BACKGROUND
over the past few decades, gene duplications have been recognized as one of the main forces capable of generating large gene families with novel functions.
in fact, duplications are thought to be one of the primary sources of adaptive evolution given that they generate new genetic material, and in doing so, create substrates that can undergo divergence through mutations  <cit> .
thus, gene duplications have the potential to affect genomic evolution dependent on the results of these mutations.
duplicated genes are subject to the same forces that affect the evolution of single copy genes.
these include genetic drift, which is opposed by negative or purifying selection, and positive selection, which can fix advantageous changes faster than expected by chance  <cit> .
additionally, other forces affect the evolution of duplicated genes.
functional redundancy can permit the accumulation of changes in one copy of the gene without negative consequences to an organism's proteome.
although this often leads to silencing or deletion of one gene copy, it can also lead to improvements on the ancestral functions or the development of new functions  <cit> .
subfunctionalization  <cit> , where an ancestral gene's functions are shared between the descendant genes, and neofunctionalization  <cit> , where one copy acquires a novel gene function, are possible consequences of divergence following gene duplication.
most duplicated genes tend to diverge over time  <cit> .
however, in some instances the genes, or parts of the genes, evolve together in a process known as concerted evolution  <cit> .
essentially, instead of gene sequences becoming progressively more dissimilar, the sequences remain highly similar or even identical.
although low divergence can also be explained by strong purifying selection, these two phenomena can be distinguished by comparisons across species.
the hallmark of concerted evolution is that high sequence similarity between genes is maintained within a species  while divergence occurs between species .
this is distinct from strong purifying selection, where divergence is impeded both within and between species such that the function, which is highly susceptible to changes, is preserved.
concerted evolution between duplicated genes can be caused by ongoing genetic exchange called gene conversion  <cit> .
gene conversion is the non-reciprocal exchange of genetic material between homologous sequences.
this process can have both positive and negative consequences.
beneficially, gene conversion can decrease mutational load, eliminate deleterious mutations, and spread advantageous alleles, thus playing a role in adaptive evolution  <cit> .
conversely, gene conversion can produce harmful phenotypes, such as gaucher disease  <cit>  and shwachman-diamond syndrome  <cit> , when disruptive mutations from a pseudogene are substituted into its functional duplicate.
the duration and frequency of the exchange between duplicated sequences is thought to be variable  <cit>  and appears to depend upon several factors, including the distance between sequences.
several studies have shown a negative correlation between the frequency of gene conversion and the distance between homologous sequences  <cit> , with a drop-off in frequency at distances greater than  <dig> kb  <cit> .
additionally, regions undergoing gene conversion may be disrupted by a few key mutations, such as the insertion of mobile elements or by the gradual accumulation of single nucleotide mutations  <cit> .
gene conversion can be a stochastic process, and it is unclear what effect selection has on the duration of gene conversion.
gene conversion is also variable in the amount of gene sequence involved.
while some entire gene sequences have undergone concerted evolution, others have a mosaic evolution pattern.
under mosaic evolution, segments of the gene are homogenized and evolve in concert, while others diverge without gene conversion  <cit> .
this can complicate phylogenetic reconstruction, often producing trees that appear to indicate that multiple, independent and parallel duplications have occurred  <cit> .
characteristically, regions undergoing gene conversion have an elevated gc content relative to flanking sequences  <cit> .
intragenically, this can be seen within introns as well as at the third codon positions within exons .
an increased gc content can be explained by the biased gene conversion  model, which asserts that heteroduplexes formed during gene conversion are preferentially repaired to gc alleles over at alleles  <cit> .
this leads to an increase in gc fixation and an elevated gc content.
biased dna repair has been reported in mammalian cells, lending weight to this model  <cit> .
this characteristic increase in gc content can be used in support of other evidence that a region is undergoing gene conversion.
here we performed a whole genome analysis to look for duplicated genes undergoing concerted evolution.
previous whole genome analyses have been conducted looking for gene conversion in c. elegans  <cit> , s. cerevisiae  <cit> , rodent genomes  <cit> , bacterial genomes  <cit> , the rice genome  <cit>  and d. melanogaster  <cit> .
until recently  <cit> , there were no whole genome analyses of gene conversion in the human genome.
benovoy and drouin  <cit>  recently used a whole genome approach to identify examples of gene conversion in the human genome.
however, their analysis was restricted to multigene families with three or more members.
our study differs in that we attempt to identify gene pairs  created by duplication early in mammalian radiation that have maintained regions of high sequence similarity due to ongoing gene conversion.
our goal was to identify examples of gene conversion preserved in all, or a large majority, of mammalian species.
a small number of other genes, such as oxct2a and oxct2b, emr <dig> with cd <dig> and emr <dig>  tlr <dig> and tlr <dig>  and ccr <dig> and ccr <dig>  <cit> , have been reported to be evolving in concert by gene conversion in multiple lineages.
by looking for duplicated genes in both the human and mouse genomes with greater sequence percent identity between paralogues than between orthologues, we identified three gene pairs with signals of long enduring concerted evolution: bmp8a/b, ddx19a/b, and tubg1/ <dig>  two of these  are novel examples, while one  has been reported previously  <cit> .
we also detail a detection strategy that can be applied to identify additional examples of genes evolving by this phenomenon.
we performed extensive phylogenetic studies and analyzed the selective pressures acting on the gene pairs as well as their relative expression patterns in several human tissues.
through these examinations we show that gene conversion has been occurring between the gene pairs for over  <dig> million years, and the extended duration of this process in multiple distant lineages suggests that there has been prolonged selection for homogenization of segments within the genes.
