stbl)c
Caenorhabditis elegans
Drosophila melanogasterD. pseudoobscura
Linked duplication events(multiexon duplog pairs onlyd)
Human
Mouse
Zebrafish
Zebrafish (stbl)cC. elegans
D. melanogasterD. pseudoobscura

NOTE.—The ‘‘number of duplication events’’ actually means the summation of weight factors from the gene pairs belonging to respective subsets. For details on the weightfactors, see supplementary materials and methods (Supplementary Material online). The numbers therefore can be fractional and were rounded off to the nearest tenth. The
percentage (in parentheses) in each cell is the proportion that the relative orientation in question (column) accounts for in the species in question (row).
a
Relative transcriptional orientation of the contributing pairs. ‘‘Head-to-tail,’’ ‘‘tail-to-tail,’’ and ‘‘head-to-head’’ denote, respectively, the gene pairs of 5’-3’ 5’-3’, 5’-3’ 3’-5’, and3’-5’ 5’-3’ orientations.
b
A set of all duplication events whose resulting genes are both mapped on the same chromosomes.
cA set of ‘‘stable’’ zebrafish duplogs that are consistently annotated on both the Zv7 and Zv8 assemblies (for details, see supplementary materials and methods, Supplementary
Material online ).
dOnly contributions from the multiexon gene pairs are counted.

depends on the physical distance (fig. 6). The proportion of
inverted pairs in multiexon duplogs increases as the duplogs
get more separated, reaching approximately half for themost distant class (top 25% in the physical distance), andthe ratio between tail-to-tail and head-to-head events wasnearly 1:1 for most classes of the physical distance. This pattern was also observed in previous studies (e.g., Shoja andZhang 2006). By itself, the pattern is consistent both withtandem duplications followed by genomic rearrangements
such as inversions and with the de novo creation of relatively
distant duplogs with almost random orientations. When thepattern is combined with the age dependence of compositions of physical distance (fig. 4) and relative orientation (fig.
5), however, the latter scenario seems more plausible.
To corroborate this idea, we conducted a cross-sectional
analysis of the proportion of inverted pairs, separately foreach of the four physical distance categories (fig. 7). (Thesample sizes of the subsets, each specified by an age-group
and a physical distance class, are available on request to thefirst or last author.) We should note that most of the subsets
for D. melanogaster and D. pseudoobscura and some of thesubsets for other animals have sizes that are too small (often
less than 10) to give statistical significance. Taking accountof fluctuations due to sampling errors, the proportion of inverted pairs in each quartile appears either nearly constantthroughout the time interval 0  dS , 0.6 or gradually
decreasing with age with a few exceptions (fig. 7; forstatistical test results, see supplementary table S3,
Supplementary Material online). This is true for three vertebrates and possibly for Drosophila, except for the second
quartile in human, which exhibited a U-shaped age dependence. Especially, the proportion of inverted events in theyoungest age-group (dS , 0.01) did not differ so much fromthat in the whole set (0  dS , 0.6).
To see whether this pattern can be explained solely via
tandem duplications and subsequent rearrangements, we
compared the observed proportions of inverted duplogs in
the youngest age-group (dS , 0.01) with theoretical expectations. We restricted our analysis mainly to the intersectionof the youngest age-group (dS , 0.01) and the third quartile(50–75% from the bottom) of the physical distance. We
avoided using the fourth quartile (top 25%) because the duplog pairs in this class could have indefinite probabilities ofrearrangements due to unbound physical distance. For details on this analysis, see ‘‘Theoretical Estimation of the Proportion of Inverted Duplogs’’ subsection of supplementarymaterials and methods (Supplementary Material online).
Here, we only note that we took account of the inversion
rate disparity between duplication-rich regions and the remaining genomic regions. Based on the recent genome-wide
analyses (Newman et al. 2005; Ranz et al. 2007), we estimated that recently duplicated regions on average underwent inversions ca. 67 times more frequently than theremaining regions for human, and that the rate disparity

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FIG. 5.—Age dependence of the composition of transcriptional relative orientations between linked multiexon duplogs. Each line graph shows theage dependence of the proportions of relative orientations in the set of linked multiexon events in each species. The levels of the light blue, light orange,
and light green lines at each category represent the proportions of duplication events attributed to ‘‘head-to-tail,’’ ‘‘tail-to-tail,’’ and ‘‘head-to-head’’gene pairs, respectively, which in turn mean gene pairs with the (5’-3’ 5’-3’), (5’-3’ 3’-5’), and (3’-5’ 5’-3’) configurations, respectively.

