BACKGROUND
gene duplication followed by subsequent functional divergence is widely recognized as an important mechanism for the evolution of novelty  <cit> .
on a small scale, local tandem duplications can rapidly produce new gene families, such as the hox cluster in animals  <cit> , the olfactory receptors in vertebrate genomes  <cit> , and numerous other examples in plants  <cit> , protists  <cit>  and other lineages.
recently duplicated genes have a strong tendency to become pseudogenes, and will generally be lost due to disabling mutations unless positive selection preserves the duplicate loci.
based on the divergence of surviving gene pairs in diverse genomes, the typical lifetime of duplicated genes in a diploid background has been estimated to be several million years  <cit> .
on a grander scale, entire genomes can be duplicated by polyploidization so that the cells of the resulting organism find themselves with two copies of every gene.
again, there is presumably a strong tendency towards rapid differential loss due to mutation of superfluous copies, and the long-term effect on the genome is elimination of most of the duplicate loci  <cit> .
in the case of polyploidy, the population dynamic and stoichiometric effects are different from the case of a localized duplication in a diploid background.
loss of a copy of a locally-duplicated gene simply restores the pre-duplication genome.
in contrast, in the case of whole genome duplication the polyploid population is presumably reproductively isolated from its diploid brethren, and inactivation/loss of one of a pair of duplicate sequences puts that gene at half the copy number of the remaining loci, at least in the early stages of rediploidization.
as haploinsufficiency is relatively rare  <cit> , reduced copy number is not by itself an overwhelming impediment to large scale loss, as is evident from analysis of surviving duplicates in the arabidopsis, rice, teleost, and yeast genomes  <cit> .
early thoughts on the selective forces leading to duplicate gene retention centered on divergence in protein function.
this suggests that one or both copies could acquire novel  <cit>  and/or complementary  <cit>  biochemical functions that would render both copies indispensable.
it was further recognized that novel or complementary organismal functions could arise from differential regulatory mutations  <cit> .
thus, if duplicate genes become expressed in different cell types or developmental stages, they might become indispensable and resistant to loss even if their associated peptides remain interchangeable.
through this mechanism, novel spatiotemporal roles can emerge, with numerous individual examples of cis- or trans-regulatory subfunctionalization known, for example, in teleost fish  <cit> .
the well-studied amphibian xenopus laevis has chromosome number  and genome size , roughly double that of its congener xenopus  tropicalis   <cit> .
this difference is attributed to a merger of two diploid progenitors originating ~ <dig> million years ago  <cit> .
allotetraploidy is suggested by the ease with which modern xenopus species can form hybrids via unreduced gametes  <cit> .
however, we cannot rule out an autotetraploid origin.
in this latter case, the duplicated pairs would be identical at the duplication event, whereas in the allotetraploid case such pairs would represent orthologs from the speciation event of the progenitors and might have separated at slightly different epochs prior to their last common ancestor, depending on the level of polymorphism at speciation.
however, the differences in measurable terms are subtle, and in the following we refer to polyploidization events as genome duplications regardless of their origin.
the x. laevis genome duplication is significantly more recent than the teleost-specific duplication )  <cit>  and the ancient vertebrate-specific duplications   <cit> .
however, it is older than the typical lifetime of duplicated genes in a diploid background   <cit> .
thus, by comparing x. laevis and x. tropicalis gene pairs, we can analyze an animal gene complement relatively soon after rediploidization, taking advantage of large-scale genome sequence data.
RESULTS
to study the evolution of duplicate gene pairs in x. laevis relative to their unique orthologs in x. tropicalis, we identified  <dig> x. laevis open reading frames  from an assembly of over half a million expressed sequence tags  and transcripts  <cit> , and compared them with each other and with a set of  <dig> predicted transcripts from the x. tropicalis genome project .
over half of the x. laevis orfs in our set appear to be complete – that is, with a plausible start and stop codon.
to measure the evolutionary divergence between x. laevis and x. tropicalis orthologs, and between gene pairs within x. laevis arising from the whole genome duplication event , we determined the transversion rate at four-fold synonymous codon positions, denoted  <dig> dtv .
we used transversions rather than total substitutions as  they occur at a slower rate than transitions,  they provide a simpler molecular clock as no assumptions or modeling of transition/transversion rates are needed for multiple-substitution correction, and  transversions are insensitive to local variations in gc content and unaffected by methylation effects.
figure 1a shows that  <dig> dtv distributions are sharply peaked for both x. laevis-x.
tropicalis intergenomic mutual best aligned pairs  and for x. laevis-x.