RESULTS
identification of gene pairs
using our search criteria, outlined in methods , we were able to identify three gene pairs as candidate loci for concerted evolution.
due to the filtering steps we utilized, each gene pair is less than  <dig> mb apart and shares high sequence similarity  in at least two consecutive exons and their intervening intron.
the three gene pairs we identified are: bmp8a/b, ddx19a/b, and tubg1/ <dig>  figure  <dig> shows the gene structure and orientation of these gene pairs in the human genome.
bmp8a and bmp8b are members of the bone morphogenesis protein family, a subfamily of the transforming growth factor type beta  supergene family, found on chromosome 1p <dig> approximately  <dig> kb apart in an inverted orientation.
each gene has seven exons with an open reading frame  of  <dig> bps encoding a protein containing  <dig> amino acids.
bone morphogenesis proteins are known to be involved in vertebrate development  <cit> .
while little has been reported about their function in humans, this gene pair has been linked to involvement in reproductive system development in the mouse   <cit> .
concerted evolution of this gene pair was previously reported  <cit> .
ddx19a and ddx19b are dead box helicase genes that are found on chromosome 16q <dig> .
these genes are tandemly duplicated   <dig> kb apart.
both genes have  <dig> exons but differ by  <dig> bps  in their orfs.
while ddx19a's orf transcript size is  <dig> bps , ddx19b has an additional codon .
using chicken, which has a single orthologue of ddx <dig>  as an outgroup, it is apparent that ddx19a has undergone a deletion of  <dig> bps near the start of exon  <dig>  this deletion is seen in all investigated mammalian orthologues of ddx19a, including opossum.
although the functions of these genes are not well characterized, both genes are thought to be atp-dependent rna helicases that are involved in mrna transport from the nucleus  <cit> .
tubg <dig> and tubg <dig> are gamma tubulin genes on chromosome 17q <dig>  and are situated in direct orientation approximately  <dig> kb apart.
both genes have  <dig> exons with an orf size of  <dig> bps, encoding a protein with  <dig> amino acids.
the gamma tubulin genes are components of the microtubule organizing centers where they play a central role in the nucleation of microtubules  <cit> .
identification of orthologues
using various techniques, including blat  <cit> , blast  <cit>  and rt-pcr, we identified homologues of each gene in additional organisms.
in all three cases, orthologues of both copies within the gene pair were identified in eutherian mammals, while only a single orthologue was found in non-mammalian vertebrates.
also, in each mammalian species investigated, only two copies of each gene could be identified, indicating that these multigene families have a consistent copy number of two in mammals.
interestingly, only a single orthologue of bmp8a/b was found in the opossum, while orthologues appear to exist for both genes from the ddx19a/b and tubg1/ <dig> gene pairs in this organism.
although the low sequence coverage and incomplete assembly of the opossum genome could prevent the identification of a second bmp <dig> orthologue, another explanation involves different timings for the origins of these gene pairs.
in each case, since two orthologues are present in all eutherian mammals examined, the most parsimonious explanation is that a duplication event occurred early in mammalian evolution.
for bmp8a/b, the absence of the duplication in opossum suggests that the duplication event occurred after the divergence of the opossum lineage  from the armadillo .
this would place the duplication event somewhere between  <dig> and  <dig> million years ago   <cit> .
for ddx19a/b and tubg1/ <dig>  the presence of the duplication in opossum suggests that the duplication event is older, occurring before the divergence of opossum lineage .
phylogenetic analyses
we created phylogenetic trees using two different methods, neighbor-joining  and maximum likelihood , to examine the evolution of the duplicated gene pairs in the mammalian lineage.
comparison of the trees created by these two different methods showed that the branching pattern and node structure were highly similar in every case.
generally, duplicated genes that undergo divergent evolution display a tree in which the orthologues cluster and are monophyletic.
in contrast, duplicated genes that evolve in a concerted fashion display trees where paralogues cluster.
examining the phylogenetic trees in figure  <dig>  it is apparent that these trees show signatures of both types of evolution.
for example, in figure 2b, ddx19a and its three primate orthologues are monophyletic, as is ddx19b and its primate orthologues.
a similar phylogeny is also seen in the rodent lineage.
this pattern is typical of divergent evolution.
in contrast, the pattern for dog, cow, horse and opossum is distinct in that the paralogues group together.
this could be indicative of concerted evolution.
an alternate explanation for this pattern involves multiple independent duplications.
ddx19a/b's phylogeny could be explained by six independent duplications .
however, a single duplication that predates the mammalian divergence would be a more parsimonious explanation.
in this hypothesis, mosaic evolution, where some exons evolve divergently and others evolve concertedly, would explain the mixture of evolutionary patterns seen in the phylogenetic trees.
to test this hypothesis, we performed a sliding window analysis on the gene pairs  then built new phylogenetic trees  after separating the exons into divergently or concertedly evolving categories .
both analyses favor the conclusion that these gene pairs are evolving in a mosaic pattern.
the exons were categorized as divergently or concertedly evolving using a combination of the mvista identities shown in figure  <dig>  the sliding window plots , and a visual inspection of the multiple alignments.
figure  <dig> shows that categorizing the exons before building phylogenetic trees is sufficient to separate the two signatures of evolution observed in figure  <dig> .
the divergent gene trees for ddx19a/b and tubg1/ <dig>  show monophyletic clades in which all the eutherian mammal orthologues cluster.
this phylogeny is not seen for bmp8a/b, except for in the rodent lineage, but can be explained by the greater stretch of high similarity between paralogues in non-rodent mammals .
in contrast, the convergent gene trees for all three gene pairs  have paralogues grouping together in almost all instances.
the only exceptions are the human and chimpanzee branches in ddx19a/b and tubg1/ <dig>  these phylogenetic trees illustrate that these gene pairs have evolved in a mosaic pattern.
a summary of the percent similarity between the human genes in divergent and concerted regions was also generated .
evidence of gene conversion
one cause of concerted evolution is gene conversion.
to look for statistically significant evidence of homogenization, we used the program geneconv  <cit> .
multiple alignments, both of the orfs and the genomic sequences, were used as input for geneconv in order to detect candidate fragments of aligned gene conversion.
the results identify several statistically significant fragments in both the orf alignments and the genomic sequence alignments .
from the table, it is clear that the regions detected by geneconv overlap to a large degree with the peaks of high sequence identity between paralogues .
