Epigenetic Changes and Repositioning Determine the Evolutionary Fate of Duplicated Genes

Consideration of epigenetic silencing, perhaps by DNA methylation, led to an epigenetic complementation (EC) model for evolution by gene duplication (Rodin and Riggs (2003) J. Mol. Evol., 56, 718-729). This and subsequent work on genome-wide analyses of gene duplicates in several eukaryotic species pointed to a fundamental link between localization in the genome, epigenetic regulation of expression, and the evolutionary fate of new redundant gene copies, which can be either non- or neo-functionalization. Our main message in this report is that repositioning of a new duplicate to an ectopic site epigenetically alters its expression pattern, and concomitantly the rate and direction of mutations. Furthermore, comparison of syntenic vs. non-syntenic pairs of gene duplicates of different age unambiguously indicates that repositioning saves redundant gene duplicates from pseudogenization and hastens their evolution towards a new development-time and tissue-specific pattern of function.     

Rapid divergence of gene duplicates on the Drosophila melanogaster X chromosome

The recent sequencing of several eukaryotic genomes has generated considerable interest in the study of gene duplication events. The classical model of duplicate gene evolution is that recurrent mutation ultimately results in one copy becoming a pseudogene, and only rarely will a beneficial new function evolve. Here, we study divergence between coding sequence duplications in Drosophila melanogaster as a function of the linkage relationship between paralogs. The mean K(a)/K(s) between all duplicates in the D. melanogaster genome is 0.2803, indicating that purifying selection is maintaining the structure of duplicate coding sequences. However, the mean K(a)/K(s) between duplicates that are both on the X chromosome is 0.4701, significantly higher than the genome average. Further, the distribution of K(a)/K(s) for these X-linked duplicates is significantly shifted toward higher values when compared with the distributions for paralogs in other linkage relationships. Two models of molecular evolution provide qualitative explanations of these observations-relaxation of selective pressure on the duplicate copies and, more likely, positive selection on recessive adaptations. We also show that there is an excess of X-linked duplicates with low K(s), suggesting a larger proportion of relatively young duplicates on the D. melanogaster X chromosome relative to autosomes.     

The evolutionary fate of recently duplicated retrogenes in mice

Inferences about the evolutionary impact of gene duplications often rely on the analysis of their long-term outcome. The fate of the majority of them must, however, be decided shortly after duplication. Here we analysed the evolutionary pattern of 10 mouse genes very recently duplicated by retrotransposition, by sequencing the retroposed copy in five to 10 closely related mouse species. In all cases the retroposed copy experienced accelerated nonsynonymous evolution whereas the divergence pattern of the source copy appeared unaffected by the duplication, consistent with the neofunctionalization model. The analysis further revealed that most retrogenes, including pseudogenes, did not experience a period of relaxed neutral evolution, but have been submitted to purifying selection ever since their retroposition. We propose that these duplicates play a biochemical role but are not indispensable. Purifying selection prevents them from acquiring a negative role until they are lost or silenced. This period of unnecessary redundancy could in rare cases give the time for new functions to evolve.     

Comparative analysis of teleost fish genomes reveals preservation of different ancient clock duplicates in different fishes

Clock (Circadian locomotor output cycle kaput) was the first vertebrate circadian clock gene identified in a mouse forward genetics mutagenesis screen. It encodes a bHLH-PAS protein that is highly conserved throughout evolution. Tetrapods also have the second Clock gene, Clock2 or Npas2 (Neuronal PAS domain protein 2). Conversely, the fruit fly, an invertebrate, has only one clock gene. Interrogation of the five teleost fish genome databases revealed that the zebrafish and the Japanese pufferfish (fugu) each have three clock genes, whereas the green spotted pufferfish (tetraodon), the Japanese medaka fish and the three-spine stickleback each have two clock genes. Phylogenetic and splice site analyses indicated that zebrafish and fugu each have two clock1 genes, clock1a and clock1b and one clock2; tetraodon also have clock1a and clock1b but do not have clock2; and medaka and stickleback each have clock1b and one clock2. Genome neighborhood analysis further showed that clock1a/clock1b in zebrafish, fugu and tetraodon is an ancient duplicate. While the dN/dS ratios of these three fish clock duplicates are all <1, indicating that purifying selection has acted upon them; the Tajima relative rate test showed that all three fish clock duplicates have asymmetric evolutionary rates, implicating that one of these duplicates have been under positive selection or relaxed functional constraint. These results support the view that teleost fish clock genes were generated from an ancient genome-wide duplication, and differential gene loss after the duplication resulted in retention of different ancient duplicates in different teleost fishes, which could have contributed to the evolution of the distinct fish circadian clock mechanisms.     

