Evolutionary Patterns of RNA-Based Duplication in Non-

Mammalian Chordates
Ming Chen1,4., Ming Zou1,4., Beide Fu1,4., Xin Li2, Maria D. Vibranovski3, Xiaoni Gan1,4, Dengqiang
Wang1,4,5, Wen Wang2, Manyuan Long3*, Shunping He1*
1 Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, People’s
Republic of China, 2 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan Province, People’s Republic of China, 3 Department of Ecology and
Evolution, The University of Chicago, Chicago, Illinois, United States of America, 4 Graduate University of Chinese Academy of Sciences, Beijing, People’s Republic of China,
5 Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Wuhan, People’s Republic of China

Abstract
The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and
Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates.
In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary
patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies
revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate
species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong
evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression
profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression
patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the
nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our
analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution
to the evolution of non-mammalian genomes.

Citation: Chen M, Zou M, Fu B, Li X, Vibranovski MD, et al. (2011) Evolutionary Patterns of RNA-Based Duplication in Non-Mammalian Chordates. PLoS ONE 6(7):
e21466. doi:10.1371/journal.pone.0021466
Editor: Michael Freitag, Oregon State University, United States of America
Received January 21, 2011; Accepted June 1, 2011; Published July 11, 2011
Copyright: ß 2011 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from National Natural Science Foundation of China [2007CB411601] (http://www.nsfc.gov.cn/Portal0/default124.
htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: clad@ihb.ac.cn (SH); mlong@uchicago.edu (ML)
. These authors contribute equally to this work.

Introduction Two features characterize the retrogenes of mammals and

Drosophila. They often show the ‘‘expressed in testis’’ [2] and ‘‘out
RNA-based duplication is a molecular process in which RNA is of the X’’ patterns [3,15]. Numerous studies [1,2,15,16,18] have
reverse-transcribed into cDNA and inserted at a new position in the revealed a bias toward retrogene expression in the testis. For
genome. The newly created ‘‘retrocopy’’ usually contains the example, one study [2] showed that the proportion of testis ESTs
untranslated and coding regions of the parental gene but does not that map to retrocopies is higher than that of multi-exon genes,
carry a promoter. It has three alternative evolutionary fates: (i) it and that a higher proportion of intact retrocopies is expressed in
may recruit a new regulatory sequence, thus likely acquiring a new the testis when compared to retropseudogenes. These observations
expression pattern and forming a new expressed duplicate copy, or revealed that retrogenes are often transcribed and functional in the
‘‘retrogene’’; (ii) it may occasionally recruit a regulatory sequence testis. In the ‘‘out of the X’’ pattern, a disproportionately large
and a new coding region from the insertion site to be translated into number of retrogenes are derived from parental genes on the X
a chimeric protein; (iii) it may, more often, lose its coding potential, chromosome [2–4,15]. These autosomal retrogenes compensate
become a pseudogene, and eventually disappear from the genome. for the silencing of parental X-linked genes during and after male
It has been shown that most mammalian retrocopies have become meiotic sex chromosome inactivation [4]. This out-of-X gene
‘‘retropseudogenes’’ [1–4]. However, it has long been expected that traffic cannot be explained by mutation bias and was driven by
retrocopies will be shown to play a significant role in evolution [5]. natural selection to facilitate male germline function [3].
Many functional retrogenes have been reported in mammals, birds, Chordates (phylum Chordata) are a broad class of animals that
and invertebrates [1,3,6–10]. It seems that there are very few RNA- have in common a notochord with a hollow dorsal nerve cord
based duplicates in the chicken genome [11]. The reverse [19]. The phylum Chordata consists of three subphyla Urochordata,
transcriptases of the CR1 elements present in chicken have been Cephalochordata, and Craniata. Subphylum Urochordata is represented
found to be responsible for the deficiency of retrocopies in that by the tunicates and Cephalochordata by the lancelets. Craniata
genome [12–14]. In contrast, in Drosophila melanogaster, about 100 includes the Vertebrata, which in turn includes cyclostomes, fish,
candidate retrogenes have been identified [15–17]. amphibians, reptiles, birds, and mammals. Retrogene origination

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 RNA-Based Duplication in Nonmammalian Chordates