laevis intragenomic pairs , consistent with synchronous gene divergence due to speciation and gene duplication, respectively.
no corresponding recent peak was found in the x. tropicalis self comparison .
for comparison, the distributions of  <dig> dtv distances between mouse-rat, mouse-human, and mouse-frog orthologs are shown in figure 1b.
from this analysis we identified  <dig> likely x. laevis-x.
tropicalis  orthologous genes.
a simple molecular clock estimate puts the divergence of the x. laevis and x. tropicalis lineages at ~ <dig> mya, and the genome duplication event at ~ <dig> mya, consistent with mitochondrial data  <cit>  and a previous analysis of a dozen duplicated genes  <cit> .
guided by figure  <dig>  we conservatively identified pairs of x. laevis paralogs for  <dig> of the lt genes.
these define high confidence llt triplets such that  the x. laevis pair arose during the whole genome duplication event and is retained in the modern pseudotetraploid genome within the expressed gene dataset, and  the single x. tropicalis gene is the unique ortholog.
x. laevis paralogs are arbitrarily designated l <dig> and l2; both are "co-orthologs"  <cit>  of the corresponding x. tropicalis gene.
this set represents the largest collection of such triplets from any whole genome duplication event in animals – three to four times larger than in teleost fish  <cit>  and four to five times larger than in previous work on xenopus  <cit> .
zebrafish duplicates from the much older teleost genome duplication show near-saturation at the synonymous codon positions   <cit> .
how many of the ancient duplicated x. laevis gene pairs have subsequently lost one of the copies?
this number cannot be accurately determined with only a partial collection of x. laevis genes based on ests.
nevertheless, we can crudely estimate a likely loss range of 50–75%, as discussed in the methods section.
in some scenarios of duplicate gene evolution, one paralog experiences relaxed constraint and/or positive selection for a novel function, while the other evolves under negative selection.
to test for such asymmetric evolution, we identified amino acid positions in each llt triplet that were identical between the x. tropicalis peptide and one of its x. laevis co-orthologs , but changed in the other x. laevis sequence.
figure  <dig> compares the number of such changes per aligned position for each of the  <dig> x. laevis doublets with  <dig> or more total changes.
in general, changes are evenly distributed between l <dig> and l <dig>  with  <dig> pairs  showing significant asymmetry at the 1% level relative to a simple neutral model.
for this sample size, we would have expected only around six such outliers.
hence, while a few genes do show detectable asymmetric evolution, figure  <dig> is generally consistent with a hypothesis of symmetric change under purifying selection  <cit> .
this is in agreement with earlier results published by chain and evans  <cit>  who detected asymmetric evolution in <dig> of  <dig> x. laevis paralog pairs  based on somewhat different statistical criteria.
gene duplicates have been proposed to exhibit accelerated  <cit>  or slowed  <cit>  evolution, but the effects are subtle and hard to distinguish at the individual gene level.
to investigate this effect in bulk we compared amino acid and nucleotide changes between lt orthologs and ll paralogs over an alignment of half a million amino acids.
p-distances,  <dig> dtv , and the dn/ds ratio  are shown in table  <dig>  assuming that synonymous substitution rates are comparable across the genus xenopus, the nucleotide variation provides a simple molecular clock.
while ll pairs experienced only ~73% of the nucleotide change of the lt pairs , they accumulated ~94% as many amino acid changes.
thus, paralogous ll pairs exhibit a relative acceleration of 25–30% more amino acid substitutions  than orthologous lt pairs.
a similar acceleration is detected with the traditional dn/ds ratio .
this is demonstrated using both the p-dist/ <dig> dtv measure described in the text, and the conventional ratio of non-synonymous to synonymous substitution rates dn/ds.
both show a 25–30% enhancement in amino acid change.
these results are derived from a  <dig>  <dig> amino acid concatenated, gap-free, multiple sequence alignment  produced from the  <dig>  <dig> triplets possessing at least a  <dig> amino acid aligned block.
human-mouse-rat  and llt nucleotide divergences are roughly comparable , suggesting that the mammalian sequences can provide a control for variations in the evolutionary pattern of change across different gene families.
to compare evolutionary patterns in mammals to those in frogs, we identified  <dig> orthologous llthmr sextuplets from these five species totaling  <dig> aligned amino acids in conserved blocks .
figure  <dig> shows the amino acid substitution per synonymous transversion for different subsets of genes, normalized to the human-mouse value.