was ca. 162 times for D. melanogaster (supplementarymaterials and methods, Supplementary Material online). As
the small P values in supplementary table S4 (Supplementary
Material online) show, the theoretical estimation via themodel of tandem duplications and subsequent chromosomal
rearrangements alone fails to explain the observed proportion of inverted duplogs, which is about 4–15 times larger
than the expectation, for any of the six animals tested.
It must be noted that the pattern of C. elegans duplogs infigure 7 totally differed from that of vertebrate duplogs. For
this species, we observed a high proportion (.80%) of inverted duplogs in the bottom 25% of physical distance in dS, 0.03 followed by a plunge in the proportion (down to,20%) during 0.03  dS , 0.10 (panel D in fig. 7). The
second class (bottom 25–50% in the physical distance) also
seems to show a similar but weak trend.

Reanalyses on Zebrafish Using a Stable Set of
Duplogs
Our cross-sectional analyses showed that zebrafish duplogs
behave similarly to mammalian duplogs. One caveat to theseresults is that the zebrafish genome assembly we used is stilla draft assembly, and, in the worst case, all our findings on
zebrafish duplogs could be artifacts stemming from the poor-

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quality portion of the assembly. After the first round of ouranalysis, an improved version of the draft zebrafish genome
assembly, build Zv8, came out (The Danio rerio Sequencing
Project 2008). So, we constructed a ‘‘stable’’ set of zebrafish duplogs consisting only of those duplogs that are mapped on both assemblies and whose annotations remainunchanged in the two assemblies (supplementary materials and methods, Supplementary Material online). This stable set should mostly consist of duplogs mapped on theclone-based portion of the genome assembly, and should
therefore represent an almost random sampling of the duplogs from the finished assembly. Although the total numberof duplication events reduced to approximately one-third(2,831 for Zv7 vs. 1,044 for ‘‘stable’’; see table 1), the observed patterns remained almost unchanged (supplementary
fig. S3, Supplementary Material online). We therefore believe that our observation was not an artifact and that zebrafish duplogs also share the patterns displayed by mammalian
duplogs.

Cross-sectional Analyses Based on Mid-Intron
Sequence Divergence
In this study, we used the dS between duplogs as a proxy to
the duplication date. Previous studies revealed that

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FIG. 6.—Physical distance dependence of the composition of transcriptional relative orientations between linked multiexon duplogs. Each line
graph shows the physical distance dependence of the proportions of relative orientations in the set of linked multiexon events in each species. The
meanings of the three colors are the same as in figure 5.

synonymous substitutions are also under weak selection
over translation efficiency (Stenico et al. 1994; Akashi
1995), splicing efficiency (Parmley et al. 2006; Warneckeand Hurst 2007), and so on (e.g., Chuang and Li 2007). For human, mouse, and Drosophila, effects of selections on synonymous substitutions were shown to be so weak that dS can wellapproximate the neutral nucleotide divergence (e.g., Parmleyetal.2006;Cutter2008).ForC.elegans,incontrast,dSstrongly
correlates with the codon usage bias (Cutter 2008), questioning the use of raw dS values as proxies for the divergence dates.
Regarding zebrafish, we do not know any such studies on dS.
Because data on these two species contribute important conclusions in this study, we reconducted the cross-sectional analyses using the mid-intron sequence divergence (dI) as analternative proxy tothe divergence date (for details, see supplementary materials and methods for details, Supplementary
Material online). Although dI is not completely free from selection either, the nature of (weak) selection on dI is different fromthat on dS. Consistent features between the two crosssectional analyses, one based on dS and the other on dI, will
therefore suggest the authenticity of the features. Supplementary figures S4 and S5 (Supplementary Material online) showthat the main features for zebrafish remain valid and so do
those for C. elegans, indicating that our conclusions are biologically significant. It should be noted, however, that the sample
size for C. elegans is quite small (supplementary fig. S5A, Supplementary Material online). This may have obscured thetiming of the switching from the inverted-predominance tothe direct-predominance in the first quartile of the physical distance (supplementary fig. S5E, Supplementary Material online).