by RNA-based duplication has been reported and analyzed only in the 34% to 87% figure for non-mammalian chordates. Moreover,
mammals, and little is known about retroposition in non- for Sea squirt, stickleback and zebrafish, the total estimated
mammalian chordates [20]. To assess the generality of retrocopies number of retrogenes was only a little smaller than that for human.
(or retrogenes) in non-mammalian chordates, including the In amphioxus, fugu, Medaka, Western clawed frog and Lizard, the
distribution and evolutionary patterns, we identified retrocopies estimated number of retrogenes was even larger than that for
(or retrogenes) in ten non-mammalian chordate species. These human (Table 1).
species included five fish species: the zebrafish (Danio rerio), Medaka Thirdly, for those species that have sufficient expression data,
(Oryzias latipes), stickleback (Gasterosteus aculeatus), fugu (Takifugu we studied retrocopy expression in them. In Western clawed frog,
rubripes), and Tetraodon (Tetraodon nigroviridis); one amphibian: the Sea squirt, zebrafish, stickleback, and Medaka, more than 40% of
Western clawed frog (Xenopus tropicalis); one bird: the chicken the retrocopies were expressed, whereas in human, only 27% of
(Gallus gallus); one reptile: the lizard (Anolis carolinensis); one retrocopies were expressed. Furthermore, there was a significant
Urochordate: the Sea squirt (Ciona intestinalis); and one Cephalochordate: excess of expressed intact retrocopies relative to expressed
amphioxus (Branchiostoma floridae). Two mammals, human (Homo retropseudogenes in these five genomes (one-tailed Fisher’s exact
sapiens) and platypus (Ornithorhynchus anatinus), were used for test, p,0.01, Table 2). This suggests that intact retrocopies were
comparison. After conducting a systemic evolutionary analysis, more likely to be expressed than retropseudogenes. Taken
we discovered distinct patterns associated with the evolution of together, this evidence suggests that a larger fraction of the
retrocopies (or retrogenes) in these non-mammalian chordate retrocopies is likely to be functional in the eight non-mammalian
species. chordates studied (Table 2) than in the two mammals studied.

Results Retrogene expression in the gonads and brains of non-

Distribution of retrocopies in various chordate genomes mammalian chordates
We identified retrocopies in 12 chordate species (phylogenetic We analyzed the EST information (http://genome.ucsc.edu/)
relationships are shown in Figure 1) by using the modified of seven species under study and summarized the relevant statistics
computational pipelines in earlier studies [1]. We classified these as in Table 3. Given the total number of ESTs, the human genome
either intact retrocopy or retropseudogene according to whether expresses a relatively small proportion of its retrocopies (27%),
or not they contained frameshift mutations or premature stop whereas Medaka, stickleback, zebrafish, and Western clawed frog
codons when compared with their parental genes. In Amphioxus, express about 40% or more of their retrocopies, even though fewer
we found a relatively large number of retrocopies (337), total EST sequences are available than for human (Table 3). At the
considering the small genome size of this species (Table 1). In extreme, 89% of the retrocopies in Sea squirt are transcribed.
the Sea squirt genome and five fish genomes, we identified Only 18% of the retrocopies in the lizard genome appeared to be
relatively fewer retrocopies than in non-mammalian tetrapods expressed perhaps because there is much less total expression
(other than chicken) such as lizard and Western clawed frog information available (Table 3). Except in human, most of the
(Table 1). However, the number of retrocopies in lizard and expressed retrocopies were found to be intact.
Western clawed frog is lower than that of human and platypus, We further analyzed the tissue distributions of the expressed
where 4738 and 542 retrocopies were found. retrogenes (Table 4). In most of the species under study, many
functional retrogenes were expressed in the brain. In Western
Higher proportions of the retrocopies were found to be clawed frog, lizard, Medaka, zebrafish and Sea squirt, many
functional retrogenes were expressed in the testis or ovary. We
functional in non-mammalian chordates explored whether retrogenes were expressed more often in the
To deduce retrocopy functionality, we first compared the
brain and gonad than in other tissues. Table 4 shows statistics
fraction of intact retrocopies between non-mammalian chordates
suggesting that this is true in the human, Western clawed frog,
and mammals. In non-mammalian chordates, the proportion of
Medaka, and Sea squirt genomes.
intact retrocopies ranged from 54% to 87%, significantly (one-
tailed Fisher’s exact test; p,0.01) higher than the proportion of
intact retrocopies in the two mammalian species studied here Gene traffic in the Medaka genome
(Table 1), suggesting that a higher percentage of retrocopies are In this study, we tested the ‘‘out of the X’’ hypothesis in the non-
likely to be functional in non-mammalian chordates than in mammalian chordate genomes. The sex-determining system of
human or platypus. Medaka is XX–XY [21], but the differentiation of the sex
Secondly, we calculated the ratios of the nonsynonymous chromosomes seems to be in an early stage. Chromosome 1 acts as
substitutions to the synonymous substitutions per site (Ka/Ks) the X chromosome, whereas the Y chromosome is a variant form
between each retrocopy and its parental gene. Intact retrocopies of chromosome 1 with a 250-kb Y-specific region that contains the
had different Ka/Ks distributions than retropseudogenes: a higher male-determining gene, DMY [22]. This suggests an early stage in
proportion of intact retrocopies had Ka/Ks,0.5 relative to the the evolution of sex chromosomes [23]. We identified 131
proportion of retropseudogenes (one-tailed Fisher’s exact test, functional retrogenes in the Medaka genome. Of these, five genes
Table 2). In other words, intact retrocopies were found to be more were from the sex chromosome. About 3.6 autosomal retrogenes
likely to be under functional constraints. For example, 66% of the were expected from the X chromosome, which is not significantly
intact retrocopies and only 39% of the retropseudogenes had Ka/ different from the observed value (five, Fisher’s exact test, two-tail,
Ks,0.5 in Amphioxus (Figure 2). There were 27% more intact p = 0.75), revealing no excess of autosomal functional retrogenes
retrocopies than retropseudogenes were observed with Ka/ from the X chromosome in Medaka.
Ks,0.5. Also, if we defined intact retrocopies with Ka/Ks
significantly smaller than 0.5 (see Materials and Methods) as Age distribution of retrocopies
functional retrogenes, only 3% and 17% (Table 1) of retrocopies Figure 3 shows the Ks distribution of retrocopies in all these
could be considered to be functional retrogenes in human and species. It also shows that, for tetrapods other than chicken, there
platypus, respectively. These proportions are much less than that are many young retrocopies. However, no burst of young