<dig> exons were included if > <dig> bps were within the positive fragment.
<dig> introns were included if > <dig> bps were within the positive fragment.
h human, c chimp, o orangutan, rh rhesus macaque, r rat, a all primates or rodents.
hits enclosed by brackets are positive pairwise fragments only.
p-value for that region in the global analysis is not significant .
hits within genomic dna span both introns and exons.
increased gc content at the third codon position and in introns has also been used as evidence for gene conversion  <cit> .
gc content was calculated for each gene in both the divergent and concerted regions in multiple species.
the average gc content was calculated at each of the codon positions and plotted with p-values ascertained within the human sequences using a fisher exact test and/or yates-corrected chi-square test .
for bmp8a/b, only rodent sequences were used because in other species all of the exons appear to be evolving in concert.
from these plots, it is clear that there is a significant difference  in the third codon position for ddx19a/b and tubg1/ <dig> .
however, although there is an increase in the third codon position of the rodent bmp8a/b , the difference is not significant.
a similar comparison was undertaken using the introns from these three gene pairs.
again, a significant increase in gc content is seen in the introns that appear to be evolving under concerted evolution, as opposed to the introns evolving divergently as well as  <dig> kb of upstream and downstream flanking sequence .
taken together with the results from geneconv, the increase in gc content suggests that gene conversion is the cause of the concerted evolution in these three gene pairs.
sequence evolution and functional domains
we investigated the selective pressures working on these gene pairs by calculating the dn/ds ratios between the orthologues.
of interest was whether the regions evolving divergently were under different selective pressures than the regions evolving in concert.
average dn/ds values, calculated using pairs of orthologues, are shown in table  <dig>  values of dn/ds are calculated for the whole gene, as well as the regions evolving divergently and concertedly .
as you can see from this table, five of the six values of dn/ds for the concerted regions are lower than the dn/ds values for the divergent regions.
this suggests that either these regions are under more negative selective pressure or it could indicate that a consequence of gene conversion is lower nonsynonymous divergence between sequences.
we also performed sliding window and paml analyses to further examine the dn/ds values in the gene pairs .
although the results from some analyses show suggestions of positive selection, no significant evidence was found.
therefore, there does not appear to be an overlap between the regions undergoing concerted evolution and positive selection.
conversely, there is some evidence indicating that the regions undergoing concerted evolution have lower dn/ds values, suggesting that both gene conversion and purifying selection are acting at these sites.
additionally, we looked to see if the regions undergoing concerted evolution contained or overlapped with specific functional domains within these genes.
however, in each case there did not appear to be any significant overlap of the functional domains with the regions undergoing concerted evolution .
although parts of some domains overlap regions of concerted evolution, there is no clear relationship that could indicate the selection for gene conversion limited to a particular domain.
in some cases, the exons involved in concerted evolution fall outside of the known functional domains.
this could indicate that there is another functional domain in these exons that has not been previously described.
gene expression analysis
we performed gene expression analyses to determine whether there is differential tissue expression within the gene pairs.
we used qpcr and pyrosequencing assays to attain a ratio of expression between the two members of the gene pair.
however, the bmp8a/b gene pair was resistant to both assays.
therefore, although we were able to obtain measurements of expression for ddx19a/b and tubg1/ <dig>  the expression analysis of bmp8a/b is limited to previously published reports in mice.
for ddx19a/b, we were able to use both qpcr  and pyrosequencing  to obtain expression ratios .
although the values for each assay are slightly different, the overall trend is consistent.
there appears to be several tissues where the expression level of the two genes is similar .
however, in several other tissues the expression level of ddx19a is higher than the expression level of ddx19b .
in these tissues, it appears that ddx19b is expressed at about one half to three quarters the level of ddx19a, with the highest degree of differential expression seen in skeletal muscle.
although the expression level difference between ddx19a and ddx19b is not considerable in most tissues, the expression ratio for tubg <dig> and tubg <dig> is more remarkable .
again we used both qpcr  and pyrosequencing  to obtain expression ratios.
similarly, both assays are relatively consistent in their findings, although there are some small differences in their absolute values.
interestingly, for this gene pair there are several tissues where there is a significant difference in the level of expression between the two genes.
unlike ddx19a/b, where either the expression was equal or one gene  was higher in expression, there are some tissues where tubg <dig> shows higher expression , some tissues where tubg <dig> shows higher expression  and some tissues where expression is relatively equal .
also, the magnitudes of the expression differences are considerably larger in this gene pair, ranging from four to five times higher expression of tubg <dig> in liver and bone marrow, to five to six times higher expression of tubg <dig> in brain.
thus, the tubg1/ <dig> gene pair is differentially expressed in several tissues.
we performed additional analysis looking for regulatory regions upstream of each gene to try to assess whether gene pairs share promoters or enhancers.
however, in general it appears that the upstream regulatory regions have diversified throughout the gene pair evolution, even though regions within the genes have evolved in concert .
discussion
we have identified three duplicated gene pairs arising early in mammalian evolution that have undergone concerted evolution via gene conversion.
while one of these gene pairs has been previously reported to be evolving in concert  <cit> , the other two pairs are novel.
these findings, identified using a genome-wide strategy, increase by 50% the number of identified cases of human gene pairs undergoing this process for such an extended period of time.
in each case, the two copies of the gene are separated by less than  <dig> kb of sequence in the human genome and we have inferred that duplication occurred over  <dig> mya.
phylogenetic analyses demonstrate that each pair of genes has regions evolving in concert in all examined mammalian species .