An ancient frame-shifting event in the highly conserved KPNA gene family has undergone extensive compensation by natural selection in vertebrates

One of the prevailing arguments in evolutionary theory is that the duplicates of genes can acquire novel functionality. This is because only one of the paralogs need maintain the ancestral function, leaving room for natural experimentation due to a respite in purifying selection. Although many duplicates can subsequently become disabled by nullifying mutations, a few may also go on to diverge along a novel evolutionary trajectory. Here, evidence is provided that demonstrates how this scenario may not always be true. Rather, in the case of the highly conserved KPNA importin family, an initial relaxation in selection induced a frameshift that was later suppressed and heavily compensated for as part of a reparative and optimizing process. Despite a resulting divergence, there remains a distinct preservation of both sequence and functionality among the paralogs. This would indicate that duplicates can be retained by selection for reasons related to their redundant functionality. It also shows that, even when positive selection is inferred in duplicate genes, this may be of a compensatory nature rather than one representing any biochemical innovation. Generally, this development would perhaps be a more common outcome for gene duplication than is currently maintained.     

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, “marking” one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.     

Not born equal: increased rate asymmetry in relocated and retrotransposed rodent gene duplicates

Duplicated genes frequently evolve at different rates. This asymmetry is evidence of natural selection's ability to discriminate between the 2 copies, subjecting them to different levels of purifying selection or even permitting adaptive evolution of one or both copies. However, if gene duplication creates pairs of protein-coding sequences that are initially identical, this raises the question of how selection tells the 2 copies apart. Here, we investigated asymmetric sequence divergence of recently duplicated genes in rodents and related this to 2 possible sources of such asymmetry: gene relocation as a consequence of duplication and retrotransposition as a mechanism of gene duplication. We found that most young rodent duplicates that have been relocated were created by retrotransposition. The degree of rate asymmetry in gene pairs where one copy has been relocated (either by retrotransposition or DNA-based duplication) is greater than in pairs formed by local DNA-based duplication events. Furthermore, by considering the direction of transposition for distant duplicates, we found a consistent tendency for retrogenes to undergo accelerated protein evolution relative to their static paralogs, whereas DNA-based transpositions showed no such tendency. Finally, we demonstrate that the faster sequence evolution of retrogenes correlates with the profound alteration of their expression pattern that is precipitated by retrotransposition.     

Excess of amino acid substitutions relative to polymorphism between X-linked duplications in Drosophila melanogaster

We have obtained sequence polymorphism data from 13 genes belonging to 5 gene families in Drosophila melanogaster where the K(a)/K(s) between copies is greater than 1. Twelve of these 13 loci are X-linked. In general, there is evidence of purifying selection in all families, as inferred both from levels of silent and replacement variation and insertion/deletion variation, suggesting that the loci are likely functional. Shared polymorphisms indicative of gene conversion between paralogs are rare among the X-linked families, in contrast to available data from autosomal duplicates. McDonald-Kreitman tests between duplicates reveal an excess of amino-acid fixations between copies in the X-linked families, suggesting that the divergence between these loci was driven by positive selection. In contrast, available data from autosomal duplicates show a deficit of fixations, consistent with gene conversion being a strong homogenizing force.     

Duplicated genes evolve slower than singletons despite the initial rate increase

Background

Gene duplication is an important mechanism that can lead to the emergence of new functions during evolution. The impact of duplication on the mode of gene evolution has been the subject of several theoretical and empirical comparative-genomic studies. It has been shown that, shortly after the duplication, genes seem to experience a considerable relaxation of purifying selection.

Results

Here we demonstrate two opposite effects of gene duplication on evolutionary rates. Sequence comparisons between paralogs show that, in accord with previous observations, a substantial acceleration in the evolution of paralogs occurs after duplication, presumably due to relaxation of purifying selection. The effect of gene duplication on evolutionary rate was also assessed by sequence comparison between orthologs that have paralogs (duplicates) and those that do not (singletons). It is shown that, in eukaryotes, duplicates, on average, evolve significantly slower than singletons. Eukaryotic ortholog evolutionary rates for duplicates are also negatively correlated with the number of paralogs per gene and the strength of selection between paralogs. A tally of annotated gene functions shows that duplicates tend to be enriched for proteins with known functions, particularly those involved in signaling and related cellular processes; by contrast, singletons include an over-abundance of poorly characterized proteins.

Conclusions

These results suggest that whether or not a gene duplicate is retained by selection depends critically on the pre-existing functional utility of the protein encoded by the ancestral singleton. Duplicates of genes of a higher biological import, which are subject to strong functional constraints on the sequence, are retained relatively more often. Thus, the evolutionary trajectory of duplicated genes appears to be determined by two opposing trends, namely, the post-duplication rate acceleration and the generally slow evolutionary rate owing to the high level of functional constraints.