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 RNA-Based Duplication in Nonmammalian Chordates

Figure 1. Percentages of LSPs of retrocopies in different species. The percentage of LSPs in a particular lineage (shown above each branch) is
the ratio that the number of lineage-specific parent families (LSPs) in the lineage account for the total numbers of parent families the lineage has.
Branch A is the lineage Teleostei.
doi:10.1371/journal.pone.0021466.g001

retrocopies has been found in ancient chordates such as with a Ks,0.23 (0.292650/63.7), originated within about 50
Amphioxus, Sea squirt or fish. For example, assuming a neutral million years. However, for zebrafish, the divergence of the Danio
mutation rate of 1–1.361029 substitutions per site per year in rerio and Cyprinus carpio species occurred about 50 million years ago
primates [24], about 1352 retrocopies were generated in the [27]. We compared 38 pairs of orthologous genes [28] between D.
human genome within 38–50 million years. The Western clawed rerio and C. carpio and obtained an overall Ks value of 0.413. Only
frog, Xenopus tropicalis, and the African clawed frog, X. laevis 32 retrocopies had a Ks,0.413 and originated within 50 million
diverged about 63.7 million years ago [25]. A Ks value of 0.292 years. For fugu and Tetraodon, the amount of neutral substitution
corresponds to the divergence between these two species [26]. (Ks) since the Tetraodon–Fugu divergence was 0.35 [29], there are
There are about 85 retrocopies in the Western clawed frog, which only 18 retrocopies in Fugu originated within the last 50 million

Table 1. Identification of retrocopies in 12 species of Chordata.

Retrocopies Total protein Intact Retro- Retrogene Genome size

Species number number Pa (%) retrocopies pseudogenes Pb (%) number Pc (%) (Mb)

Amphioxus 337 50817 0.6% 235 102 70% 176 52% 520
Sea squirt 110 19858 0.6% 96 14 87% 96 87% 173
Zebrafish 195 31743 0.6% 151 44 77% 119 61% 1527
Tetraodon 90 23118 0.4% 66 24 73% 60 67% 342
Fugu 182 47841 0.4% 148 34 81% 142 78% 393
Medaka 218 24661 0.8% 159 59 73% 131 60% 700
Stickleback 132 27576 0.5% 119 13 90% 111 84% 447
Western clawed 398 27711 1.4% 216 182 54% 140 35% 1511
frog
Lizard 404 17732 2.2% 217 187 54% 136 34% 1770
Chicken 78 22194 0.4% 57 21 73% 51 65% 1051
Platypus 542 26836 2.0% 146 396 27% 92 17% 1918
Human 4738 47509 10% 565 4173 12% 131 3% 3253

a
Percentage of retrocopies per protein.
b
Percentage of intact retrocopies among the total retrocopies.
c
Percentage of retrogenes among the total retrocopies.
doi:10.1371/journal.pone.0021466.t001

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 RNA-Based Duplication in Nonmammalian Chordates

Table 2. Higher fraction of the retrocopies may be functional in 8 nonmammalian chordates.

Intact and Pseudo and Fisher’s exact Intact with Pseudo with Fisher’s
Species Ka/Ks,0.5a Ka/Ks,0.5 Ka/Ks,0.5 testb EST support EST support EST support exact testc

Western clawed frog 188 117 71 ,0.01 150 109 41 ,0.01

Zebrafish 97 82 15 0.01 90 82 8 ,0.01
Sea squirt 65 60 5 0.05 98 92 6 ,0.01
Amphioxus 195 155 40 ,0.01 - - - -
Medaka 136 111 25 ,0.01 86 79 7 ,0.01
Chick 47 42 5 ,0.01 - - - -
Fugu 141 121 20 0.01 - - - -
Lizard 248 151 97 ,0.01 73 63 10 ,0.01

a
calculated by using an LPB method.
b
Excess of intact retrocopies with Ka/Ks,0.5 relative to retropseudogenes.
c
Excess of expressed intact retrocopies relative to retropseudogenes.
doi:10.1371/journal.pone.0021466.t002