concerted evolution in multigene families is thought to occur by two mechanisms: unequal crossing over and gene conversion.
while unequal crossing over is often invoked to explain concerted evolution within tandemly-arrayed gene families, gene conversion is used to explain concerted evolution of duplicated genes  and mosaic evolution  <cit> .
this is because gene conversion can homogenize parts of a gene without changing the copy number.
since all of the eutherian mammals examined have evidence of only two copies of these genes, it is unlikely that unequal crossing over is the basis for the observed concerted evolution.
for additional proof of gene conversion, we used the program geneconv and also looked for a significant increase in gc content.
although homogenization or increased gc by themselves do not prove gene conversion is acting within these regions, in combination with mosaic evolution  and a consistent copy number , this evidence supports gene conversion, rather than unequal crossing over, as the cause of the concerted evolution in these gene pairs.
also, although the homogenization of the gene pairs could have been achieved by a single gene conversion event in each mammal, our evidence is more in line with multiple gene conversion events throughout evolution leading to concerted evolution of the gene pairs.
it is interesting that gene conversion between these gene pairs appears to have been active for over  <dig> million years and has resulted in concerted evolution in every mammal examined.
the duration of gene conversion is thought to be variable and often stochastic in nature  <cit> .
therefore, although gene conversion could occur between two loci for over  <dig> million years by chance, it is expected to do so only in rare instances.
thus, it appears highly unlikely that gene conversion would be maintained coincidentally for over  <dig> million years in multiple lineages.
consequently, we speculate that if gene conversion is seen consistently at the same loci for an extended length of time, there may have been selection for this process and the maintenance of homogenized gene sequences.
the duration and prevalence of gene conversion within these gene pairs is suggestive of selection for homogenization and co-evolution.
however, the basis for this proposed selection is unclear.
our analyses do not show any obvious overlap of the regions evolving in concert with protein domains or known functional sequences.
nevertheless, a few previous reports have identified similar patterns of concerted evolution which could provide clues as to why gene conversion can be maintained for such extended periods of time.
three of the reports describing similar gene pairs suggest that protein binding co-evolution between two genes might be the basis for the conserved gene conversion.
the vazquez-salat et al  <cit>  study, which looks at gene conversion between ccr <dig> and ccr <dig>  suggests that along with homodimerization, these proteins also heterodimerize, creating a synergistic effect which enhances their function.
similarly, kruithof  <cit>  and kwakkenbos  <cit>  looked at gene conversion between tlr <dig> and tlr <dig> and between emr <dig>  cd <dig> and emr <dig>  respectively, and suggest that gene conversion may be used to conserve or co-evolve binding regions.
these papers propose that mosaic evolution maintains structural or binding regions, while receptor, ligand binding or other functional domains are allowed to diverge.
a similar proposal can be applied to explain the mosaic evolution identified in the three gene pairs we investigated.
additionally, the conserved boundaries of the mosaic evolution in multiple species further supports the idea that while some regions are co-evolving to maintain a specific function, other regions are diverging in both sequence and function.
interestingly, if we look at the protein binding of the gene pairs we identified, there are some indications these proteins have conserved binding partners.
we speculate that gene conversion has been utilized to maintain perfect homology at binding sites.
additionally, our results indicate that the regions undergoing gene conversion tend to have lower dn/ds values than the sequences evolving divergently .
this suggests a second process, stronger negative selection in combination with gene conversion, has been utilized to conserve these sites.
an example of binding site co-evolution occurs between bmp8a and bmp8b, which, like other bmp <dig> proteins, work as homodimers  <cit> .
these proteins could theoretically interact and form heterodimers, similar to the interaction of bmps - <dig> and - <dig>  <cit> .
perhaps a heterodimer would perform an additional or enhanced function.
similarly, by assessing tubg <dig> and tubg <dig> binding partners using ncbi's entrez database  <cit>  it is clear from in vivo evidence, based on affinity capture experiments, that both proteins interact with pxn, rnf19a, and tubgcp <dig>  in this case, although there is no evidence that the protein products of tubg1/ <dig> interact with each other directly, they may interact with the same partners and thus need to share a highly similar or identical binding domain.
although similar evidence is not available for ddx19a/b, shared binding partners may be identified by functional interaction analyses of these proteins.
however, our assertion that gene conversion is being selected to maintain binding sites is purely speculative.
while it appears that selection may be acting to maintain gene conversion within these regions, the purpose and evolutionary implications of this process are still largely unknown.
our gene expression analyses of ddx19a/b and tubg1/ <dig>  indicate that although these genes could share some functions, they may act in different tissues.
this is a form of subfunctionalization.
an example occurs in rodent bmp8a/b, which have been shown to have differential expression in the mouse reproductive system  <cit> .
the most striking example of subfunctionalization was the differences seen in the expression between tubg <dig> and tubg <dig>  these genes appear to have significantly different expression in six of the ten examined tissues .
these results agree with previously reported expression analyses of these genes, such as the increased expression of tubg <dig> in liver and tubg <dig> in brain  <cit> .
this could indicate that the genes have different functional relevance in different tissues.
we attribute the subfunctionalization of tissue expression patterns observed in the three gene pairs to the divergence of regulatory regions upstream of each gene.
when we examine  <dig> kb of sequence upstream of each gene, we see little to no overlap in predicted or known enhancers and conserved transcription binding sites.
these results again show the mosaic pattern of evolution within these gene pairs, where some regions evolve in concert to maintain function.
other regions, including regulatory regions, are not preserved, leading to diverging functions and subfunctionalization.
