BACKGROUND
genes that are unnecessary for existence in a new environment are often eliminated during the course of evolution, but genomes also acquire novel genetic elements as a source of functional and phenotypic diversity  <cit> .
retrocopies, one type of novel genetic element, are genome segments that are reverse-transcribed from intronless mrna and then inserted into new positions in the genome   <cit> .
retrocopies have long been considered evolutionary dead ends, because it was expected that these segments lack regulatory elements and originate from rna-based duplication  <cit> .
however, many recent studies revealed that retrocopies can be used to generate new genes, called retrogenes, by fusion with other genes  or via acquisition of new exons or introns from de novo genome sequences  <cit> .
furthermore, retrogenes, which are functional retrocopies, have often evolved functional roles in male germ lines  <cit> .
thus, “dead on arrival” copies of parental genes are currently considered to serve as “seeds of evolution”  <cit> .
despite the substantial contribution to molecular evolution revealed by studies on retrocopies in humans and fruit flies , the patterns of retrocopy formation and effects on genomic dynamics of lower vertebrates remain undetermined.
the african coelacanth is the second closest living fish relative of tetrapods.
thus, genomic data on this species, which have recently been made available, are useful for researching successful land adaptations during vertebrate evolution  <cit> .
the african coelacanth is well known for its morphological resemblance to its fossil ancestors; as anticipated, protein-coding sequence evolution in the coelacanth genome was shown to be relatively slow  <cit> .
because retrogenes can serve as “seeds of evolution,” investigation into the pattern and rate of retrogene formation and implications of the role of these retrogenes in the african coelacanth genome can provide novel insight into coelacanth evolution and water-to-land vertebrate adaptations.
in this study, we identified and investigated  <dig> parent-retrocopy pairs in the african coelacanth genome.
based on the obtained results, we suggest that retrocopies attained biological relevance and were subject to natural selection following fusion with existing genes or acquisition of new gene elements, although many retrocopies have ultimately been eliminated from the genome during coelacanth evolution.
analysis of these potentially functional retrocopies  provides new insight into the dynamics of functional organization of the genome during coelacanth evolution and vertebrate transition from water to land.
RESULTS
retrocopies and potential retrogenes in the african coelacanth genome
although retrocopies were long thought to be pseudogenes, it is now known that many are functional and considered retrogenes following fusion with other genes  <cit>  or acquisition of regulatory elements and exons from de novo sequences  <cit> .
to evaluate whether a retrocopy is functional, multiple criteria, such as a dn/ds ratio < <dig>  or transcriptional evidence, were developed in previous studies .
in this study,  <dig> one-to-one parent-retrocopy pairs were identified in the african coelacanth genome .
to assess retrocopy functionality, we first compared the sequences of each retrocopy and its parent gene, which revealed a total of  <dig> intact retrocopies with no frameshift mutations or premature stop codons .
these retrocopies possess intact orfs  and thus have the potential to become functional protein sequences  <cit> .
additionally,  <dig> retrocopies with a dn/ds ratio significantly < <dig>  were also identified .
dn/ds < <dig>  is indicative of purifying selection, which reflects potential for retrocopy functionality  <cit> .
second, we mapped retrocopies to ensembl genes  to detect retrocopies showed overlap with ensembl genes.
in this step,  <dig> retrocopies were detected, which included  <dig> exon-overlapping retrocopies and  <dig> non-exon-overlapping retrocopies .
these retrocopies may represent the main direct way by which retrocopies can influnce the genome, fusing with an existing gene, or acquiring new gene elements from a de novo sequence  <cit> .
finally, we mapped the rna-seq data  accessions drp <dig> and srx189186; see methods) to the african coelacanth genome and identified  <dig> retrocopies with transcriptional evidence  values, additional file 1: table s3).fig.
<dig> venn diagram of the unique and common retrocopies among different categories.
red refers to retrocopies that overlap with ensembl genes.
yellow refers to retrocopies with intact orfs.
green refers to retrocopies with evidence of transcription.
blue refers to retrocopies with dn/ds ratios significantly < <dig>
the common and unique retrocopies identified by the previously discussed different criteria are summarized in fig.
<dig>  pearson chi-square tests revealed whether an expressed retrocopy  was intact, showed a dn/ds value < <dig> , or overlapped with an ensembl gene .
these results indicate that the analyzed retrocopies met the criteria for functional retrocopies.
moreover,  <dig> retrocopies were identified by at least one of the four criteria, and  <dig> retrocopies were identified by all criteria; this indicates that 85– <dig> retrocopies are potential retrogenes .
retrocopy age distribution
to investigate the origin and evolution of these retrocopies, we assessed the retrocopy age distribution based on the increase in ds values, which were estimated by comparing the parent genes and retrocopies.
the distribution revealed a burst pattern of young retrocopies .
notably, similar age distribution patterns of retrocopies were revealed in humans, platypuses, and western clawed frogs in a previous study  <cit> .
however, this pattern was not generated in ray-finned fish, including sticklebacks, zebrafish, medaka, tetraodon, and fugu  <cit> .
moreover, the associated dn/ds ratios with the retrocopies tended to decrease as ds increased , which indicates a greater constraint of selection on older retrocopies.fig.