years, which is the approximate time of divergence of these two spanning the whole coding region. One chimeric retrogene
species [28,29]. Notably, there is only one retrocopy in Tetraodon matched one EST sequence that spanned both the recruited
with Ks,0.35. coding sequence and retrosequence. Figure 4 shows an exempli-
fied chimeric retrogene in the Western clawed frog. The parental
Chimeric retrogenes identified in the Zebrafish and gene ENSXETT00000014486 has nine exons. Of these, eight
Western clawed frog exons were reverse-transcribed and formed a retrocopy. This
To identify chimeric retrogenes, we defined Ensembl-annotated retrocopy inserted into the first exon of a host gene and formed the
genes sharing 30%,70% of their coding sequences with our chimeric retrogene ENSXETT00000014488.
retrocopies as a chimeric retrogenes. By this criterion, we found
nine chimerical retrogenes in the zebrafish and sixteen in the In non-mammalian chordates retrocopies may be mainly
Western clawed frog (Table 5, for more information, please see produced by LINE1 elements
supplemental Table S1 and Table S2); 89% and 50% of chimeric Retrocopies have been shown to be generated by LINE1
coding structures were confirmed by mRNA or EST sequences in elements in human [30–32]. However, it is not known whether
zebrafish and Western clawed frog respectively (Table 5). For retrocopies are mainly produced by LINE1 or other LINE
example, out of nine chimeric retrogenes in zebrafish, seven genes elements in non-mammalian chordates. We used RepeatMasker
matched at least one mRNA sequence with .98% identity, [33] to identify different kinds of LINE elements in all these species

Figure 2. Ka/Ks distributions for intact retrocopies and retropseudogenes in Amphioxus. The Ka/Ks values were obtained through
comparing retrocopies and corresponding parental genes.
doi:10.1371/journal.pone.0021466.g002

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 RNA-Based Duplication in Nonmammalian Chordates

Table 3. Total EST analysis of retrocopies.

Species Number of EST Number (e)a Intact (e) Pseudo (e) Percentage (%)b

Lizard 156802 73 63 10 18%

Sea squirt 1213772 98 92 6 89%
Medaka 666358 86 79 7 39%
Stickleback 279365 56 54 2 42%
Zebrafish 1511074 90 82 8 46%
Human 9217591 1268 342 926 27%
Western clawed frog 1290068 150 109 41 38%

a
These data are of expressed (e) retrocopies.
b
The percentage of expressed retrocopies in the total retrocopies of each.
doi:10.1371/journal.pone.0021466.t003

(except Amphioxus and lizard, and the data for human and parent families (LSPs) of the retrocopies onto the species tree
platypus came from [34] and [35], respectively). We found the (Figure 1). We can see terminal branches of branch A, whose
number of retrocopies correlated with the number of LINE1 divergence times are not as long as those of other branches, as the
copies (p,0.001, Pearson correlation test; Table 6) but not with species listed there have lower proportions of LSPs (27.3%–37.6%
any other type of LINE element. Furthermore, in the chicken in Fugu). On the contrary, the proportion of LSPs is over 40% on
genome, the total number of retroelements was not small, all the other branches, increasing to 87.9% in human. This high
although only 78 retrocopies were detected. We analyzed the proportion of LSPs in the human genome results in higher
LINE elements in the chicken genome, and found most to be CR1 proportions in the related internal branches.
elements, which seemed likely to have generated negligible
number of retrocopies [11]. As in the chicken, we found that Discussion
CR1 elements also dominate the LINE elements of the Western
clawed frog genome. In contrast, we found 4074 LINE1-like In this study, we identified numerous retrocopies in ten non-
elements and 398 retrocopies in the Western clawed frog genome. mammalian chordate species. We observed obvious differences in
Two pufferfish, fugu and Tetraodon, diverged only 50 million the evolution of RNA-based duplication between mammalian and
years ago [28], and the number of retrocopies found in fugu was non-mammalian chordates. In mammals, most retrocopies are
about twice that of Tetraodon, which is consistent with the fact retropseudogenes [1,2]. In non-mammalian chordates, most
that there are more LINE1 elements in fugu than in Tetraodon. retrocopies are intact. Amphioxus, Sea squirt, two pufferfish,
Medaka, and stickleback have small genomes (Table 1), and the
Gene family of parental genes retropseudogenes in small genomes may degenerate faster than
Pan and Zhang [36] recently identified retrofamilies of more those of species with large genomes [37,38]. For example, given
than one retrocopy present in only one lineage. These they called that, in pufferfish, the rate of DNA loss per nucleotide substitution
‘‘lineage-specific retrofamilies’’ (LSRs). Because most of the is approximately five times faster and the rate of neutral mutation
retrocopies that we identified have not been annotated by is about 2.5 times faster than in mammals, the retropseudogenes
Ensembl, they were not assigned to any LSRs. However, to should have degenerated more than ten times faster in the
investigate the characteristics of the parental genes that generated pufferfish genomes than in mammalian genomes [29]. The Ks
the retrocopies, we classified them according to the Ensembl gene distribution of retropseudogenes (supplemental Figure S1) also
family annotation, and mapped the percentages of lineage-specific supports this conclusion in that there are rare, old retro-

Table 4. Tissue distribution of functional expressed retrogenes.