CONCLUSIONS
in conclusion, it appears that gene or domain homogenization caused by extended periods of gene conversion can result in concerted evolution that may be maintained, perhaps by selection, throughout mammalian evolution.
we speculate that this process is being used to maintain homo- or hetero- binding domains within gene pairs.
we have identified additional examples of this relatively rare form of evolution and, although it is rare, postulate that it has relevance within gene duplication evolution

 over the past few decades, gene duplications have been recognized as one of the main forces capable of generating large gene families with novel functions. in fact, duplications are thought to be one of the primary sources of adaptive evolution given that they generate new genetic material, and in doing so, create substrates that can undergo divergence through mutations. thus, gene duplications have the potential to affect genomic evolution dependent on the results of these mutations. duplicated genes are subject to the same forces that affect the evolution of single copy genes. these include genetic drift, which is opposed by negative or purifying selection, and positive selection, which can fix advantageous changes faster than expected by chance. additionally, other forces affect the evolution of duplicated genes. functional redundancy can permit the accumulation of changes in one copy of the gene without negative consequences to an organism's proteome. although this often leads to silencing or deletion of one gene copy, it can also lead to improvements on the ancestral functions or the development of new functions. subfunctionalization, where an ancestral gene's functions are shared between the descendant genes, and neofunctionalization, where one copy acquires a novel gene function, are possible consequences of divergence following gene duplication. most duplicated genes tend to diverge over time. however, in some instances the genes, or parts of the genes, evolve together in a process known as concerted evolution. essentially, instead of gene sequences becoming progressively more dissimilar, the sequences remain highly similar or even identical. although low divergence can also be explained by strong purifying selection, these two phenomena can be distinguished by comparisons across species. the hallmark of concerted evolution is that high sequence similarity between genes is maintained within a species while divergence occurs between species. this is distinct from strong purifying selection, where divergence is impeded both within and between species such that the function, which is highly susceptible to changes, is preserved. concerted evolution between duplicated genes can be caused by ongoing genetic exchange called gene conversion. gene conversion is the non-reciprocal exchange of genetic material between homologous sequences. this process can have both positive and negative consequences. beneficially, gene conversion can decrease mutational load, eliminate deleterious mutations, and spread advantageous alleles, thus playing a role in adaptive evolution. conversely, gene conversion can produce harmful phenotypes, such as gaucher disease and shwachman-diamond syndrome, when disruptive mutations from a pseudogene are substituted into its functional duplicate. the duration and frequency of the exchange between duplicated sequences is thought to be variable and appears to depend upon several factors, including the distance between sequences. several studies have shown a negative correlation between the frequency of gene conversion and the distance between homologous sequences, with a drop-off in frequency at distances greater than kb. additionally, regions undergoing gene conversion may be disrupted by a few key mutations, such as the insertion of mobile elements or by the gradual accumulation of single nucleotide mutations. gene conversion can be a stochastic process, and it is unclear what effect selection has on the duration of gene conversion. gene conversion is also variable in the amount of gene sequence involved. while some entire gene sequences have undergone concerted evolution, others have a mosaic evolution pattern. under mosaic evolution, segments of the gene are homogenized and evolve in concert, while others diverge without gene conversion. this can complicate phylogenetic reconstruction, often producing trees that appear to indicate that multiple, independent and parallel duplications have occurred. characteristically, regions undergoing gene conversion have an elevated gc content relative to flanking sequences. intragenically, this can be seen within introns as well as at the third codon positions within exons. an increased gc content can be explained by the biased gene conversion model, which asserts that heteroduplexes formed during gene conversion are preferentially repaired to gc alleles over at alleles. this leads to an increase in gc fixation and an elevated gc content. biased dna repair has been reported in mammalian cells, lending weight to this model. this characteristic increase in gc content can be used in support of other evidence that a region is undergoing gene conversion. here we performed a whole genome analysis to look for duplicated genes undergoing concerted evolution. previous whole genome analyses have been conducted looking for gene conversion in c. elegans, s. cerevisiae, rodent genomes, bacterial genomes, the rice genome and d. melanogaster. until recently, there were no whole genome analyses of gene conversion in the human genome. benovoy and drouin recently used a whole genome approach to identify examples of gene conversion in the human genome. however, their analysis was restricted to multigene families with three or more members. our study differs in that we attempt to identify gene pairs created by duplication early in mammalian radiation that have maintained regions of high sequence similarity due to ongoing gene conversion. our goal was to identify examples of gene conversion preserved in all, or a large majority, of mammalian species. a small number of other genes, such as oxct2a and oxct2b, emr with cd and emr tlr and tlr and ccr and ccr, have been reported to be evolving in concert by gene conversion in multiple lineages. by looking for duplicated genes in both the human and mouse genomes with greater sequence percent identity between paralogues than between orthologues, we identified three gene pairs with signals of long enduring concerted evolution: bmp8a/b, ddx19a/b, and tubg1/ two of these are novel examples, while one has been reported previously. we also detail a detection strategy that can be applied to identify additional examples of genes evolving by this phenomenon. we performed extensive phylogenetic studies and analyzed the selective pressures acting on the gene pairs as well as their relative expression patterns in several human tissues. through these examinations we show that gene conversion has been occurring between the gene pairs for over million years, and the extended duration of this process in multiple distant lineages suggests that there has been prolonged selection for homogenization of segments within the genes.  