Discussion‘‘Fourth Mode’’ of Gene Duplication
In this study, we characterized the short-term evolution of
duplogs using cross-sectional analyses. We analyzed six animals with high-quality genome assemblies: human, mouse,zebrafish, C. elegans, D. melanogaster, and D. pseudoobscura. Albeit with one or two exceptions, the duplog sets inthe studied six animals shared the following evolutionary
patterns, mainly among multiexon duplogs and mostly
among all duplogs as well:(i) Except for C. elegans, the proportions of the third andfourth quartiles (50–100%) of physical distance in thelinked duplogs are almost unchanged across age-groups or
decrease gradually as the age increases (fig. 4);(ii) The proportion of inverted duplication events is almost
unchanged through the age interval of 0  dS , 0.6 ordecreases gradually as the events age (fig. 5);(iii) The proportion of inverted events, with all ages mixed
up, increases as the physical distance increases. Inverted
events account for ca. 10–20% and approximately halfin the bottom 25% and the top 25% of the physicaldistance, respectively (fig. 6); andGenome Biol. Evol. 3:1119–1135. doi:10.1093/gbe/evr074 Advance Access publication August 22, 2011

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FIG. 7.—Proportions of inverted duplication events as functions of duplication date (in dS) in different classes of the physical distance (for linked
multiexon duplog pairs). Each line graph shows the age (in dS) dependence of the proportion of inverted duplication events in a quartile classified by thephysical distance between duplogs, contributed from linked multiexon duplog pairs. The blue, magenta, orange, and cyan lines represent, respectively,
the first, second, third, and fourth quartiles of linked duplication events. Because Drosophila melanogaster and D. pseudoobscura experienced only
small numbers of duplication events, stochastic fluctuations are so large that the proportions in panels E and F are not statistically meaningful inmany data points (see also supplementary table S3, Supplementary Material online).

(iv) For the three vertebrates, the proportion of inverted
events in each class of the physical distance remains
almost constant across the age-groups or gradually
decreases with age. The proportion hovers around 10–
20% and around half in the bottom 25% and the top
25% of the physical distance, respectively (fig. 7).
Caenorhabditis elegans posed an exception against thepatterns (i), (ii), and (iv), which will be discussed later. For
the two Drosophila species, the patterns corresponding
to (iv) were unclear, likely due to small sample sizes (fig.
7E and F).
The ‘‘static’’ pattern (iii) has been repeatedly observed
also in the previous genome-wide analyses (e.g., Shoja
and Zhang 2006). As far as we know, however, this study
is the first to report patterns (i), (ii), and (iv) of the age dependence for animal duplogs. We emphasize that patterns(i)–(iv) were observed even for multiexon duplogs. This precludes the explanation via retrotransposition because suchmechanism mostly creates single-exon duplogs (see, e.g.,Babushok et al. 2007), unless premature long RNA transcripts were reverse-transcribed. Pattern (iii) is consistent
with the classical view of tandem duplication followed bygenomic rearrangements (e.g., Friedman and Hughes
2003, 2004; Conceicxão and Aguadé 2008; Hu et al.

11302008). In contrast, it is difficult to explain patterns (i), (ii),and (iv) with this classical view. Patterns (i) and (ii) show thatthe most distant linked class (top 25% in the physical distance) and inverted pairs were already present in considerable proportions almost as soon as the duplications
occurred. And pattern (iv) indicates that inverted pairs remain accounting for around half of the most distant linked
class (top 25% in the physical distance) throughout the timeinterval of 0  dS , 0.6 we studied. Our statistical test
showed that pattern (iv) cannot be explained by tandem
duplication and subsequent chromosomal rearrangements
alone (supplementary table S4, Supplementary Material online; see also Results). By analogy, we expect that sucha mechanism alone cannot explain the patterns (i) and(ii), either.
The most natural interpretation for these observations
would be ‘‘yet another mode of gene duplication’’ that isdifferent from the three well-known duplication mechanisms: tandem duplication, retrotransposition, and wholegenome duplication. Here, we term this duplication modeas ’’drift’’ duplication. Its physical distance distribution appears to peak around a few hundred kilobase pairs for vertebrates and a few dozen kilobase pairs for invertebrates,
which is in between those of tandem duplication (short

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FIG. 8.—Evolution of the duplication mode composition in vertebrates. The pie charts at the exterior nodes of the phylogenetic tree represent theestimated compositions of the duplication modes in extant vertebrate species: human, mouse, and zebrafish. The inset above the key (on the bottom
right) illustrates the color code of the duplication modes. A thin horizontal line represents a chromosome, and solid short rectangles (exons) connectedby sharply bent lines represent genes, with black ones original and four colored ones duplicate copies.