x. laevis paralogs show roughly double the rate of peptide change compared with both the human-mouse and mouse-rat matched controls.
the intermediate level of lt divergence within retained duplicates is consistent with this effect being confined to the x. laevis genes rather than a general feature of frogs that would also accelerate x. tropicalis genes.
this effect is subtle and requires our large dataset to detect, as it amounts to a little more than one additional amino acid substitution per peptide in x. laevis paralogs.
to test whether the acceleration found in x. laevis is a feature of retained gene duplicates or simply a feature of all genes in that lineage, we compared genes possessing observed paralogs with apparent single copy genes by identifying two mutually exclusive sets of orthologs from the five species.
set 5a consists of the original sextuplets with one of the x. laevis paralogs randomly removed from each gene.
the 5b quintuplets each have only a single laevis gene with no known recent  paralogs.
significantly accelerated evolution in x. laevis peptides is found only in genes with a confirmed paralog .
for the x. laevis genes without recent observed paralogs, the normalized peptide vs nucleotide ratio is  <dig>  ±  <dig> , much closer to the ratio of  <dig> seen between the other species.
due to the incompleteness of the est-derived x. laevis gene set we expect some of the 5b genes to have unobserved paralogs in the available x. laevis expressed gene set.
the observed ratio can be explained if ~20% of the 5b genes have as yet undetected paralogs with the same pattern of evolution as those in 5a.
to study the peptide evolution in x. laevis paralogs further, we identified  <dig> highly-constrained positions in the six-way llthmr multiple alignments, defined as positions with an identical amino acid in human, mouse, rat and at least two of the three frog orthologs.
the vast majority of these sites  were identical across all six peptides, but  <dig> sites  varied in just a single frog sequence.
of these, 26%  occurred in x. tropicalis, with ~37% in each of the x. laevis paralogs.
thus, even at highly-conserved positions duplicate x. laevis genes appear to be accepting additional substitutions eliminated by purifying selection in other species.
similar observations have been made in a number of previous studies .
are the extra copies of duplicated genes lost in a random fashion following tetraploidization or do different types of genes show different propensities for rentention or loss due, perhaps, to selective constraints?
to address this question, we assigned panther classification terms to the set of annotated x. tropicalis genes based on hhm models  <cit> .
we then grouped these genes into high-level categories of molecular function, biological function, and pathways and compared the relative frequencies of genes within these categories in genes with two retained copies  to that of a reference set, using tools and methods developed by thomas et al  <cit>  and described in detail herein.
as our reference set we chose all x. tropicalis genes with orthologs in x. laevis, whether or not a second x. laevis co-ortholog is present.
no significant difference in frequencies of genes in any of the molecular function categories were found between the two sets .
this is also true for the biological function and pathway classifications  and is in agreement with a similar comparison performed by morin et al  <cit> .
genes were categorized by assigning panther classification terms to our x. tropicalis gene set by hmm scanning of the peptides and then grouping the terms into high-level categories of molecular function.
we were able to assign categories to  <dig> genes in the reference set and to  <dig> genes in the subset.
out null hypothesis is that the second copy of genes following the x. laevis tetraploidization are lost in a random fashion.
none of the  <dig> categories shown here show significant deviation from the null hypothesis, and the remaining molecular categories all have p-values close to  <dig>  all p-values were bonferroni-corrected for multiple tests.
in addition to sequence evolution, the spatiotemporal expression of duplicated genes could become altered rapidly, generating strong selective pressure to retain both duplicates.
this issue has not been addressed in previous studies  <cit> .
to begin to investigate expression differences between ll paralogs, we analyzed est data.
while most x. laevis genes in our set do not have sufficient counts in any one est library for a statistically significant determination of differential expression, two large est sets are available that allow us to address this question: the osada anterior neuroectoderm library  <cit>   and the nibb early gastrulation library .
under a simple null model for equal expression rates,  <dig> of the  <dig> pairs in ane  have p-values less than  <dig> , with only ~ <dig>  expected under the null hypothesis.
at a p-value less than  <dig> , we expect  <dig>  false positives but observe  <dig> significant deviations from equal expression.
thus, 40–50% of x. laevis genes with sufficient est data show differential expression in the anterior neuroectoderm, and similarly in the early gastrulation dataset .
this suggests that many x. laevis paralogs have accumulated differential regulatory changes such that they are no longer functionally redundant  <cit>  in terms of their organismal/developmental role.
note that our fraction of doublets showing differential expression is considerably higher than the ~14% found by morin et al  <cit> .