identification of gene pairs using our search criteria, outlined in methods, we were able to identify three gene pairs as candidate loci for concerted evolution. due to the filtering steps we utilized, each gene pair is less than mb apart and shares high sequence similarity in at least two consecutive exons and their intervening intron. the three gene pairs we identified are: bmp8a/b, ddx19a/b, and tubg1/ figure shows the gene structure and orientation of these gene pairs in the human genome. bmp8a and bmp8b are members of the bone morphogenesis protein family, a subfamily of the transforming growth factor type beta supergene family, found on chromosome 1p approximately kb apart in an inverted orientation. each gene has seven exons with an open reading frame of bps encoding a protein containing amino acids. bone morphogenesis proteins are known to be involved in vertebrate development. while little has been reported about their function in humans, this gene pair has been linked to involvement in reproductive system development in the mouse. concerted evolution of this gene pair was previously reported. ddx19a and ddx19b are dead box helicase genes that are found on chromosome 16q. these genes are tandemly duplicated kb apart. both genes have exons but differ by bps in their orfs. while ddx19a's orf transcript size is bps, ddx19b has an additional codon. using chicken, which has a single orthologue of ddx as an outgroup, it is apparent that ddx19a has undergone a deletion of bps near the start of exon this deletion is seen in all investigated mammalian orthologues of ddx19a, including opossum. although the functions of these genes are not well characterized, both genes are thought to be atp-dependent rna helicases that are involved in mrna transport from the nucleus. tubg and tubg are gamma tubulin genes on chromosome 17q and are situated in direct orientation approximately kb apart. both genes have exons with an orf size of bps, encoding a protein with amino acids. the gamma tubulin genes are components of the microtubule organizing centers where they play a central role in the nucleation of microtubules. identification of orthologues using various techniques, including blat, blast and rt-pcr, we identified homologues of each gene in additional organisms. in all three cases, orthologues of both copies within the gene pair were identified in eutherian mammals, while only a single orthologue was found in non-mammalian vertebrates. also, in each mammalian species investigated, only two copies of each gene could be identified, indicating that these multigene families have a consistent copy number of two in mammals. interestingly, only a single orthologue of bmp8a/b was found in the opossum, while orthologues appear to exist for both genes from the ddx19a/b and tubg1/ gene pairs in this organism. although the low sequence coverage and incomplete assembly of the opossum genome could prevent the identification of a second bmp orthologue, another explanation involves different timings for the origins of these gene pairs. in each case, since two orthologues are present in all eutherian mammals examined, the most parsimonious explanation is that a duplication event occurred early in mammalian evolution. for bmp8a/b, the absence of the duplication in opossum suggests that the duplication event occurred after the divergence of the opossum lineage from the armadillo. this would place the duplication event somewhere between and million years ago. for ddx19a/b and tubg1/ the presence of the duplication in opossum suggests that the duplication event is older, occurring before the divergence of opossum lineage. phylogenetic analyses we created phylogenetic trees using two different methods, neighbor-joining and maximum likelihood, to examine the evolution of the duplicated gene pairs in the mammalian lineage. comparison of the trees created by these two different methods showed that the branching pattern and node structure were highly similar in every case. generally, duplicated genes that undergo divergent evolution display a tree in which the orthologues cluster and are monophyletic. in contrast, duplicated genes that evolve in a concerted fashion display trees where paralogues cluster. examining the phylogenetic trees in figure it is apparent that these trees show signatures of both types of evolution. for example, in figure 2b, ddx19a and its three primate orthologues are monophyletic, as is ddx19b and its primate orthologues. a similar phylogeny is also seen in the rodent lineage. this pattern is typical of divergent evolution. in contrast, the pattern for dog, cow, horse and opossum is distinct in that the paralogues group together. this could be indicative of concerted evolution. an alternate explanation for this pattern involves multiple independent duplications. ddx19a/b's phylogeny could be explained by six independent duplications. however, a single duplication that predates the mammalian divergence would be a more parsimonious explanation. in this hypothesis, mosaic evolution, where some exons evolve divergently and others evolve concertedly, would explain the mixture of evolutionary patterns seen in the phylogenetic trees. to test this hypothesis, we performed a sliding window analysis on the gene pairs then built new phylogenetic trees after separating the exons into divergently or concertedly evolving categories. both analyses favor the conclusion that these gene pairs are evolving in a mosaic pattern. the exons were categorized as divergently or concertedly evolving using a combination of the mvista identities shown in figure the sliding window plots, and a visual inspection of the multiple alignments. figure shows that categorizing the exons before building phylogenetic trees is sufficient to separate the two signatures of evolution observed in figure. the divergent gene trees for ddx19a/b and tubg1/ show monophyletic clades in which all the eutherian mammal orthologues cluster. this phylogeny is not seen for bmp8a/b, except for in the rodent lineage, but can be explained by the greater stretch of high similarity between paralogues in non-rodent mammals. in contrast, the convergent gene trees for all three gene pairs have paralogues grouping together in almost all instances. the only exceptions are the human and chimpanzee branches in ddx19a/b and tubg1/ these phylogenetic trees illustrate that these gene pairs have evolved in a mosaic pattern. a summary of the percent similarity between the human genes in divergent and concerted regions was also generated. evidence of gene conversion one cause of concerted evolution is gene conversion. to look for statistically significant evidence of homogenization, we used the program geneconv. multiple alignments, both of the orfs and the genomic sequences, were used as input for geneconv in order to detect candidate fragments of aligned gene conversion. the results identify several statistically significant fragments in both the orf alignments and the genomic sequence alignments. from the table, it is clear that the regions detected by geneconv overlap to a large degree with the peaks of high sequence identity between paralogues. exons were included if > bps were within the positive fragment. introns were included if > bps were within the positive fragment. h human, c chimp, o orangutan, rh rhesus macaque, r rat, a all primates or rodents. hits enclosed by brackets are positive pairwise fragments only. p-value for that region in the global analysis is not significant. hits within genomic dna span both introns and exons. increased gc content at the third codon position and in introns has also been used as evidence for gene conversion. gc content was calculated for each gene in both the divergent and concerted regions in multiple species. the average gc content was calculated at each of the codon positions and plotted with p-values ascertained within the human sequences using a fisher exact test and/or yates-corrected chi-square test. for bmp8a/b, only rodent sequences were used because in other species all of the exons appear to be evolving in concert. from these plots, it is clear that there is a significant difference in the third codon position for ddx19a/b and tubg1/. however, although there is an increase in the third codon position of the rodent bmp8a/b, the difference is not significant. a similar comparison was undertaken using the introns from these three gene pairs. again, a significant increase in gc content is seen in the introns that appear to be evolving under concerted evolution, as opposed to the introns evolving divergently