range) and retrotransposition (long range, i.e., mostly unlinked). Drift duplications are almost randomly oriented,
with the frequency ratio of head-to-tail : tail-to-tail :head-to-head  2:1:1, as opposed to tandem duplications
due to unequal crossing-overs, which are mostly head-totail. A drift duplication can also create multiexon duplogs,
as opposed to retrotransposition, whose products aremostly intronless. Retrotransposition is also drifting ina sense; however, it always passes through the RNA stage.
This is the clear difference from drift duplication. With this
name, ‘‘drift,’’ we also implied that even some interchromosomal duplications may be attributed from drift duplication,
though RNA-mediated duplications may be more frequent
among interchromosomal duplications. DNA molecules fordrift duplications are usually much larger than those forRNA-mediated duplications and may not be able to moveto different chromosomes easily. This conjecture should
be examined in future studies.
It is not certain at this point whether this mode, namely drift
duplication,is duetoasingleduplicationmechanismor not.We
should note that this might also be explained via a considerable
proportion of duplication events in extremely unstable regions
undergoing tremendously frequent rearrangements (Eichler
and Sankoff 2003; Pevzner and Tesler 2003; Murphy et al.
2005).Whenestimating thetheoretical proportions of inverted
duplogs in supplementary table S4 (Supplementary Material
online), we took account of the rearrangement rate disparity
between recently duplicated regions and the remaining genomic regions. Still, it is possible that the duplicated regions in fact

consist of relatively stable regions and extremely unstable regions. In the future, analyses of finished genome assemblies
in closely related species or analyses of structural variations
within species with finished reference genomes will reveal
whether our observation is due to rearrangements in extremely
unstable regions or to genuine creation mechanisms of relatively distant and randomly orientedduplogs. Here,we will simply assume the latter and continue our discussion.
Recent analyses of the human genome revealed rich instances of recent SDs nonrandomly distributed across the human genome (; Bailey et al. 2002; Bailer and Eichler 2006).
A substantial proportion of such SDs was found to be of interspersed type (Bailey et al. 2002, 2003; Bailey and Eichler
2006). The finished mouse genome assembly analysis also revealed rich instances of recent SDs (She et al. 2008; Church
et al. 2009). Although these authors emphasized the differences of mouse SDs from human SDs such as the enrichmentof tandem duplications, their classification of linked SDs wasquite coarse grained at low physical distance resolutions of 1
Mb. In contrast, our cross-sectional analysis strongly indicatesthat a considerable fraction of mouse SDs classified as ‘‘tandem’’ so far, as well as a majority of interspersed SDs, are created de novo via the drift duplication, and that de novo
creation of duplogs via the drift duplication is a common nature of mammalian genomes. This in turn suggests that thedrift duplication has been actively operating in the genome
since the common ancestor of placental mammals (fig. 8).
Actually, we could further extend the period during
which the drift duplication has been active. We observed

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that zebrafish duplogs also exhibit the patterns similar tomouse (rather than human) duplogs (figs. 4–7), indicatinga substantial contribution of the drift duplication in this species. Hence, we can infer that the drift duplication has been
generating duplicate genes in rates comparable with thoseof tandem duplications at least since the common ancestorof tetrapods/ray-finned fish (fig. 8). So far, tandem duplications due to unequal crossing-over have often been regarded as a dominant mechanism to produce recent
duplicate genes (e.g., Shoja and Zhang 2006; Pan andZhang 2007, 2008). To the best of our knowledge, the present study is the first to point out that the drift duplication
also has produced as many duplicate genes as, or even morethan, tandem duplications at least in vertebrate genomes.
Various modes of duplications among three vertebrate
species are compared in figure 8. The drift duplication,
shown in red, constitutes a major part in all three species,
especially in human. While retroposed duplication is mostfrequent in mouse, other unlinked duplication is more than
one-third of the total in zebrafish. Although we restricted
the drift duplications to intrachromosomes, it may be possible that the same molecular mechanisms are also involved
in interchromosomal duplications classified as ‘‘other unlinked’’ in this figure. If so, this type may be the major mechanism for duplog generation.
Let us now discuss the mechanism causing the drift duplication. In the human genome, over one-fourth of the recent
interspersed SDs seem to be explained by Alu-mediatedmechanisms (Bailey et al. 2003), but about a half of theevents seem unaccounted for. For mouse, many but not mostof the SDs are bounded by LINEs (long interspersed elements)
or LTRs (long terminal repeats), suggesting mechanisms mediated by such repeats (She et al. 2008). As for zebrafish or
C. elegans, we do not know previous studies on the mechanisms potentially causing the drift duplication. Regarding
fruit flies, a recent large-scale experimental screening ofeight genomes in the D. melanogaster subgroup identified
17 duplicates generated with the mediation of repetitive elements (Yang et al. 2008). It would be interesting to carefully
examine the boundaries of the SDs resulting from the drift
duplication in zebrafish and C. elegans, and if many of themturn out to be mediated by species-specific repeat sequences. We have to note, however, that a majority of the SDsseem to have been caused by repeat-independent mechanisms (Zhou and Mishra 2005). Determining the specificmechanisms responsible for the drift duplication would require correlating features in the flanking sequences of duplicated regions (Bailey et al. 2003; Zhou and Mishra 2005) withpositional relationships between the duplicated regions.