a total of  <dig> matched pairs of antisense "probes" were computed as described in methods, and applied to the est data by in silico hybridization.
genes with  <dig> or more hits to ests were used to test the null hypothesis that expression levels are the same between paralogs.
the four libraries are: ane   <cit> ; nibbegast ; nich_brain <dig> ; and xgc_kid <dig> .
higher spatiotemporal resolution of gene expression can be obtained with in situ hybridization.
using antisense probes to the highly variable 3' end of transcripts, we examined the expression patterns of four gene triplets in both frog species: the cyclin-associated protein skp1a, forkhead box transcription factor foxa <dig>  the metabolic enzyme isocitrate dehydrogenase , and the calcium binding protein sorcin.
the spatial expression patterns of the paralogs in x. laevis differ from one another and from the pattern of their unique ortholog in x. tropicalis .
a striking example is skp1a, whose amino acid sequence is 100% identical in all three frog peptides.
this peptide is therefore under strong selection across its entire length.
one paralog is expressed in the kidney and multiple head structures where the other paralog is either not expressed or only weakly so.
these data support studies of other gene pairs in x. laevis  that show subdivided expression patterns relative to single copy counterparts in mammals  <cit> .
CONCLUSIONS
the duplication of an entire genome is a spectacular natural experiment in which tens of thousands of genes are effectively duplicated synchronously, so that each gene has a matched "paralogous" partner with a highly similar or identical sequence and chromosomal context.
subsequent divergence, loss, and rearrangement then gradually erode the signs of duplication.
whole genome duplication can be a powerful evolutionary force, but the polyploidies and subsequent rediploidization that occurred early in the vertebrate and teleost lineages are so ancient  that the immediate evolutionary response is obscured in modern genomes.
genome tetraploidization occurred more recently in the evolution of x. laevis and with extensive genomic and cdna sequencing available this provides a unique opportunity to analyze a genome in the process of reacting to a recent tetraploidization.
we identify more than  <dig> cases in which a single gene in x. tropicalis possesses precisely two co-orthologous genes in x. laevis, both of which have survived until the present – the largest such collection of orthologs from an animal whole genome duplication.
analysis of such triplets reveals an accelerated evolution, or relaxation of constraint, in the peptides of the x. laevis duplicates compared to their orthologs in x. tropicalis and other vertebrates.
in contrast, x. laevis genes for which only one duplicate is retained do not appear to show such acceleration.
this is a subtle effect for any single gene, affecting on average only ~1– <dig> amino acids per peptide, and can only be confidently established by means of the large number of genes available for analysis.
the relaxed constraint experienced by retained duplicates is consistent with overlapping/redundant biochemical functions.
the response to genome duplication, however, is more complex than simply relaxing sequence constraints.
in one notable example, duplicate x. laevis genes produce identical peptides that are also identical to their  x. tropicalis ortholog.
in this case, and in other examples studied with in situ hybridization, the x. laevis duplicates were found to be expressed in different patterns during development.
we looked for other examples of differential gene expression by considering est counts in deeply sequenced cdna libraries, and found that a significant fraction  of duplicate genes show divergent expression levels in specific tissues.
these results are consistent with the subfunctionalization model for the retention of duplicated genes  <cit> , in which paralogs acquire complementary coding and/or cis-regulatory mutations that leave both copies subject to purifying selection.
these changes must occur rapidly, as the lifetime of truly redundant duplicates would be short  due to  the ease with which single nucleotide mutations across a gene can generate a null allele, and  the expected nearly neutral selection on such a null allele in the presence of a second locus of identical function.
while whole genome duplications are found in the ancestry of vertebrates, teleost fishes, yeasts, and multiple angiosperm lineages, there are relatively few cases in which a duplicated genome has a natural unduplicated sister sequence that can provide a recent comparative reference.
for example, tetrapods can serve as a sister taxon for the study of the teleost duplication, but with a divergence of ~ <dig> million years; for arabidopsis, the related taxa all share either more ancient duplications or their own unique duplications that complicate analysis.
the x. tropicalis/x.
laevis system provides an ideal testing ground for ideas about whole genome duplication, as the timing of the x. laevis tetraploidization is neither "too recent" compared with the lifetime of a duplicated locus, nor "too ancient" for measures of nucleotide variation to have reached saturation.
the x. tropicalis genome is available in draft form .
as we have shown, the divergence of the two x. laevis sub-genomes is extensive, comparable to the divergence between mouse and rat.
this suggests that whole genome shotgun approaches would successfully capture the genic regions of the x. laevis genome and provide a unique comparative reference for the study of genome evolution.