Species Tissue Na (%) Tissue N (%) Tissue N (%) Tissue N (%) Tissue N (%) Test1b Test2c

Sea squirt blood cells 54.9 gonad 45.1 digestive gland 31.9 heart 18.9 neural complex 17.6 p,0.05 -
Medaka brain 28.0 testis 22.7 ovary 21.3 liver 10.7 eye 2.7 p,0.01 p,0.01
Stickleback brain 63.6 gills 36.3 eyes 29.1 skin 12.7 - NA
Zebrafish heart 12.3 gills 9.6% testis 8.2 ovary 8.2 brain 8.2 - -
Lizard testes 41.7 brain 25.0 ovary 22.2% Regenerating tail 19.4 Dewlap 13.9 NA NA
Western clawed brain 43.9 testis 41.5 Liver 14.6 Lung 14.6 Intestine 9.8 p,0.05 p,0.05
frog
Human testis 58.3 brain 55.0 hippocampus 33.3% placenta 26.7 Melanotic 21.7 p,0.01 p,0.01
melanoma

a
percentage of expressed retrogenes in every tissue among total expressed retrogenes.
b
test whether there are more retrogenes expressed in gonad, binary logistic regression.
c
test whether there are more retrogenes expressed in brain.
doi:10.1371/journal.pone.0021466.t004

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 RNA-Based Duplication in Nonmammalian Chordates

Figure 3. Ks distribution of retrocopies in 12 chordates. The Ks values were obtained through comparing retrocopies and corresponding
parental genes.
doi:10.1371/journal.pone.0021466.g003

pseudogenes in these compact genomes. Moreover, in compact frog have large genomes of about 1.5 Gb, but the fractions of
genomes, there is usually a stronger selection against deleterious intact retrocopies in these species are also high (above 54% to
insertions [39]. Only the functional beneficial retrocopies are likely 77%). Interestingly, the size of the platypus genome is similar to
to be retained and fixed. Notably, zebrafish and Western clawed that of the lizard, zebrafish and Western clawed frog (Table 1), but

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 RNA-Based Duplication in Nonmammalian Chordates

Figure 4. A chimerical retrocopy in Western clawed frog. Red boxes represent exons of parental and retrocopy; light blue boxes represent
exons of chimeric gene, and blue lines represent introns.
doi:10.1371/journal.pone.0021466.g004

most of the retrocopies in the platypus genome are retro- [42]. These results, in conjunction with previous tests in Populus
pseudogenes (as many as 73%). [43], indicate that ‘‘out of the X’’ patterns are not detectable for
The duplicated retrocopies might be a result of ‘‘subfunctiona- the nascent sex chromosomal systems.
lization’’ [40]. Further analysis indicated that a higher fraction of Our observations also showed that the number of retrocopies of
the retrocopies was likely to be functional in the non-mammalian these chordates correlated with the number of LINE1 copies in
chordates than the in the two mammals studied, as supported by these species, suggesting an experimentally testable prediction: that
the analyses of evolutionary constraints and expression profiles. the retrocopies in the non-mammalian chordates may also be
Moreover, the number of functional retrogenes in the eight non- mainly produced by LINE1 elements as mammalian retrocopies
mammalian chordate species (excepting chicken and Tetraodon) are.
was close to the number of functional retrogenes in the human We identified nine chimerical genes in zebrafish and sixteen
genome, although the total number of retrocopies in these species chimerical genes in the Western clawed frog. The drastic changes
was found to be an order of magnitude lower than in human. in the protein structures in these genes likely brought up the novel
Retrogenes have evolved some common tissue-biased expres- functions, as has been previously observed in the Drosophila new
sion patterns. In general, they are preferentially expressed in the gene, jingwei [44]. This provides evidence that the non-mammals
testis, brain and ovary. Previous work has shown that retrogenes evolved under positive selection for new gene functionality.
tend to be expressed in the testis in both mammals and Drosophila This study identified large numbers of retrogenes in the non-
[1,2,15,16,18]. Our research shows that many retrogenes are mammalian chordates. Further investigation of these retrogenes
expressed in the testis not only in human, but also in Western revealed some common evolutionary patterns. A similar rate of
clawed frog, Medaka and Sea squirt. Two hypotheses could functional retrogene origination was found throughout the
explain this observation [14,39,41]. The first is that a hypertran- evolution of chordates, in spite of the fact that the processed
scription state exists in meiotic and postmeiotic spermatogenic pseudogenes evolved in diverse rates. Many retrogenes evolved
cells. This state allows the transcription of retrocopies in the testis gonad- and brain-based expression patterns. Moreover, we
that would not usually be transcribed. Some retrocopies then performed an analysis on two non-mammal species, the Western
acquire a beneficial function and evolve into functional retrogenes.
The second is that retrocopies are preferentially inserted into or
close to germline-expressed genes. The leaky expression of Table 6. The relationships between retrocopy number and
germline-expressed genes allows some retrogenes to be expressed the copy numbers of different kinds of LINE elements.
in the germline [14]. As in the testis, we also found that many
retrogenes were expressed in the brain, in accordance with
Species Retrocopies LINE1 LINE2 CR1 RTE
previous observation in primates [1].
In Medaka, the hypothetical ‘‘out of the X’’ movement was not Zebrafish 195 4653 54088 0 6105
observed. This result is consistent with the fact that the Western clawed 398 4074 0 73281 0
differentiation of the sex chromosomes in Medaka is primitive frog
Platypus 572 60 19109700 437600 856900