Recent ‘‘Expansion’’ of Histone Tandem Array in
Drosophila melanogaster
We observed that, regarding all duplogs in the two fruit
flies, whereas D. pseudoobscura duplogs seem to conform1132

to the general patterns (i)–(iv) discussed above, D. melanogaster duplogs were eccentric in the sense that they
did not follow any of patterns (i)–(iv). Especially, the proportion of inverted events was quite small (ca. 9%) in dS , 0.01
for the top 25% of the physical distance, and this seemed tobe causing most of the eccentric patterns. When restricted
to multiexon events, however, the proportion of inverted
events was around half in the subclass in question (fig.
7E). So, we carefully examined the single-exon pairs thathave dS , 0.01 and belong to the top 25% in the physicaldistance. We found that tandem repeats of the histone genecluster (Celniker et al. 2002) make an overwhelming contribution of 63 events to this subclass. When we reconducted
the cross-sectional analyses after removing these histone
tandem repeats, the remaining D. melanogaster duplogs
largely followed the patterns commonly observed in otheranimals (except C. elegans) (data not shown). This implies
that, aside from the huge tandem array of the histone genecluster, duplogs of the two fruit flies also follow the generalrules (i)–(iv). We are not saying that the huge tandem arrayof histones is specific to D. melanogaster. Such repeat might
be missing in the D. pseudoobscura genome because it isstill a WGS-based draft assembly. Whether this is true or
not will be revealed if a clone-based analysis is conducted.
Rather, we can say that the effect of this tandem repeatstood out because the whole set of D. melanogaster duplication events is pretty small (table 1 and fig. 3E), which
made the patterns of D. melanogaster duplogs ‘‘appear’’ eccentric. Tandem duplication may be a predominant duplication mechanism in the fruitfly genomes. Still, it seems thatthese genomes are also undergoing the drift duplication tosome degree. This raises the possibility that the drift duplication have been operating since the common ancestor ofbilateral multicellular animals. It would be premature, however, to conclude this conjecture now. The sample size tosupport this is too small, totaling ca. 11 multiexon eventswith dS , 0.01 belonging to the top 25% of the physicaldistance in the D. melanogaster and the D. pseudoobscuragenomes.
At face value, the large tandem array of D. melanogasterhistone gene clusters appears to indicate that a burst of tandem duplications occurred very recently to amplify the histone cluster. This apparent evolutionary pattern is, however,also consistent with the strong concerted evolution, as observed for the tandem clusters of ribosomal RNA genes (forreview, see Eickbush and Eickbush 2007). The pattern could
also be explained with strong selective pressures on synonymous substitutions, maybe due to the requirements to conserve mechanisms for histone gene regulation. To determine
which mechanism actually generated the observed patternof the histone gene clusters in D. melanogaster, it would be
inevitable to conduct evolutionary analyses of the histone
clusters based on clone-based sequences from closely related Drosophila species.