 gene duplication followed by subsequent functional divergence is widely recognized as an important mechanism for the evolution of novelty. on a small scale, local tandem duplications can rapidly produce new gene families, such as the hox cluster in animals, the olfactory receptors in vertebrate genomes, and numerous other examples in plants, protists and other lineages. recently duplicated genes have a strong tendency to become pseudogenes, and will generally be lost due to disabling mutations unless positive selection preserves the duplicate loci. based on the divergence of surviving gene pairs in diverse genomes, the typical lifetime of duplicated genes in a diploid background has been estimated to be several million years. on a grander scale, entire genomes can be duplicated by polyploidization so that the cells of the resulting organism find themselves with two copies of every gene. again, there is presumably a strong tendency towards rapid differential loss due to mutation of superfluous copies, and the long-term effect on the genome is elimination of most of the duplicate loci. in the case of polyploidy, the population dynamic and stoichiometric effects are different from the case of a localized duplication in a diploid background. loss of a copy of a locally-duplicated gene simply restores the pre-duplication genome. in contrast, in the case of whole genome duplication the polyploid population is presumably reproductively isolated from its diploid brethren, and inactivation/loss of one of a pair of duplicate sequences puts that gene at half the copy number of the remaining loci, at least in the early stages of rediploidization. as haploinsufficiency is relatively rare, reduced copy number is not by itself an overwhelming impediment to large scale loss, as is evident from analysis of surviving duplicates in the arabidopsis, rice, teleost, and yeast genomes. early thoughts on the selective forces leading to duplicate gene retention centered on divergence in protein function. this suggests that one or both copies could acquire novel and/or complementary biochemical functions that would render both copies indispensable. it was further recognized that novel or complementary organismal functions could arise from differential regulatory mutations. thus, if duplicate genes become expressed in different cell types or developmental stages, they might become indispensable and resistant to loss even if their associated peptides remain interchangeable. through this mechanism, novel spatiotemporal roles can emerge, with numerous individual examples of cis- or trans-regulatory subfunctionalization known, for example, in teleost fish. the well-studied amphibian xenopus laevis has chromosome number and genome size, roughly double that of its congener xenopus tropicalis. this difference is attributed to a merger of two diploid progenitors originating ~ million years ago. allotetraploidy is suggested by the ease with which modern xenopus species can form hybrids via unreduced gametes. however, we cannot rule out an autotetraploid origin. in this latter case, the duplicated pairs would be identical at the duplication event, whereas in the allotetraploid case such pairs would represent orthologs from the speciation event of the progenitors and might have separated at slightly different epochs prior to their last common ancestor, depending on the level of polymorphism at speciation. however, the differences in measurable terms are subtle, and in the following we refer to polyploidization events as genome duplications regardless of their origin. the x. laevis genome duplication is significantly more recent than the teleost-specific duplication ) and the ancient vertebrate-specific duplications. however, it is older than the typical lifetime of duplicated genes in a diploid background. thus, by comparing x. laevis and x. tropicalis gene pairs, we can analyze an animal gene complement relatively soon after rediploidization, taking advantage of large-scale genome sequence data.  to study the evolution of duplicate gene pairs in x. laevis relative to their unique orthologs in x. tropicalis, we identified x. laevis open reading frames from an assembly of over half a million expressed sequence tags and transcripts, and compared them with each other and with a set of predicted transcripts from the x. tropicalis genome project. over half of the x. laevis orfs in our set appear to be complete – that is, with a plausible start and stop codon. to measure the evolutionary divergence between x. laevis and x. tropicalis orthologs, and between gene pairs within x. laevis arising from the whole genome duplication event, we determined the transversion rate at four-fold synonymous codon positions, denoted dtv. we used transversions rather than total substitutions as they occur at a slower rate than transitions, they provide a simpler molecular clock as no assumptions or modeling of transition/transversion rates are needed for multiple-substitution correction, and transversions are insensitive to local variations in gc content and unaffected by methylation effects. figure 1a shows that dtv distributions are sharply peaked for both x. laevis-x. tropicalis intergenomic mutual best aligned pairs and for x. laevis-x. laevis intragenomic pairs, consistent with synchronous gene divergence due to speciation and gene duplication, respectively. no corresponding recent peak was found in the x. tropicalis self comparison. for comparison, the distributions of dtv distances between mouse-rat, mouse-human, and mouse-frog orthologs are shown in figure 1b. from this analysis we identified likely x. laevis-x. tropicalis orthologous genes. a simple molecular clock estimate puts the divergence of the x. laevis and x. tropicalis lineages at ~ mya, and the genome duplication