as well as kb of upstream and downstream flanking sequence. taken together with the results from geneconv, the increase in gc content suggests that gene conversion is the cause of the concerted evolution in these three gene pairs. sequence evolution and functional domains we investigated the selective pressures working on these gene pairs by calculating the dn/ds ratios between the orthologues. of interest was whether the regions evolving divergently were under different selective pressures than the regions evolving in concert. average dn/ds values, calculated using pairs of orthologues, are shown in table values of dn/ds are calculated for the whole gene, as well as the regions evolving divergently and concertedly. as you can see from this table, five of the six values of dn/ds for the concerted regions are lower than the dn/ds values for the divergent regions. this suggests that either these regions are under more negative selective pressure or it could indicate that a consequence of gene conversion is lower nonsynonymous divergence between sequences. we also performed sliding window and paml analyses to further examine the dn/ds values in the gene pairs. although the results from some analyses show suggestions of positive selection, no significant evidence was found. therefore, there does not appear to be an overlap between the regions undergoing concerted evolution and positive selection. conversely, there is some evidence indicating that the regions undergoing concerted evolution have lower dn/ds values, suggesting that both gene conversion and purifying selection are acting at these sites. additionally, we looked to see if the regions undergoing concerted evolution contained or overlapped with specific functional domains within these genes. however, in each case there did not appear to be any significant overlap of the functional domains with the regions undergoing concerted evolution. although parts of some domains overlap regions of concerted evolution, there is no clear relationship that could indicate the selection for gene conversion limited to a particular domain. in some cases, the exons involved in concerted evolution fall outside of the known functional domains. this could indicate that there is another functional domain in these exons that has not been previously described. gene expression analysis we performed gene expression analyses to determine whether there is differential tissue expression within the gene pairs. we used qpcr and pyrosequencing assays to attain a ratio of expression between the two members of the gene pair. however, the bmp8a/b gene pair was resistant to both assays. therefore, although we were able to obtain measurements of expression for ddx19a/b and tubg1/ the expression analysis of bmp8a/b is limited to previously published reports in mice. for ddx19a/b, we were able to use both qpcr and pyrosequencing to obtain expression ratios. although the values for each assay are slightly different, the overall trend is consistent. there appears to be several tissues where the expression level of the two genes is similar. however, in several other tissues the expression level of ddx19a is higher than the expression level of ddx19b. in these tissues, it appears that ddx19b is expressed at about one half to three quarters the level of ddx19a, with the highest degree of differential expression seen in skeletal muscle. although the expression level difference between ddx19a and ddx19b is not considerable in most tissues, the expression ratio for tubg and tubg is more remarkable. again we used both qpcr and pyrosequencing to obtain expression ratios. similarly, both assays are relatively consistent in their findings, although there are some small differences in their absolute values. interestingly, for this gene pair there are several tissues where there is a significant difference in the level of expression between the two genes. unlike ddx19a/b, where either the expression was equal or one gene was higher in expression, there are some tissues where tubg shows higher expression, some tissues where tubg shows higher expression and some tissues where expression is relatively equal. also, the magnitudes of the expression differences are considerably larger in this gene pair, ranging from four to five times higher expression of tubg in liver and bone marrow, to five to six times higher expression of tubg in brain. thus, the tubg1/ gene pair is differentially expressed in several tissues. we performed additional analysis looking for regulatory regions upstream of each gene to try to assess whether gene pairs share promoters or enhancers. however, in general it appears that the upstream regulatory regions have diversified throughout the gene pair evolution, even though regions within the genes have evolved in concert. discussion we have identified three duplicated gene pairs arising early in mammalian evolution that have undergone concerted evolution via gene conversion. while one of these gene pairs has been previously reported to be evolving in concert, the other two pairs are novel. these findings, identified using a genome-wide strategy, increase by 50% the number of identified cases of human gene pairs undergoing this process for such an extended period of time. in each case, the two copies of the gene are separated by less than kb of sequence in the human genome and we have inferred that duplication occurred over mya. phylogenetic analyses demonstrate that each pair of genes has regions evolving in concert in all examined mammalian species. concerted evolution in multigene families is thought to occur by two mechanisms: unequal crossing over and gene conversion. while unequal crossing over is often invoked to explain concerted evolution within tandemly-arrayed gene families, gene conversion is used to explain concerted evolution of duplicated genes and mosaic evolution. this is because gene conversion can homogenize parts of a gene without changing the copy number. since all of the eutherian mammals examined have evidence of only two copies of these genes, it is unlikely that unequal crossing over is the basis for the observed concerted evolution. for additional proof of gene conversion, we used the program geneconv and also looked for a significant increase in gc content. although homogenization or increased gc by themselves do not prove gene conversion is acting within these regions, in combination with mosaic evolution and a consistent copy number, this evidence supports gene conversion, rather than unequal crossing over, as the cause of the concerted evolution in these gene pairs. also, although the homogenization of the gene pairs could have been achieved by a single gene conversion event in each mammal, our evidence is more in line with multiple gene conversion events throughout evolution leading to concerted evolution of the gene pairs. it is interesting that gene conversion between these gene pairs appears to have been active for over million years and has resulted in concerted evolution in every mammal examined. the duration of gene conversion is thought to be variable and often stochastic in nature. therefore, although gene conversion could occur between two loci for over million years by chance, it is expected to do so only in rare instances. thus, it appears highly unlikely that gene conversion would be maintained coincidentally for over million years in multiple lineages. consequently, we speculate that if gene conversion is seen consistently at the same loci for an extended length of time, there may have been selection for this process and the maintenance of homogenized gene sequences. the duration and prevalence of gene conversion within these gene pairs is suggestive of selection for homogenization and co-evolution. however, the basis for this proposed selection is unclear. our analyses do not show any obvious overlap of the regions evolving