<dig> the ds distribution of the  <dig> retrocopies in the genome of the african coelacanth
fig.
<dig> dot plot of the dn/ds ratios with the ds values for each of the  <dig> retrocopies
to assess the proportions of retrogenes with different age ranks, we subdivided the retrocopies into bins based on ds intervals and then estimated the percentage of functional retrocopies for each bin.
regardless of the frequency burst of young retrocopies, the retrocopy bins with lower ds values  tended to contain a smaller percentage of retrocopies that exhibited intact orfs, dn/ds ratios significantly < <dig> , overlap with ensembl genes, or expression compared with those that had elevated ds values  .
additionally, estimation of the mean log <dig> values for the retrocopies in each ds bin showed that elevated mean values tended to be associated with relatively old retrocopies .
finally, the “expression frequency” was considered the number of tissues that expressed a specific retrocopy, and an elevated expression frequency tended to be associated with relatively old retrocopies .fig.
<dig> percentages of retrocopies with different indications of functionality in each ds bin, distributed according to the increase in ds values.
the red line corresponds to the right y-axis and represents the number of retrocopies.
the lines in black, grey, blue and green correspond to the left y-axis and represent the percentages of retrocopies showing intact orfs, dn/ds ratios significantly < <dig> ,overlap with ensembl genes and transcriptional evidence, respectively
fig.
<dig> mean expression levels of retrocopies in different ds bins.
expression levels were measured as log values
fig.
<dig> mean expression frequency for retrocopies in different ds bins
expression patterns
a total of  <dig> retrocopies showed evidence of expression when we mapped the rna-seq data to the african coelacanth genome.
.
in this study, we refer to the “expression frequency” as the number of tissues showing expression of a retrocopy; retrocopies with higher expression frequencies  tended to exhibit higher log values for each tissue  and higher ds values , but lower dn/ds ratios  than the retrocopies with lower expression frequencies .
young retrocopies were more likely than old retrocopies to be expressed with lower fpkm values and lower expression frequencies .table  <dig> mean and median value for ds, dn/ds and log of retrocopies with different express frequency
alog per retrocopy per tissue
in previous studies that examined retrocopies in humans and fruit flies  <cit> , the preferential retrogene expression showed bias in the testis.
in this study,  <dig> of the  <dig> retrocopies were expressed in the testis and were the most overrepresented retrocopies expressed in tissues .
additionally, the retrocopies expressed in the testis was also the most overrepresented of retrocopies that were uniquely expressed in one tissue , which indicates that this expression pattern bias also occurs in the coelacanth.
however, despite the overrepresentation of retrocopy expression in the testis, no ds value or dn/ds ratio bias was detected in the retrocopy expression patterns in any tissue .
retrocopy-overlapped genes
the primary mechanisms by which a retrocopy can be transcribed and become functional are fusion  with an existing gene or acquisition of new gene elements from a de novo sequence  <cit> .
in this study,  <dig> retrocopies overlapped with  <dig> coelacanth ensembl genes.
we defined the  <dig> ensembl genes as “retrocopy-overlapped genes” , and these ensembl genes might harbor inserted retrocopies or have originated from the de novo exon/intron acquisition of retrocopies .
these retrocopy-overlapped genes  might represent the most direct pathway for retrocopies to influence the genome dynamics of the coelacanth.
to further examine this direct influence, we compared the go enrichment results for the retrocopy parent genes with those of the  <dig> retrocopy-overlapped genes.
the results revealed that the gene functions that were overrepresented in the parent genes were most likely related to the synthesis and metabolism of biological molecules, whereas those in the retrocopy-overlapped genes were most likely related to transmembrane transport .
this result might indicate functional organization of the genome during coelacanth evolution, which was most likely produced by retrotransposition.table  <dig> comparison of over-represented go terms between parent genes of the retrocopies and the retrocopy-overlapped genes
to further determine retrocopy influence on coelacanth and lower vertebrate evolution, we investigated potential orthologous sequences of the retrocopy-overlapped genes in ray-finned fish, including elephant sharks, fugu, zebrafish, gar, and some tetrapods, such as clawed frogs, anole lizards, zebra finches, mice, and humans.
in particular, we focused on two retrocopy groups: those that were coelacanth-specific and those that were lost in tetrapods.
a total of  <dig> retrocopy-overlapped genes were identified as new retrogenes or lineage-specific genes for the coelacanth, and no orthologous sequence was found in any of the species investigated.