event at ~ mya, consistent with mitochondrial data and a previous analysis of a dozen duplicated genes. guided by figure we conservatively identified pairs of x. laevis paralogs for of the lt genes. these define high confidence llt triplets such that the x. laevis pair arose during the whole genome duplication event and is retained in the modern pseudotetraploid genome within the expressed gene dataset, and the single x. tropicalis gene is the unique ortholog. x. laevis paralogs are arbitrarily designated l and l2; both are "co-orthologs" of the corresponding x. tropicalis gene. this set represents the largest collection of such triplets from any whole genome duplication event in animals – three to four times larger than in teleost fish and four to five times larger than in previous work on xenopus. zebrafish duplicates from the much older teleost genome duplication show near-saturation at the synonymous codon positions. how many of the ancient duplicated x. laevis gene pairs have subsequently lost one of the copies? this number cannot be accurately determined with only a partial collection of x. laevis genes based on ests. nevertheless, we can crudely estimate a likely loss range of 50–75%, as discussed in the methods section. in some scenarios of duplicate gene evolution, one paralog experiences relaxed constraint and/or positive selection for a novel function, while the other evolves under negative selection. to test for such asymmetric evolution, we identified amino acid positions in each llt triplet that were identical between the x. tropicalis peptide and one of its x. laevis co-orthologs, but changed in the other x. laevis sequence. figure compares the number of such changes per aligned position for each of the x. laevis doublets with or more total changes. in general, changes are evenly distributed between l and l with pairs showing significant asymmetry at the 1% level relative to a simple neutral model. for this sample size, we would have expected only around six such outliers. hence, while a few genes do show detectable asymmetric evolution, figure is generally consistent with a hypothesis of symmetric change under purifying selection. this is in agreement with earlier results published by chain and evans who detected asymmetric evolution in of x. laevis paralog pairs based on somewhat different statistical criteria. gene duplicates have been proposed to exhibit accelerated or slowed evolution, but the effects are subtle and hard to distinguish at the individual gene level. to investigate this effect in bulk we compared amino acid and nucleotide changes between lt orthologs and ll paralogs over an alignment of half a million amino acids. p-distances, dtv, and the dn/ds ratio are shown in table assuming that synonymous substitution rates are comparable across the genus xenopus, the nucleotide variation provides a simple molecular clock. while ll pairs experienced only ~73% of the nucleotide change of the lt pairs, they accumulated ~94% as many amino acid changes. thus, paralogous ll pairs exhibit a relative acceleration of 25–30% more amino acid substitutions than orthologous lt pairs. a similar acceleration is detected with the traditional dn/ds ratio. this is demonstrated using both the p-dist/ dtv measure described in the text, and the conventional ratio of non-synonymous to synonymous substitution rates dn/ds. both show a 25–30% enhancement in amino acid change. these results are derived from a amino acid concatenated, gap-free, multiple sequence alignment produced from the triplets possessing at least a amino acid aligned block. human-mouse-rat and llt nucleotide divergences are roughly comparable, suggesting that the mammalian sequences can provide a control for variations in the evolutionary pattern of change across different gene families. to compare evolutionary patterns in mammals to those in frogs, we identified orthologous llthmr sextuplets from these five species totaling aligned amino acids in conserved blocks. figure shows the amino acid substitution per synonymous transversion for different subsets of genes, normalized to the human-mouse value. x. laevis paralogs show roughly double the rate of peptide change compared with both the human-mouse and mouse-rat matched controls. the intermediate level of lt divergence within retained duplicates is consistent with this effect being confined to the x. laevis genes rather than a general feature of frogs that would also accelerate x. tropicalis genes. this effect is subtle and requires our large dataset to detect, as it amounts to a little more than one additional amino acid substitution per peptide in x. laevis paralogs. to test whether the acceleration found in x. laevis is a feature of retained gene duplicates or simply a feature of all genes in that lineage, we compared genes possessing observed paralogs with apparent single copy genes by identifying two mutually exclusive sets of orthologs from the five species. set 5a consists of the original sextuplets with one of the x. laevis paralogs randomly removed from each gene. the 5b quintuplets each have only a single laevis gene with no known recent paralogs. significantly accelerated evolution in x. laevis peptides is found only in genes with a confirmed paralog. for the x. laevis genes without recent observed paralogs, the normalized peptide vs nucleotide ratio is ±, much closer to the ratio of seen between the other species. due to the incompleteness of the est-derived x. laevis gene set we expect some of the 5b genes to have unobserved paralogs in the available x. laevis expressed gene set. the observed ratio can be explained if ~20% of the 5b genes have as yet undetected paralogs with the same pattern of evolution as those in 5a. to study the peptide evolution in x. laevis paralogs further, we