Table 5. Evidence of chimeric coding structure in Zebrafish, Medaka 218 698 0 0 29

Western clawed frog. Human 4738 516000 315000 0 0
Fugu 182 1411 13283 0 4150
Tetraodon 90 324 2043 0 1974
Chimeric
b Stickleback 132 16 0 0 1
Species retrogenes mRNA EST Merged Percentage
Sea squirt 110 7597 5007 0 0
Zebrafish 9 7a 1 8 89%
Chicken 78 0 10000 205000 0
Western clawed frog 16 7 1 8 50%
a b
significance p,0.001; NS NS NS
a
mRNA or EST sequences that span both recruited coding sequence and r = 0.994
retrosequence.
b a
the percentage of chimeric retrogenes with evidence of chimeric coding Pearson correlation test; r is correlation coefficient.
b
structure. Not Significant.
doi:10.1371/journal.pone.0021466.t005 doi:10.1371/journal.pone.0021466.t006

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 RNA-Based Duplication in Nonmammalian Chordates

clawed frog and zebrafish, and found sixteen and nine chimerical downloaded tissue information about the expressed functional
genes reside in their genomes, respectively. This may suggest that retrogenes from NCBI using Batch Entrez (http://www.ncbi.nlm.
the acquisition of drastically new protein functions accompany the nih.gov/). We downloaded Ensembl gene family information using
evolution of these chordate organisms. BIOMART (http://www.ensembl.org/).

Materials and Methods Chimeric retrogene screen

For the zebrafish and western clawed frog, there were abundant
Retrocopy identification mRNA and EST sequences that could be considered evidence of
To identify retrocopies in the twelve genomes studied (Table 1), chimeric structure, so we only identified chimeric retrocopies in
we adapted an approach previously used in humans [1]. All these two genomes. After we obtained the retrocopies, we
genome sequences and annotated protein datasets for these species compared the gene position of Ensembl annotated genes to our
except those for amphioxus were downloaded from Ensembl retrocopies and identified any overlapping pairs. Then we
(http://www.ensembl.org/). (For zebrafish and Medaka, the data performed a TBLASTN search using these Ensembl annotated
are release 50; humans, release 53; all others, release 52.) The genes as queries against overlapped retrocopies and their parental
amphioxus genome sequences were obtained from the website of genes. The Ensembl annotated genes with at least 30% coding
the Joint Genome Institute (http://genome.jgi-psf.org/). sequences that not matching the retrocopies or parental genes
For each species, a TBLASTN [45] analysis was performed (with flanking 50,000 bp) were regarded as chimeric retrogenes.
using all the protein sequences as queries against the whole-
genome sequences. Homologous HSPs (high-scoring segment
pairs) were chained together using a dynamic programming LINE elements and retrocopies numbers
algorithm. Homologous chains that had more than 60% alignable The LINE elements of the human and the platypus [35] were
regions and more than 40% identity to the query protein were obtained from published articles, and we performed a repeat
considered homologous genes. Using GeneWise [46], we identified analysis of the different chordate genomes using RepeatMasker
homologous genes without introns (or gaps more than 40 bp) from and the RepBase database [33]. To avoid false-positive LINE1
the exon coordinates as candidate genes. hits, a Smith–Waterman score of 250 was chosen as the cut-off
Next, all the candidate genes were aligned with all the Ensembl value.
proteins using FASTA [47]. We only retained those alignments with
.40% identity and an alignment length of at least 40 amino acids. Statistics
The candidate genes were regarded as candidate retrocopies if the In this study, we used Fisher’s exact test to determine whether
best hit was a gene with multiple coding exons (having introns larger an excess of intact retrocopies with Ka/Ks,0.5 or existed or were
than 70 bp). We then checked whether the introns of the parental expressed relative to retropseudogenes. Binary logistic regression
gene (the best hit) had been lost or retained in the retrocopies. If was used to determine whether there were more retrogenes
introns were retained, the retrocopy we identified may be false- expressed in the gonads or brain relative to other tissues. The
positive and should be discarded. To further reduce the number of Pearson correlation test was used to determine whether the
false-positive candidates, we removed candidate retrocopies with number of retrocopies correlated with different kinds of LINE
only one less intron than the parental gene. We also used elements. The expected number of retrogenes from the X
RepeatMasker to remove all candidates with more than 50% chromosome was determined according the method described
repeat elements. The identified retrocopies were further classified as by Vinckenbosch et al. [2].
intact retrocopies or retropseudogenes according to whether their
open reading frames were disrupted (by frameshift mutations or Supporting Information
premature stop codons) compared with those of the parental genes.
Figure S1
(PPT)
Ka and Ks estimation and functional retrogenes
The retrocopies were aligned with their parental genes. The Ka Table S1
and Ks substitution rates and the Ka/Ks ratios were calculated (XLS)
with KaKs_calculator_1.2 [48] using the LPB [49,50] method. We
Table S2
defined the intact retrocopies with Ka/Ks,0.5 (p,0.01) as
(XLS)
functional retrogenes via the codeml program in PAML3.14
[51,52]. This method compares a model in which Ka/Ks is fixed
to 0.5 (null model) to a model in which Ka/Ks is estimated from Acknowledgments
the data. Twice the log likelihood difference was compared to a x2 We thank Huifeng Jiang for technical help and Lei Yang for editing this
distribution with one degree of freedom. manuscript. Thanks are also extended to the Wuhan sub-center of
supercomputer environment, Chinese Academy of Science.
Expression and functional analyses
The expression data were downloaded from the UCSC (http:// Author Contributions
hgdownload.cse.ucsc.edu/downloads.html). Our retrocopy se- Conceived and designed the experiments: SH WW ML. Performed the
quences were then mapped onto them using BLAST. If a experiments: MC MZ BF XG DW MDV. Analyzed the data: MC MZ BF.
retrocopy had an overlap of more than 200 bp and more than Contributed reagents/materials/analysis tools: MC XL. Wrote the paper:
98% identity, we considered it to be expressed. We also MC.