these new genes most likely originated recently through the de novo acquisition of gene elements from retrocopies and not from host gene-retrocopy fusion.
thus, within the approximately  <dig> my coelacanth evolutionary history  <cit> , the rate of new retrogene formation has been relatively slow compared with that in humans   <cit> .
this result is consistent with the morphological resemblance and slow pace of evolution of protein-coding sequences in coelacanth genome.
comparison of new retrogenes with the remainder of retrocopy-overlapped genes showed that these genes tended to contain fewer exons, were more likely overlap at an exon site, and were more underexpressed .
additionally, the retrocopies that were mapped to these genes tended to be younger, have a lower expression frequency, and were less constrained by natural selection .
the go analysis indicated that the overrepresented functional categories were most likely related to cell response, modification of biomolecules, and the immune function .
in addition to the  <dig> coelacanth-specific retrocopy-overlapped genes, we identified  <dig> genes that were specifically lost in tetrapods .
this indicates that these genes might be unnecessary for living on land.
in previous studies on these genes using zebrafish data , some associations with important developmental categories were reported , including caudal fin development, eye morphology, vasculature development, and nervous system development  <cit> .
compared with the retrocopy-overlapped genes that still exist in tetrapod lineages, the  <dig> specifically lost genes did not show any difference in genetic structure or expression.
however, the “lost” retrocopy-overlapped genes had lower dn/ds values than those that were retained, which indicates different selection pressures .
comparison of the go enrichment results showed that the overrepresented functional categories for the lost genes were most likely to be related to the nervous system, whereas those in the “retained” gene set were most likely related to circadian rhythms and ionic transmembrane transport .
discussion
we explored the evolutionary fate and implications of retrocopies in the coelacanth genome and provided a novel perspective to understand the evolution of lower vertebrates and their adaptations in the transition from water to land.
in total, we screened  <dig> retrocopies in the african coelacanth genome.
the age distribution of the retrocopies based on obtained ds values showed a burst pattern of young retrocopies that accumulated in the genome .
notably, similar patterns for retrocopy age distribution were found in humans, platypuses, and western clawed frogs, but not ray-finned fish  <cit> .
this finding shows that the coelacanth’s birth pattern of retrocopies is more similar to that of tetrapods than that of ray-finned fish, which indicates that this pattern change might be related to the vertebrate evolutionary transition from water to land.
because retropositions require rna as a mediator, reverse transcriptase that stems from retrotransposons might be also necessary for retropositions.
reverse transcriptase encoded by long interspersed nuclear elements , which possess endonucleolytic activity that can recognize any polyadenylated mrna, seems to be responsible for retrotranspositions in mammals  <cit> .
additionally, as previously demonstrated  <cit> , the line- <dig>  element subfamily of lines can generate processed genes, which indicates that l <dig> retrotransposon activity has generated retroposed gene copies in mammals  <cit> .
as in non-mammalian chordates, it was also reported that the number of retrocopies correlated with the number of l <dig> copies, but not with any other type of line element, such as l <dig>  <cit> .
additionally, the difference between retrocopy birth pattern ray-finned fish and that in tetrapods coincides with the dramatic difference in l <dig> retrotransposon diversity between mammals and fish  <cit> .
these results indicate that l <dig> may also be responsible for the retrotranspositions in non-mammalian chordates and represent a genetic signal that reflects the vertebrate evolutionary transition from water to land.
despite the burst pattern of young retrocopies that accumulated in the genome, retrocopies with indications of function  were much more overrepresented among the old than young retrocopies .
the overrepresentation of the retrogenes among the old retrocopies and decrease in frequency of relatively old retrocopies indicated that many of the retrocopies might have been eliminated during evolution, which was consistent with our predictions based on the expected lack of regulatory elements and introns in the retrocopies.
such features clearly indicate a course of rapid diversity within the evolutionary fate of the retrocopies before their elimination or incorporation into a pathway.
however, the retrocopies could be affected by natural selection associated with the host genes in which the retrocopies are inserted, or the retrocopies could be constrained by natural selection after the acquisition of exons/new regulatory elements .
such retrocopies might attain biological relevance and become evolutionarily stable and, thus, ultimately be overrepresented as other unstable retrocopies are eliminated.
this hypothesis was also supported by our analysis of retrocopy-overlapped genes.
the newly originated coelacanth-specific retrocopy-overlapped genes were younger than the other retrocopy-overlapped genes.
as indicated by their lower expression level , the younger genes formed from the retrocopies might be less evolutionarily stable compared with the other retrocopy-overlapped genes.
moreover, these newly formed genes tended to contain fewer exons , which indicated that the new retrogenes might continue to recruit exons or introns during their course of evolution, or, as a complementary mechanism, these retrocopies might be preferentially inserted into host genes with more exons.