in concert with protein domains or known functional sequences. nevertheless, a few previous reports have identified similar patterns of concerted evolution which could provide clues as to why gene conversion can be maintained for such extended periods of time. three of the reports describing similar gene pairs suggest that protein binding co-evolution between two genes might be the basis for the conserved gene conversion. the vazquez-salat et al study, which looks at gene conversion between ccr and ccr suggests that along with homodimerization, these proteins also heterodimerize, creating a synergistic effect which enhances their function. similarly, kruithof and kwakkenbos looked at gene conversion between tlr and tlr and between emr cd and emr respectively, and suggest that gene conversion may be used to conserve or co-evolve binding regions. these papers propose that mosaic evolution maintains structural or binding regions, while receptor, ligand binding or other functional domains are allowed to diverge. a similar proposal can be applied to explain the mosaic evolution identified in the three gene pairs we investigated. additionally, the conserved boundaries of the mosaic evolution in multiple species further supports the idea that while some regions are co-evolving to maintain a specific function, other regions are diverging in both sequence and function. interestingly, if we look at the protein binding of the gene pairs we identified, there are some indications these proteins have conserved binding partners. we speculate that gene conversion has been utilized to maintain perfect homology at binding sites. additionally, our results indicate that the regions undergoing gene conversion tend to have lower dn/ds values than the sequences evolving divergently. this suggests a second process, stronger negative selection in combination with gene conversion, has been utilized to conserve these sites. an example of binding site co-evolution occurs between bmp8a and bmp8b, which, like other bmp proteins, work as homodimers. these proteins could theoretically interact and form heterodimers, similar to the interaction of bmps - and -. perhaps a heterodimer would perform an additional or enhanced function. similarly, by assessing tubg and tubg binding partners using ncbi's entrez database it is clear from in vivo evidence, based on affinity capture experiments, that both proteins interact with pxn, rnf19a, and tubgcp in this case, although there is no evidence that the protein products of tubg1/ interact with each other directly, they may interact with the same partners and thus need to share a highly similar or identical binding domain. although similar evidence is not available for ddx19a/b, shared binding partners may be identified by functional interaction analyses of these proteins. however, our assertion that gene conversion is being selected to maintain binding sites is purely speculative. while it appears that selection may be acting to maintain gene conversion within these regions, the purpose and evolutionary implications of this process are still largely unknown. our gene expression analyses of ddx19a/b and tubg1/ indicate that although these genes could share some functions, they may act in different tissues. this is a form of subfunctionalization. an example occurs in rodent bmp8a/b, which have been shown to have differential expression in the mouse reproductive system. the most striking example of subfunctionalization was the differences seen in the expression between tubg and tubg these genes appear to have significantly different expression in six of the ten examined tissues. these results agree with previously reported expression analyses of these genes, such as the increased expression of tubg in liver and tubg in brain. this could indicate that the genes have different functional relevance in different tissues. we attribute the subfunctionalization of tissue expression patterns observed in the three gene pairs to the divergence of regulatory regions upstream of each gene. when we examine kb of sequence upstream of each gene, we see little to no overlap in predicted or known enhancers and conserved transcription binding sites. these results again show the mosaic pattern of evolution within these gene pairs, where some regions evolve in concert to maintain function. other regions, including regulatory regions, are not preserved, leading to diverging functions and subfunctionalization.  in conclusion, it appears that gene or domain homogenization caused by extended periods of gene conversion can result in concerted evolution that may be maintained, perhaps by selection, throughout mammalian evolution. we speculate that this process is being used to maintain homo- or hetero- binding domains within gene pairs. we have identified additional examples of this relatively rare form of evolution and, although it is rare, postulate that it has relevance within gene duplication evolution 
the extended duration of concerted evolution in multiple distant lineages suggests that there has been prolonged homogenization of specific segments within these gene pairs. these genes were required to be present in multiple mammalian genomes, suggesting duplication early in mammalian divergence. to eliminate genes that have been conserved due to strong purifying selection, our analysis also required at least one intron to have retained high sequence similarity between paralogues. through these analyses, our results demonstrate additional examples of a process that plays a definite, although unspecified, role in molecular evolution. here we identify human gene pairs that have undergone concerted evolution, caused by ongoing gene conversion, since at least the human-mouse divergence. we identified three human gene pairs undergoing concerted evolution. although we speculate that selection for homogenization could have been utilized in order to maintain crucial homo- or hetero- binding domains, it remains unclear why gene conversion has persisted for such extended periods of time. concerted evolution occurs in multigene families and is characterized by stretches of homogeneity and higher sequence similarity between paralogues than between orthologues.
 concerted evolution occurs in multigene families and is characterized by stretches of homogeneity and higher sequence similarity between paralogues than between orthologues. here we identify human gene pairs that have undergone concerted evolution, caused by ongoing gene conversion, since at least the human-mouse divergence. our strategy involved the identification of duplicated genes with greater similarity within a species than between species. these genes were required to be present in multiple mammalian genomes, suggesting duplication early in mammalian divergence. to eliminate genes that have been conserved due to strong purifying selection, our analysis also required at least one intron to have retained high sequence similarity between paralogues.  we identified three human gene pairs undergoing concerted evolution. phylogenetic investigations reveal that in each case the duplication appears to have occurred prior to eutherian mammalian radiation, with exactly two paralogues present in all examined species. this indicates that all three gene duplication events were established over million years ago.  the extended duration of concerted evolution in multiple distant lineages suggests that there has been prolonged homogenization of specific segments within these gene pairs. although we speculate that selection for homogenization could have been utilized in order to maintain crucial homo- or hetero- binding domains, it remains unclear why gene conversion has persisted for such extended periods of time. through these analyses, our results demonstrate additional examples of a process that plays a definite, although unspecified, role in molecular evolution. 