identified highly-constrained positions in the six-way llthmr multiple alignments, defined as positions with an identical amino acid in human, mouse, rat and at least two of the three frog orthologs. the vast majority of these sites were identical across all six peptides, but sites varied in just a single frog sequence. of these, 26% occurred in x. tropicalis, with ~37% in each of the x. laevis paralogs. thus, even at highly-conserved positions duplicate x. laevis genes appear to be accepting additional substitutions eliminated by purifying selection in other species. similar observations have been made in a number of previous studies. are the extra copies of duplicated genes lost in a random fashion following tetraploidization or do different types of genes show different propensities for rentention or loss due, perhaps, to selective constraints? to address this question, we assigned panther classification terms to the set of annotated x. tropicalis genes based on hhm models. we then grouped these genes into high-level categories of molecular function, biological function, and pathways and compared the relative frequencies of genes within these categories in genes with two retained copies to that of a reference set, using tools and methods developed by thomas et al and described in detail herein. as our reference set we chose all x. tropicalis genes with orthologs in x. laevis, whether or not a second x. laevis co-ortholog is present. no significant difference in frequencies of genes in any of the molecular function categories were found between the two sets. this is also true for the biological function and pathway classifications and is in agreement with a similar comparison performed by morin et al. genes were categorized by assigning panther classification terms to our x. tropicalis gene set by hmm scanning of the peptides and then grouping the terms into high-level categories of molecular function. we were able to assign categories to genes in the reference set and to genes in the subset. out null hypothesis is that the second copy of genes following the x. laevis tetraploidization are lost in a random fashion. none of the categories shown here show significant deviation from the null hypothesis, and the remaining molecular categories all have p-values close to all p-values were bonferroni-corrected for multiple tests. in addition to sequence evolution, the spatiotemporal expression of duplicated genes could become altered rapidly, generating strong selective pressure to retain both duplicates. this issue has not been addressed in previous studies. to begin to investigate expression differences between ll paralogs, we analyzed est data. while most x. laevis genes in our set do not have sufficient counts in any one est library for a statistically significant determination of differential expression, two large est sets are available that allow us to address this question: the osada anterior neuroectoderm library and the nibb early gastrulation library. under a simple null model for equal expression rates, of the pairs in ane have p-values less than, with only ~ expected under the null hypothesis. at a p-value less than, we expect false positives but observe significant deviations from equal expression. thus, 40–50% of x. laevis genes with sufficient est data show differential expression in the anterior neuroectoderm, and similarly in the early gastrulation dataset. this suggests that many x. laevis paralogs have accumulated differential regulatory changes such that they are no longer functionally redundant in terms of their organismal/developmental role. note that our fraction of doublets showing differential expression is considerably higher than the ~14% found by morin et al. a total of matched pairs of antisense "probes" were computed as described in methods, and applied to the est data by in silico hybridization. genes with or more hits to ests were used to test the null hypothesis that expression levels are the same between paralogs. the four libraries are: ane; nibbegast; nich_brain; and xgc_kid. higher spatiotemporal resolution of gene expression can be obtained with in situ hybridization. using antisense probes to the highly variable 3' end of transcripts, we examined the expression patterns of four gene triplets in both frog species: the cyclin-associated protein skp1a, forkhead box transcription factor foxa the metabolic enzyme isocitrate dehydrogenase, and the calcium binding protein sorcin. the spatial expression patterns of the paralogs in x. laevis differ from one another and from the pattern of their unique ortholog in x. tropicalis. a striking example is skp1a, whose amino acid sequence is 100% identical in all three frog peptides. this peptide is therefore under strong selection across its entire length. one paralog is expressed in the kidney and multiple head structures where the other paralog is either not expressed or only weakly so. these data support studies of other gene pairs in x. laevis that show subdivided expression patterns relative to single copy counterparts in mammals.  