as previously reported, the retrogenes might be preferentially expressed in the testis.
in this study, more retrocopies were expressed in the testis than in other tissues.
however, no ds or dn/ds bias, which reflects retrocopy age range, was associated with any tissue-specific retrocopies .
moreover, expression pattern analysis revealed that older retrocopies tended to be expressed with higher fpkm values and in more tissues than the young retrocopies .
the association of increased retrocopy expression distribution among tissues with increased age indicated that retrocopy biological functions might evolve to become essential over time  <cit> .
the most direct ways in which a retrocopy can affect the genome are through retrocopy insertion into a gene and acquisition of new gene elements by the retrocopy.
we identified  <dig> retrocopy-overlapped genes.
go enrichment analysis revealed that the overrepresented go terms differed between the  <dig> retrocopy-overlapped genes and the parent genes from which those retrocopies originated.
the gene functions that were overrepresented in the parent genes were most likely related to the synthesis and metabolism of biological molecules, whereas those of the  <dig> retrocopy-overlapped genes were most likely related to transmembrane transport.
by retrotransposition of a father gene’s mrna copy insertion into a gene, these father genes may contribute to the evolution of retrocopy-overlapped genes, whose functional organization differs from that of the father’s gene.
this result indicates that retrotranspositions may contribute to functional organization dynamics of the genome.
among the  <dig> retrocopy-overlapped genes,  <dig> genes that were specifically lost in the tetrapod lineage were identified, including myh <dig>  which is related to eye morphology; ntn <dig>  which is related to olfactory processes; and esrrga, which is related to caudal fin malformations .
comparisons between these genes and the retrocopy-overlapped genes with orthologous sequences in all of the investigated tetrapods revealed no differences in gene structure or expression level; however, as predicted from the retrocopies with which they overlapped, these lost genes tended to be more constrained by natural selection in the coelacanth genome.
this result indicates that the genes were not lost in the tetrapod lineage because of any structural defect, but because during the adaptation of vertebrates from water to land, these genes were not necessary.
furthermore, the overrepresentation of functional categories related to the nervous system in these genes might indicate that, unlike the system of ionic homeostasis regulation , the sensory system of tetrapods may have experienced remodeling, which modified it from the system observed in fish.
the birth of new genes can contribute to the formation of lineage- or species-specific genes  <cit> , as found in this study.
we identified  <dig> retrocopy-overlapped genes that were specific to coelacanths.
the overrepresented gene functions related to the immune function indicated that the immune system was reinforced during coelacanth evolution .
additionally, the slow rate of new retrogene formation corresponded to the slow evolution of protein-coding genes in the coelacanth genome  <cit> , which provided novel insight into the morphological resemblance of the coelacanth to its fossil ancestors.
together, these results might indicate stability of the deep sea environment in which the coelacanth evolved.
CONCLUSIONS
our study revealed a burst pattern of young retrocopies in the coelacanth genome.
this pattern is similar to that in tetrapods rather than that in ray-finned fish, which indicates a possible genomic change related to water-to-land adaptations.
many retrocopies may have been eliminated during coelacanth genome evolution because of disrupted genetic structure defects.
however, some might have been randomly inserted into existing genes or acquired regulatory elements, exons, or introns from de novo genetic sequences that facilitated overcoming of these defects and acquiring functions .
these retrogenes were revealed to have an effect on functional organization of the genome, which provides novel insight into coelacanth evolution and the transformations involved in the transition from water to land.
however, the results of this study were only sufficient for revealing some of the effects of retrotransposition on the genome; the response of the host gene to retrocopy insertion remains obscure, and retrocopies may function as noncoding rnas that do not demand protein coding gene’s structures   <cit> .
thus, our studies in the future will further explore these issues.