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Unique Features
Duplogs

inCaenorhabditis

elegans

Caenorhabditis elegans duplogs displayed quite different patterns than the duplogs of other five animals studied, posing
exceptions against (i), (ii), and (iv) (figs. 4, 5, and 7). Especiallyagainst (iv), the closest class (bottom 25% in the physical distance) of linked duplogs switched from inverted-rich states in
0  dS , 0.03 to direct (head-to-tail)-rich ones in dS  0.1(panel D of fig. 7). The predominance of inverted pairs among
recent (0  dS , 0.03) physically close duplogs is consistent
with the finding by Katju and Lynch (2003) and, at the sametime, specific to C. elegans among the six animals studied.
This suggests some mechanisms specific to the C. elegans lineage. Katju and Lynch (2003) mainly proposed two mechanisms that may have caused this pattern: illegitimate
recombination and frequent elimination of head-to-tail duplog pairs. Here, we would like to point out another possible
mechanism: preferential homogenization between closely located inverted duplogs. Instances of intense homogenization
between inverted duplicons are known, for example, on the
male-specific region (MSR) of human Y chromosome (Rozen
et al. 2003). And it should be noted that the homologous
recombination rate of C. elegans is very low because it mostly
self-fertilizes. Whether or how C. elegans has evaded Muller’sratchet has therefore been discussed (e.g., Loewe and Cutter2008). It is possible that intense homogenization between
inverted duplogs have liberated the recombination-poor
C. elegans genome at least partially from Muller’s ratchet,as was proposed for the recombination-free MSR of human
Y chromosome (Rozen et al. 2003).
In contrast, it also merits a mention that the patterns
for C. elegans duplogs are actually quite similar to thosefor vertebrate duplogs if we focus on the age-groups with
0.1  dS , 0.6, which were not studied by Katju and Lynch(2003). To see whether this is just a coincidence or due
to shared underlying mechanisms, especially the drift
duplication, across bilateral animals would require moredetailed analyses.

a common but unexpected feature, namely substantial contributions from drift duplication. The analyses also illustrated
idiosyncrasies of duplogs in the animal species studied, especially the sharp drop with age of the proportion of inverted duplogs during 0.03  dS , 0.1 in the closest
quartile of C. elegans duplogs (fig. 7D), as well as the ‘‘burst’’of tandem duplications in the most distant quartile of
D. melanogaster duplogs. So far, tandem duplications
due to unequal crossing-over have often been invoked asa dominant mechanism to generate duplicate genes (see,e.g., Friedman and Hughes 2003, 2004; Shoja and Zhang
2006; Pan and Zhang 2008). For the first time, our crosssectional analyses revealed that the drift duplication has also
been generating duplicate genes in rates comparable withor even higher than the rates of tandem duplications at least
since the vertebrate ancestor. It would be premature, however, to conclude that the patterns we observed are universal even across animals because the number of sampled
genomes (i.e., 6) is far from large enough to make a general
conclusion, although this restriction was inevitable to keep
our analyses reliable.
We will have to analyze more animals when their finishedgenome assemblies come out. It will also be interesting toapply our cross-sectional analyses to the finished genomesof other eukaryotes (e.g., Dolinski and Botstein 2005; Haas
et al. 2005), or even to the finished genomes of procaryotes(see, e.g., Walter et al. 2009). The general features we observed were largely attributed to the creation mechanismsof duplicated genes. But mechanisms of post-duplication
evolution, such as preferential loss or homogenization ofparticular classes of duplogs (Rodin and Parkhomchuk
2004; Ezawa et al. 2006, 2010; Xu et al. 2008), might makeconsiderable contributions especially to the species-specificfeatures. Finally, although this study focused mainly on thepositional relationships between duplogs, it may also be interesting to examine the dependence of functional differences between duplogs on their positional relationships. Suchanalyses are left for future studies.

Supplementary Material
Summary and Future Tasks
Clone-based genome assemblies of finished quality are crucial to the genome-wide study of recent duplogs, or intraspecies duplicate genes (She et al. 2004; Church et al.
2009). To elucidate genome-wide trends of recent evolutionof positional relationships between duplogs, an ideal way
would be to compare the chromosomal positions of orthologs of duplicate genes using finished-quality genome
assemblies of closely related species. Unfortunately, atpresent, finished genome assemblies are too sparse in the
animal kingdom to conduct such an ideal analysis. As a practical substitute, we applied cross-sectional analyses to thesets of recent duplogs from the six animal genomes of finished or nearly finished qualities. The analyses uncovered

Supplementary materials and methods, figures S1–S5, andtables S1–S4 are available at Genome Biology and Evolution
online (http://www.gbe.oxfordjournals.org/).

Acknowledgments
This paper is dedicated to the memory of late Dr. Walter M.
Fitch, who passed away in March 2011. The authors are grateful to Dr Kenta Sumiyama for discussions on this study. This
study was conducted as a part of the Genome Network Project as well as Grant-in-Aid for Scientific Research presided by
the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported in part by the
US National Library of Medicine (grant LM010009-01 to Dan
Graur and Giddy Landan at the University of Houston).

Genome Biol. Evol. 3:1119–1135. doi:10.1093/gbe/evr074 Advance Access publication August 22, 2011

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Ezawa et al.

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Associate editor: Yoshihito Niimura

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