References
1. Marques AC, Dupanloup I, Vinckenbosch N, Reymond A, Kaessmann H 2. Vinckenbosch N, Dupanloup I, Kaessmann H (2006) Evolutionary fate of
(2005) Emergence of young human genes after a burst of retroposition in retroposed gene copies in the human genome. Proc Natl Acad Sci U S A 103:
primates. PLoS Biol 3: e357. 3220–3225.

PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e21466

 RNA-Based Duplication in Nonmammalian Chordates

3. Emerson JJ, Kaessmann H, Betran E, Long MY (2004) Extensive gene traffic on and large barbus (Barbus intermedius) showing an unusual conservation of the
the mammalian X chromosome. Science 303: 537–540. peptide binding domains. J Immunol 169: 1936–1947.
4. Potrzebowski L, Vinckenbosch N, Marques AC, Chalmel F, Jegou B, et al. 28. Steinke D, Salzburger W, Meyer A (2006) Novel relationships among ten fish
(2008) Chromosomal gene movements reflect the recent origin and biology of model species revealed based on a phylogenomic analysis using ESTs. J Mol
therian sex chromosomes. PLoS Biol 6: e80. Evol 62: 772–784.
5. Brosius J (1991) Retroposons–seeds of evolution. Science 251: 753. 29. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, et al. (2004)
6. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, et al. (2001) The Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early
sequence of the human genome. Science. pp 1304–1351. vertebrate proto-karyotype. Nature 431: 946–957.
7. Torrents D, Suyama M, Zdobnov E, Bork P (2003) A genome-wide survey of 30. Esnault C, Maestre J, Heidmann T (2000) Human LINE retrotransposons
human pseudogenes. Genome Res 13: 2559–2567. generate processed pseudogenes. Nat Genet 24: 363–367.
8. Zhang Z, Harrison PM, Liu Y, Gerstein M (2003) Millions of years of evolution 31. Pickeral OK, Makalowski W, Boguski MS, Boeke JD (2000) Frequent human
preserved: a comprehensive catalog of the processed pseudogenes in the human genomic DNA transduction driven by LINE-1 retrotransposition. Genome Res
genome. Genome Res 13: 2541–2558. 10: 411–415.
9. Betran E, Emerson JJ, Kaessmann H, Long M (2004) Sex chromosomes and 32. Wei W, Gilbert N, Ooi SL, Lawler JF, Ostertag EM, et al. (2001) Human L1
male functions: where do new genes go? Cell Cycle 3: 873–875. retrotransposition: cis preference versus trans complementation. Mol Cell Biol
10. Wang W, Zheng H, Fan C, Li J, Shi J, et al. (2006) High rate of chimeric gene 21: 1429–1439.
origination by retroposition in plant genomes. Plant Cell 18: 1791–1802. 33. Jurka J (1998) Repeats in genomic DNA: mining and meaning. Curr Opin
11. Hillier LW, Miller W, Birney E, Warren W, Hardison RC, et al. (2004) Struct Biol 8: 333–337.
Sequence and comparative analysis of the chicken genome provide unique 34. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, et al. (2001) Initial
perspectives on vertebrate evolution. Nature 432: 695–716. sequencing and analysis of the human genome. Nature 409: 860–921.
12. Haas NB, Grabowski JM, Sivitz AB, Burch JB (1997) Chicken repeat 1 (CR1) 35. Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP, et al.
elements, which define an ancient family of vertebrate non-LTR retro- (2008) Genome analysis of the platypus reveals unique signatures of evolution.
transposons, contain two closely spaced open reading frames. Gene 197: Nature 453: 175–183.
305–309. 36. Pan D, Zhang L (2009) Burst of young retrogenes and independent retrogene
13. Haas NB, Grabowski JM, North J, Moran JV, Kazazian HH, et al. (2001) formation in mammals. PLoS ONE 4: e5040.
Subfamilies of CR1 non-LTR retrotransposons have different 59UTR sequences 37. Petrov DA, Sangster TA, Johnston JS, Hartl DL, Shaw KL (2000) Evidence for
but are otherwise conserved. Gene 265: 175–183. DNA loss as a determinant of genome size. Science 287: 1060–1062.
14. Kaessmann H, Vinckenbosch N, Long M (2009) RNA-based gene duplication: 38. Petrov DA (2002) Mutational equilibrium model of genome size evolution.
mechanistic and evolutionary insights. Nat Rev Genet 10: 19–31. Theor Popul Biol 61: 531–544.
15. Betran E, Thornton K, Long M (2002) Retroposed new genes out of the X in 39. Fontanillas P, Hartl DL, Reuter M (2007) Genome organization and gene