 genes that are unnecessary for existence in a new environment are often eliminated during the course of evolution, but genomes also acquire novel genetic elements as a source of functional and phenotypic diversity. retrocopies, one type of novel genetic element, are genome segments that are reverse-transcribed from intronless mrna and then inserted into new positions in the genome. retrocopies have long been considered evolutionary dead ends, because it was expected that these segments lack regulatory elements and originate from rna-based duplication. however, many recent studies revealed that retrocopies can be used to generate new genes, called retrogenes, by fusion with other genes or via acquisition of new exons or introns from de novo genome sequences. furthermore, retrogenes, which are functional retrocopies, have often evolved functional roles in male germ lines. thus, “dead on arrival” copies of parental genes are currently considered to serve as “seeds of evolution”. despite the substantial contribution to molecular evolution revealed by studies on retrocopies in humans and fruit flies, the patterns of retrocopy formation and effects on genomic dynamics of lower vertebrates remain undetermined. the african coelacanth is the second closest living fish relative of tetrapods. thus, genomic data on this species, which have recently been made available, are useful for researching successful land adaptations during vertebrate evolution. the african coelacanth is well known for its morphological resemblance to its fossil ancestors; as anticipated, protein-coding sequence evolution in the coelacanth genome was shown to be relatively slow. because retrogenes can serve as “seeds of evolution,” investigation into the pattern and rate of retrogene formation and implications of the role of these retrogenes in the african coelacanth genome can provide novel insight into coelacanth evolution and water-to-land vertebrate adaptations. in this study, we identified and investigated parent-retrocopy pairs in the african coelacanth genome. based on the obtained results, we suggest that retrocopies attained biological relevance and were subject to natural selection following fusion with existing genes or acquisition of new gene elements, although many retrocopies have ultimately been eliminated from the genome during coelacanth evolution. analysis of these potentially functional retrocopies provides new insight into the dynamics of functional organization of the genome during coelacanth evolution and vertebrate transition from water to land.  retrocopies and potential retrogenes in the african coelacanth genome although retrocopies were long thought to be pseudogenes, it is now known that many are functional and considered retrogenes following fusion with other genes or acquisition of regulatory elements and exons from de novo sequences. to evaluate whether a retrocopy is functional, multiple criteria, such as a dn/ds ratio < or transcriptional evidence, were developed in previous studies. in this study, one-to-one parent-retrocopy pairs were identified in the african coelacanth genome. to assess retrocopy functionality, we first compared the sequences of each retrocopy and its parent gene, which revealed a total of intact retrocopies with no frameshift mutations or premature stop codons. these retrocopies possess intact orfs and thus have the potential to become functional protein sequences. additionally, retrocopies with a dn/ds ratio significantly < were also identified. dn/ds < is indicative of purifying selection, which reflects potential for retrocopy functionality. second, we mapped retrocopies to ensembl genes to detect retrocopies showed overlap with ensembl genes. in this step, retrocopies were detected, which included exon-overlapping retrocopies and non-exon-overlapping retrocopies. these retrocopies may represent the main direct way by which retrocopies can influnce the genome, fusing with an existing gene, or acquiring new gene elements from a de novo sequence. finally, we mapped the rna-seq data accessions drp and srx189186; see methods) to the african coelacanth genome and identified retrocopies with transcriptional evidence values, additional file 1: table s3).fig. venn diagram of the unique and common retrocopies among different categories. red refers to retrocopies that overlap with ensembl genes. yellow refers to retrocopies with intact orfs. green refers to retrocopies with evidence of transcription. blue refers to retrocopies with dn/ds ratios significantly < the common and unique retrocopies identified by the previously discussed different criteria are summarized in fig.  pearson chi-square tests revealed whether an expressed retrocopy was intact, showed a dn/ds value <, or overlapped with an ensembl gene. these results indicate that the analyzed retrocopies met the criteria for functional retrocopies. moreover, retrocopies were identified by at least one of the four criteria, and retrocopies were identified by all criteria; this indicates that 85– retrocopies are potential retrogenes. retrocopy age distribution to investigate the origin and evolution of these retrocopies, we assessed the retrocopy age distribution based on the increase in ds values, which were estimated by comparing the parent genes and retrocopies. the distribution revealed a burst pattern of young retrocopies. notably, similar age distribution patterns of retrocopies were revealed in humans, platypuses, and western clawed frogs in a previous study. however, this pattern was not generated in ray-finned fish, including sticklebacks, zebrafish, medaka, tetraodon, and fugu. moreover, the associated dn/ds ratios with the retrocopies tended to decrease as ds increased, which indicates a greater constraint of selection on older retrocopies.fig. the ds distribution of the retrocopies in the genome of the african coelacanth fig. dot plot of the dn/ds ratios with the ds values for each of the retrocopies to assess the proportions of retrogenes with different age ranks, we subdivided the retrocopies into bins based on ds intervals and then estimated the percentage of functional retrocopies for each bin. regardless of the frequency burst of young retrocopies, the retrocopy bins with lower ds values tended to contain a smaller percentage of retrocopies that exhibited intact orfs, dn/ds ratios significantly <, overlap with ensembl genes, or expression compared with those that had elevated ds values. additionally, estimation of the mean log values for the retrocopies in each ds bin showed that elevated mean values tended to be associated with relatively old retrocopies. finally, the “expression frequency” was considered the number of tissues that expressed a specific retrocopy, and an elevated expression frequency tended to be associated with relatively old retrocopies.fig. percentages of retrocopies with different indications of functionality in each ds bin, distributed according to the increase in ds values. the red line corresponds to the right y-axis and represents the number of retrocopies. the