     

Correlated asymmetry of sequence and functional divergence between duplicate proteins of Saccharomyces cerevisiae

The role of sequence divergence in functional divergence of duplicate genes is a topic of great interest. In this study, we compare the numbers of amino acid substitutions in each sequence since two yeast duplicates diverged, using a preduplication ancestral outgroup. Using this strategy, we explored the relationship between sequence divergence and functional divergence between duplicate partners. We show that the degree of relative functional asymmetry between duplicate proteins is proportional to the relative sequence divergence between them. Furthermore, of the two duplicates, the copy closer to their ancestral sequence (fewer number of amino acid substitutions) interacts with more proteins and affects fitness more severely when deleted. Therefore, asymmetric sequence divergence between duplicates is correlated with asymmetric functional divergence and may underlie the duplicate's role in genetic robustness against mutations. Among the functional traits considered, protein abundance appears to have the strongest correlation with the nonsynonymous divergence between duplicates. Taken together with the results from whole-genome analyses, our results indicate that within-species duplicates are subject to the same evolutionary force that acts on interspecific sequence and functional divergence. In particular, we detect signs of purifying selection on the more slowly evolving duplicate.     

Degree of Functional Divergence in Duplicates Is Associated with Distinct Roles in Plant Evolution

Gene duplication is a major mechanism to create new genes. After gene duplication, some duplicated genes undergo functionalization, whereas others largely maintain redundant functions. Duplicated genes comprise various degrees of functional diversification in plants. However, the evolutionary fate of high and low diversified duplicates is unclear at genomic scale. To infer high and low diversified duplicates in Arabidopsis thaliana genome, we generated a prediction method for predicting whether a pair of duplicate genes was subjected to high or low diversification based on the phenotypes of knock-out mutants. Among 4,017 pairs of recently duplicated A. thaliana genes, 1,052 and 600 are high and low diversified duplicate pairs, respectively. The predictions were validated based on the phenotypes of generated knock-down transgenic plants. We determined that the high diversified duplicates resulting from tandem duplications tend to have lineage-specific functions, whereas the low diversified duplicates produced by whole-genome duplications are related to essential signaling pathways. To assess the evolutionary impact of high and low diversified duplicates in closely related species, we compared the retention rates and selection pressures on the orthologs of A. thaliana duplicates in two closely related species. Interestingly, high diversified duplicates resulting from tandem duplications tend to be retained in multiple lineages under positive selection. Low diversified duplicates by whole-genome duplications tend to be retained in multiple lineages under purifying selection. Taken together, the functional diversities determined by different duplication mechanisms had distinct effects on plant evolution.     

Signatures of selection and sex-specific expression variation of a novel duplicate during the evolution of the Drosophila desaturase gene family

The tempo and mode of evolution of loci with a large effect on adaptation and reproductive isolation will influence the rate of evolutionary divergence and speciation. Desaturase loci are involved in key biochemical changes in long-chain fatty acids. In insects, these have been shown to influence adaptation to starvation or desiccation resistance and in some cases act as important pheromones. The desaturase gene family of Drosophila is known to have evolved by gene duplication and diversification, and at least one locus shows rapid evolution of sex-specific expression variation. Here, we examine the evolution of the gene family in species representing the Drosophila phylogeny. We find that the family includes more loci than have been previously described. Most are represented as single-copy loci, but we also find additional examples of duplications in loci which influence pheromone blends. Most loci show patterns of variation associated with purifying selection, but there are strong signatures of diversifying selection in new duplicates. In the case of a new duplicate of desat1 in the obscura group species, we show that strong selection on the coding sequence is associated with the evolution of sex-specific expression variation. It seems likely that both sexual selection and ecological adaptation have influenced the evolution of this gene family in Drosophila.     

Relaxed Selection Among Duplicate Floral Regulatory Genes in Lamiales

Polyploidization is a prevalent mode of genome diversification within plants. Most gene duplicates arising from polyploidization (paralogs) are typically lost, although a subset may be maintained under selection due to dosage, partitioning of gene function, or acquisition of novel functions. Because they experience selection in the presence of other duplicate loci across the genome, interactions among genes may also play a significant role in the maintenance of paralogs resulting from polyploidization. Previously, we identified duplicates of the genes LFY/FLO and AP3/DEF that directly interact in a floral regulatory pathway and are thought to be the result of ancient polyploidization in the Lamiales (> 50 mya). Although duplicates of MADS box genes including AP3/DEF are common throughout the angiosperm lineage, LFY/FLO duplicates in Lamiales are the first reported outside of tetraploid taxa. In order to explore hypotheses for the joint preservation of these interacting floral regulatory genes including novel LFY/FLO paralogs, here we clone FLO and DEF duplicates from additional Lamiales taxa and apply codon substitution models to test how selection acts on both genes following duplication. We find acceleration in the ratio of nonsynonymous-to-synonymous nucleotide substitutions for one (FLO) or both (DEF) paralogs that appears to be due to relaxed purifying selection as opposed to positive selection and shows a different pattern among functional domains of these genes. Several mechanisms are discussed that might be responsible for preservation of co-orthologs of FLO and DEF in Lamiales, including interactions among the genes of this regulatory pathway. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