the duplication of an entire genome is a spectacular natural experiment in which tens of thousands of genes are effectively duplicated synchronously, so that each gene has a matched "paralogous" partner with a highly similar or identical sequence and chromosomal context. subsequent divergence, loss, and rearrangement then gradually erode the signs of duplication. whole genome duplication can be a powerful evolutionary force, but the polyploidies and subsequent rediploidization that occurred early in the vertebrate and teleost lineages are so ancient that the immediate evolutionary response is obscured in modern genomes. genome tetraploidization occurred more recently in the evolution of x. laevis and with extensive genomic and cdna sequencing available this provides a unique opportunity to analyze a genome in the process of reacting to a recent tetraploidization. we identify more than cases in which a single gene in x. tropicalis possesses precisely two co-orthologous genes in x. laevis, both of which have survived until the present – the largest such collection of orthologs from an animal whole genome duplication. analysis of such triplets reveals an accelerated evolution, or relaxation of constraint, in the peptides of the x. laevis duplicates compared to their orthologs in x. tropicalis and other vertebrates. in contrast, x. laevis genes for which only one duplicate is retained do not appear to show such acceleration. this is a subtle effect for any single gene, affecting on average only ~1– amino acids per peptide, and can only be confidently established by means of the large number of genes available for analysis. the relaxed constraint experienced by retained duplicates is consistent with overlapping/redundant biochemical functions. the response to genome duplication, however, is more complex than simply relaxing sequence constraints. in one notable example, duplicate x. laevis genes produce identical peptides that are also identical to their x. tropicalis ortholog. in this case, and in other examples studied with in situ hybridization, the x. laevis duplicates were found to be expressed in different patterns during development. we looked for other examples of differential gene expression by considering est counts in deeply sequenced cdna libraries, and found that a significant fraction of duplicate genes show divergent expression levels in specific tissues. these results are consistent with the subfunctionalization model for the retention of duplicated genes, in which paralogs acquire complementary coding and/or cis-regulatory mutations that leave both copies subject to purifying selection. these changes must occur rapidly, as the lifetime of truly redundant duplicates would be short due to the ease with which single nucleotide mutations across a gene can generate a null allele, and the expected nearly neutral selection on such a null allele in the presence of a second locus of identical function. while whole genome duplications are found in the ancestry of vertebrates, teleost fishes, yeasts, and multiple angiosperm lineages, there are relatively few cases in which a duplicated genome has a natural unduplicated sister sequence that can provide a recent comparative reference. for example, tetrapods can serve as a sister taxon for the study of the teleost duplication, but with a divergence of ~ million years; for arabidopsis, the related taxa all share either more ancient duplications or their own unique duplications that complicate analysis. the x. tropicalis/x. laevis system provides an ideal testing ground for ideas about whole genome duplication, as the timing of the x. laevis tetraploidization is neither "too recent" compared with the lifetime of a duplicated locus, nor "too ancient" for measures of nucleotide variation to have reached saturation. the x. tropicalis genome is available in draft form. as we have shown, the divergence of the two x. laevis sub-genomes is extensive, comparable to the divergence between mouse and rat. this suggests that whole genome shotgun approaches would successfully capture the genic regions of the x. laevis genome and provide a unique comparative reference for the study of genome evolution. 
we find no significant difference in gene content in the duplicated set, versus the single-copy set based on molecular and biological function ontologies. these results support a scenario in which duplicate genes are retained through a process of subfunctionalization and/or relaxation of constraint on both copies of an ancestral gene. we identified gene triplets in which a single gene in x. tropicalis corresponds to precisely two co-orthologous genes in x. laevis – the largest such collection published from any duplication event in animals. duplicated genes can differ substantially in expression levels and patterns. ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. analysis of the considerable amount of est sequence available for this species together with the genome sequence of the related diploid xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication. analysis of these triplets reveals accelerated evolution or relaxation of constraint in the peptides of the x. laevis pairs compared with the orthologous sequences in x. tropicalis and other vertebrates.
 ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. the african clawed frog xenopus laevis experienced a relatively recent tetraploidization ~ million years ago. analysis of the considerable amount of est sequence available for this species together with the genome sequence of the related diploid xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.  we identified gene triplets in which a single gene in x. tropicalis corresponds to precisely two co-orthologous genes in x. laevis – the largest such collection published from any duplication event in animals. analysis of these triplets reveals accelerated evolution or relaxation of constraint in the peptides of the x. laevis pairs compared with the orthologous sequences in x. tropicalis and other vertebrates. in contrast, single-copy x. laevis genes do not show this acceleration. duplicated genes can differ substantially in expression levels and patterns. we find no significant difference in gene content in the duplicated set, versus the single-copy set based on molecular and biological function ontologies.  these results support a scenario in which duplicate genes are retained through a process of subfunctionalization and/or relaxation of constraint on both copies of an ancestral gene. 