lines in black, grey, blue and green correspond to the left y-axis and represent the percentages of retrocopies showing intact orfs, dn/ds ratios significantly <,overlap with ensembl genes and transcriptional evidence, respectively fig. mean expression levels of retrocopies in different ds bins. expression levels were measured as log values fig. mean expression frequency for retrocopies in different ds bins expression patterns a total of retrocopies showed evidence of expression when we mapped the rna-seq data to the african coelacanth genome.. in this study, we refer to the “expression frequency” as the number of tissues showing expression of a retrocopy; retrocopies with higher expression frequencies tended to exhibit higher log values for each tissue and higher ds values, but lower dn/ds ratios than the retrocopies with lower expression frequencies. young retrocopies were more likely than old retrocopies to be expressed with lower fpkm values and lower expression frequencies.table mean and median value for ds, dn/ds and log of retrocopies with different express frequency alog per retrocopy per tissue in previous studies that examined retrocopies in humans and fruit flies, the preferential retrogene expression showed bias in the testis. in this study, of the retrocopies were expressed in the testis and were the most overrepresented retrocopies expressed in tissues. additionally, the retrocopies expressed in the testis was also the most overrepresented of retrocopies that were uniquely expressed in one tissue, which indicates that this expression pattern bias also occurs in the coelacanth. however, despite the overrepresentation of retrocopy expression in the testis, no ds value or dn/ds ratio bias was detected in the retrocopy expression patterns in any tissue. retrocopy-overlapped genes the primary mechanisms by which a retrocopy can be transcribed and become functional are fusion with an existing gene or acquisition of new gene elements from a de novo sequence. in this study, retrocopies overlapped with coelacanth ensembl genes. we defined the ensembl genes as “retrocopy-overlapped genes”, and these ensembl genes might harbor inserted retrocopies or have originated from the de novo exon/intron acquisition of retrocopies. these retrocopy-overlapped genes might represent the most direct pathway for retrocopies to influence the genome dynamics of the coelacanth. to further examine this direct influence, we compared the go enrichment results for the retrocopy parent genes with those of the retrocopy-overlapped genes. the results revealed that the gene functions that were overrepresented in the parent genes were most likely related to the synthesis and metabolism of biological molecules, whereas those in the retrocopy-overlapped genes were most likely related to transmembrane transport. this result might indicate functional organization of the genome during coelacanth evolution, which was most likely produced by retrotransposition.table comparison of over-represented go terms between parent genes of the retrocopies and the retrocopy-overlapped genes to further determine retrocopy influence on coelacanth and lower vertebrate evolution, we investigated potential orthologous sequences of the retrocopy-overlapped genes in ray-finned fish, including elephant sharks, fugu, zebrafish, gar, and some tetrapods, such as clawed frogs, anole lizards, zebra finches, mice, and humans. in particular, we focused on two retrocopy groups: those that were coelacanth-specific and those that were lost in tetrapods. a total of retrocopy-overlapped genes were identified as new retrogenes or lineage-specific genes for the coelacanth, and no orthologous sequence was found in any of the species investigated. these new genes most likely originated recently through the de novo acquisition of gene elements from retrocopies and not from host gene-retrocopy fusion. thus, within the approximately my coelacanth evolutionary history, the rate of new retrogene formation has been relatively slow compared with that in humans. this result is consistent with the morphological resemblance and slow pace of evolution of protein-coding sequences in coelacanth genome. comparison of new retrogenes with the remainder of retrocopy-overlapped genes showed that these genes tended to contain fewer exons, were more likely overlap at an exon site, and were more underexpressed. additionally, the retrocopies that were mapped to these genes tended to be younger, have a lower expression frequency, and were less constrained by natural selection. the go analysis indicated that the overrepresented functional categories were most likely related to cell response, modification of biomolecules, and the immune function. in addition to the coelacanth-specific retrocopy-overlapped genes, we identified genes that were specifically lost in tetrapods. this indicates that these genes might be unnecessary for living on land. in previous studies on these genes using zebrafish data, some associations with important developmental categories were reported, including caudal fin development, eye morphology, vasculature development, and nervous system development. compared with the retrocopy-overlapped genes that still exist in tetrapod lineages, the specifically lost genes did not show any difference in genetic structure or expression. however, the “lost” retrocopy-overlapped genes had lower dn/ds values than those that were retained, which indicates different selection pressures. comparison of the go enrichment results showed that the overrepresented functional categories for the lost genes were most likely to be related to the nervous system, whereas those in the “retained” gene set were most likely related to circadian rhythms and ionic transmembrane transport. discussion we explored the evolutionary fate and implications of retrocopies in the coelacanth genome and provided a novel perspective to understand the evolution of lower vertebrates and their adaptations in the transition from water to land. in total, we screened retrocopies in the african coelacanth genome. the age distribution of the retrocopies based on obtained ds values showed a burst pattern of young retrocopies that accumulated in the genome. notably, similar patterns for retrocopy age distribution were found in humans, platypuses, and western clawed frogs, but not ray-finned fish. this finding shows that the coelacanth’s birth pattern of retrocopies is more similar to that of tetrapods than that of ray-finned fish, which indicates that this pattern change might be related to the vertebrate evolutionary transition from water to land. because retropositions require rna as a mediator, reverse transcriptase that stems from retrotransposons might be also necessary for retropositions. reverse transcriptase encoded by long interspersed nuclear elements, which possess endonucleolytic activity that can recognize any polyadenylated mrna, seems to be responsible for retrotranspositions in mammals. additionally, as previously demonstrated, the line- element subfamily of lines can generate