Drosophila. Genome Res 12: 1854–1859. expression shape the transposable element distribution in the Drosophila
16. Dai H, Yoshimatsu TF, Long M (2006) Retrogene movement within- and melanogaster euchromatin. PLoS Genet 3: e210.
between-chromosomes in the evolution of Drosophila genomes. Gene 385: 40. Force A, Lynch M, Pickett FB, Amores A, Yan YL, et al. (1999) Preservation of
96–102. duplicate genes by complementary, degenerative mutations. Genetics 151:
17. Bai Y, Casola C, Feschotte C, Betran E (2007) Comparative genomics reveals a 1531–1545.
constant rate of origination and convergent acquisition of functional retrogenes 41. Kleene KC (2001) A possible meiotic function of the peculiar patterns of gene
in Drosophila. Genome Biol 8: R11. expression in mammalian spermatogenic cells. Mech Dev 106: 3–23.
18. Long M, Langley CH (1993) Natural selection and the origin of jingwei, a 42. Kasahara M, Naruse K, Sasaki S, Nakatani Y, Qu W, et al. (2007) The medaka
chimeric processed functional gene in Drosophila. Science 260: 91–95. draft genome and insights into vertebrate genome evolution. Nature 447:
19. Rychel AL, Smith SE, Shimamoto HT, Swalla BJ (2006) Evolution and 714–719.
development of the chordates: collagen and pharyngeal cartilage. Mol Biol Evol 43. Zhu Z, Zhang Y, Long M (2009) Extensive structural renovation of retrogenes in
23: 541–549. the evolution of the Populus genome. Plant Physiol 151: 1943–1951.
20. Fu B, Chen M, Zou M, Long M, He S (2010) The rapid generation of chimerical 44. Zhang J, Dean AM, Brunet F, Long M (2004) Evolving protein functional
genes expanding protein diversity in zebrafish. BMC Genomics 11: 657. diversity in new genes of Drosophila. Proc Natl Acad Sci U S A 101:
21. Aida T (1921) On the Inheritance of Color in a Fresh-Water Fish, 16246–16250.
APLOCHEILUS LATIPES Temmick and Schlegel, with Special Reference 45. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped
to Sex-Linked Inheritance. Genetics 6: 554–573. BLAST and PSI-BLAST: a new generation of protein database search
22. Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, et al. (2002) DMY programs. Nucleic Acids Res 25: 3389–3402.
is a Y-specific DM-domain gene required for male development in the medaka 46. Birney E, Clamp M, Durbin R (2004) GeneWise and Genomewise. Genome Res
fish. Nature 417: 559–563. 14: 988–995.
23. Charlesworth B (1991) The evolution of sex chromosomes. Science 251: 47. Pearson WR, Wood T, Zhang Z, Miller W (1997) Comparison of DNA
1030–1033. sequences with protein sequences. Genomics 46: 24–36.
24. Yi S, Ellsworth DL, Li WH (2002) Slow molecular clocks in Old World 48. Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, et al. (2006) KaKs_Calculator:
monkeys, apes, and humans. Mol Biol Evol 19: 2191–2198. calculating Ka and Ks through model selection and model averaging. Genomics
25. Evans BJ, Kelley DB, Tinsley RC, Melnick DJ, Cannatella DC (2004) A Proteomics Bioinformatics 4: 259–263.
mitochondrial DNA phylogeny of African clawed frogs: phylogeography and 49. Li WH (1993) Unbiased estimation of the rates of synonymous and
implications for polyploid evolution. Mol Phylogenet Evol 33: 197–213. nonsynonymous substitution. J Mol Evol 36: 96–99.
26. Morin RD, Chang E, Petrescu A, Liao N, Griffith M, et al. (2006) Sequencing 50. Pamilo P, Bianchi NO (1993) Evolution of the Zfx and Zfy genes: rates and
and analysis of 10,967 full-length cDNA clones from Xenopus laevis and interdependence between the genes. Mol Biol Evol 10: 271–281.
Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling. 51. Yang Z (1997) PAML: a program package for phylogenetic analysis by
Genome Res 16: 796–803. maximum likelihood. Comput Appl Biosci 13: 555–556.
27. Kruiswijk CP, Hermsen TT, Westphal AH, Savelkoul HF, Stet RJ (2002) A 52. Yang Z (1998) Likelihood ratio tests for detecting positive selection and
novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), application to primate lysozyme evolution. Mol Biol Evol 15: 568–573.

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