processed genes, which indicates that l retrotransposon activity has generated retroposed gene copies in mammals. as in non-mammalian chordates, it was also reported that the number of retrocopies correlated with the number of l copies, but not with any other type of line element, such as l. additionally, the difference between retrocopy birth pattern ray-finned fish and that in tetrapods coincides with the dramatic difference in l retrotransposon diversity between mammals and fish. these results indicate that l may also be responsible for the retrotranspositions in non-mammalian chordates and represent a genetic signal that reflects the vertebrate evolutionary transition from water to land. despite the burst pattern of young retrocopies that accumulated in the genome, retrocopies with indications of function were much more overrepresented among the old than young retrocopies. the overrepresentation of the retrogenes among the old retrocopies and decrease in frequency of relatively old retrocopies indicated that many of the retrocopies might have been eliminated during evolution, which was consistent with our predictions based on the expected lack of regulatory elements and introns in the retrocopies. such features clearly indicate a course of rapid diversity within the evolutionary fate of the retrocopies before their elimination or incorporation into a pathway. however, the retrocopies could be affected by natural selection associated with the host genes in which the retrocopies are inserted, or the retrocopies could be constrained by natural selection after the acquisition of exons/new regulatory elements. such retrocopies might attain biological relevance and become evolutionarily stable and, thus, ultimately be overrepresented as other unstable retrocopies are eliminated. this hypothesis was also supported by our analysis of retrocopy-overlapped genes. the newly originated coelacanth-specific retrocopy-overlapped genes were younger than the other retrocopy-overlapped genes. as indicated by their lower expression level, the younger genes formed from the retrocopies might be less evolutionarily stable compared with the other retrocopy-overlapped genes. moreover, these newly formed genes tended to contain fewer exons, which indicated that the new retrogenes might continue to recruit exons or introns during their course of evolution, or, as a complementary mechanism, these retrocopies might be preferentially inserted into host genes with more exons. as previously reported, the retrogenes might be preferentially expressed in the testis. in this study, more retrocopies were expressed in the testis than in other tissues. however, no ds or dn/ds bias, which reflects retrocopy age range, was associated with any tissue-specific retrocopies. moreover, expression pattern analysis revealed that older retrocopies tended to be expressed with higher fpkm values and in more tissues than the young retrocopies. the association of increased retrocopy expression distribution among tissues with increased age indicated that retrocopy biological functions might evolve to become essential over time. the most direct ways in which a retrocopy can affect the genome are through retrocopy insertion into a gene and acquisition of new gene elements by the retrocopy. we identified retrocopy-overlapped genes. go enrichment analysis revealed that the overrepresented go terms differed between the retrocopy-overlapped genes and the parent genes from which those retrocopies originated. the gene functions that were overrepresented in the parent genes were most likely related to the synthesis and metabolism of biological molecules, whereas those of the retrocopy-overlapped genes were most likely related to transmembrane transport. by retrotransposition of a father gene’s mrna copy insertion into a gene, these father genes may contribute to the evolution of retrocopy-overlapped genes, whose functional organization differs from that of the father’s gene. this result indicates that retrotranspositions may contribute to functional organization dynamics of the genome. among the retrocopy-overlapped genes, genes that were specifically lost in the tetrapod lineage were identified, including myh which is related to eye morphology; ntn which is related to olfactory processes; and esrrga, which is related to caudal fin malformations. comparisons between these genes and the retrocopy-overlapped genes with orthologous sequences in all of the investigated tetrapods revealed no differences in gene structure or expression level; however, as predicted from the retrocopies with which they overlapped, these lost genes tended to be more constrained by natural selection in the coelacanth genome. this result indicates that the genes were not lost in the tetrapod lineage because of any structural defect, but because during the adaptation of vertebrates from water to land, these genes were not necessary. furthermore, the overrepresentation of functional categories related to the nervous system in these genes might indicate that, unlike the system of ionic homeostasis regulation, the sensory system of tetrapods may have experienced remodeling, which modified it from the system observed in fish. the birth of new genes can contribute to the formation of lineage- or species-specific genes, as found in this study. we identified retrocopy-overlapped genes that were specific to coelacanths. the overrepresented gene functions related to the immune function indicated that the immune system was reinforced during coelacanth evolution. additionally, the slow rate of new retrogene formation corresponded to the slow evolution of protein-coding genes in the coelacanth genome, which provided novel insight into the morphological resemblance of the coelacanth to its fossil ancestors. together, these results might indicate stability of the deep sea environment in which the coelacanth evolved.  our study revealed a burst pattern of young retrocopies in the coelacanth genome. this pattern is similar to that in tetrapods rather than that in ray-finned fish, which indicates a possible genomic change related to water-to-land adaptations. many retrocopies may have been eliminated during coelacanth genome evolution because of disrupted genetic structure defects. however, some might have been randomly inserted into existing genes or acquired regulatory elements, exons, or introns from de novo genetic sequences that facilitated overcoming of these defects and acquiring functions. these retrogenes were revealed to have an effect on functional organization of the genome, which provides novel insight into coelacanth evolution and the transformations involved in the transition from water to land. however, the results of this study were only sufficient for revealing some of the effects of retrotransposition on the genome; the response of the host gene to retrocopy insertion remains obscure, and retrocopies may function as noncoding rnas that do not demand protein coding gene’s structures. thus, our studies in the future will further explore these issues. 
the distribution of retrocopies based on their ds values revealed a burst pattern of young retrocopies in the genome. among these retrocopies, retrogenes were more prevalent in old than young retrocopies, which indicates that most retrocopies may have been eliminated during evolution, even though some retrocopies survived, attained biological function as retrogenes, and became old. thus, investigating retrocopies in the coelacanth genome can determine the role of retrocopies in coelacanth genome innovations and perhaps even water-to-land adaptations. we determined the ds values, dn/ds ratios, expression patterns, and enrichment of functional categories for retrocopies in the african coelacanth genome. we identified ensembl genes that overlapped with the coelacanth genome retrocopies. our study identified retrocopies in the coelacanth genome. sequence analysis of these retrocopies and their parent genes, transcriptome data, and go annotation information revealed novel insight about the potential role of genomic retrocopies in coelacanth evolution and vertebrate adaptations during the evolutionary transition from water to land. go enrichment analysis revealed different overrepresented go terms between these “retrocopy-overlapped genes” and the retrocopy parent genes, which indicates potential genomic functional organization produced by retrotranspositions. the coelacanth is known as a “living fossil” because of its morphological resemblance to its fossil ancestors. the retrocopy birth pattern was shown to be more similar to that in tetrapods than ray-finned fish, which indicates a genomic transformation that accompanied vertebrate evolution from water to land. among the retrocopy-overlapped genes, we also identified that were coelacanth-specific, which could represent a potential molecular basis for coelacanth evolution. retrocopies are a type of novel genetic element that are likely to contribute to genome or phenotype innovations. coelacanth | retrocopy | novel genetic elementsissue-copyright-statement© the author 2015
 the coelacanth is known as a “living fossil” because of its morphological resemblance to its fossil ancestors. thus, it serves as a useful model that provides insight into the fish that first walked on land. retrocopies are a type of novel genetic element that are likely to contribute to genome or phenotype innovations. thus, investigating retrocopies in the coelacanth genome can determine the role of retrocopies in coelacanth genome innovations and perhaps even water-to-land adaptations.  we determined the ds values, dn/ds ratios, expression patterns, and enrichment of functional categories for retrocopies in the african coelacanth genome. of the retrocopies, 85– were shown to be potentially functional. the distribution of retrocopies based on their ds values revealed a burst pattern of young retrocopies in the genome. the retrocopy birth pattern was shown to be more similar to that in tetrapods than ray-finned fish, which indicates a genomic transformation that accompanied vertebrate evolution from water to land. among these retrocopies, retrogenes were more prevalent in old than young retrocopies, which indicates that most retrocopies may have been eliminated during evolution, even though some retrocopies survived, attained biological function as retrogenes, and became old. transcriptome data revealed that many retrocopies showed a biased expression pattern in the testis, although the expression was not specifically associated with a particular retrocopy age range. we identified ensembl genes that overlapped with the coelacanth genome retrocopies. go enrichment analysis revealed different overrepresented go terms between these “retrocopy-overlapped genes” and the retrocopy parent genes, which indicates potential genomic functional organization produced by retrotranspositions. among the retrocopy-overlapped genes, we also identified that were coelacanth-specific, which could represent a potential molecular basis for coelacanth evolution.  our study identified retrocopies in the coelacanth genome. sequence analysis of these retrocopies and their parent genes, transcriptome data, and go annotation information revealed novel insight about the potential role of genomic retrocopies in coelacanth evolution and vertebrate adaptations during the evolutionary transition from water to land. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords coelacanth | retrocopy | novel genetic elementsissue-copyright-statement© the author 2015 