Computing the reversal distance between genomes in the presence of multi-gene families via binary integer programming

Hannenhalli and Pevzner developed the first polynomial-time algorithm for the combinatorial problem of sorting signed genomic data. Their algorithm determines the minimum number of reversals required for rearranging a genome to another -but only in the absence of gene duplicates. However, duplicates often account for 40% of a genome. In this paper, we show how to extend Hannenhalli and Pevzner's approach to deal with genomes with multi-gene families. We propose a new heuristic algorithm to compute the nearest reversal distance between two genomes with multi-gene families via binary integer programming. The experimental results on both synthetic and real biological data demonstrate that the proposed algorithm is able to find the reversal distance with high accuracy.     

The recent evolution of a pseudogene: diversity and divergence of a mitochondria-localized small heat shock protein in Arabidopsis thaliana

In this study we examined the evolution of the genes for three organelle-localized small heat shock proteins in Arabidopsis thaliana: the chloroplast-localized (CP) protein HSP21 and two mitochondria-localized (MT) proteins, HSP23.5 and HSP23.6. We found that the CP protein and one of the MT proteins, HSP23.6, are evolving under purifying selection to maintain function. In contrast, the gene for HSP23.5, the other MT protein, is highly variable within A. thaliana, and in some accessions or ecotypes this gene may be a pseudogene. HSP23.5 and HSP23.6 are related via a segmental duplication event, and the presence of orthologs of each gene in other species within the Brassicaceae indicates that the duplication generating HSP23.5 and HSP23.6 may have occurred as much as 20 million years ago. This is considerably longer than the 4 million year half-life of gene duplicates (functional genes as well as pseudogenes) reported by some studies. Our results are consistent with the prediction that after gene duplication one gene duplicate can be maintained for some time under relaxed selection while it accumulates random mutations. By capturing a pseudogene in the making our study provides important information on how pseudogenes are formed.     

The Functional Role of Pack-MULEs in Rice Inferred from Purifying Selection and Expression Profile

Gene duplication is an important mechanism for evolution of new genes. In plants, a special group of transposable elements, called Pack-MULEs or transduplicates, is able to duplicate and amplify genes or gene fragments on a large scale. Despite the abundance of Pack-MULEs, the functionality of these duplicates is not clear. Here, we present a comprehensive analysis of expression and purifying selection on 2809 Pack-MULEs in rice (Oryza sativa), which are derived from 1501 parental genes. At least 22% of the Pack-MULEs are transcribed, and 28 Pack-MULEs have direct evidence of translation. Chimeric Pack-MULEs, which contain gene fragments from multiple genes, are much more frequently expressed than those derived only from a single gene. In addition, Pack-MULEs are frequently associated with small RNAs. The presence of these small RNAs is associated with a reduction in expression of both the Pack-MULEs and their parental genes. Furthermore, an assessment of the selection pressure on the Pack-MULEs using the ratio of nonsynonymous (Ka) and synonymous (Ks) substitution rates indicates that a considerable number of Pack-MULEs likely have been under selective constraint. The Ka/Ks values of Pack-MULE and parental gene pairs are lower among Pack-MULEs that are expressed in sense orientations. Taken together, our analysis suggests that a significant number of Pack-MULEs are expressed and subjected to purifying selection, and some are associated with small RNAs. Therefore, at least a subset of Pack-MULEs are likely functional and have great potential in regulating gene expression as well as providing novel coding capacities.     

Evolutionary mechanisms underlying the functional divergence of duplicate genes involved in vertebrates' circadian rhythm pathway

Circadian rhythms, that are governed physiologically and behaviorally by endogenous clock, have been described in many species. Living organisms use this endogenous circadian clock to anticipate environmental transitions, perform activities at biologically advantageous times during the day, and undergo characteristic seasonal responses. Gene duplication is one of the most important mechanisms in the evolution of gene diversity. After duplication, one or both of duplicates can accumulate amino acid changes, thereby promoting functional divergence through the action of natural selection. The circadian system, like many other multigene families, has undergone this genetic revolution, and so circadian genes that are found in single copies in insects are duplicated in vertebrates. We analyzed six groups of genes involved in vertebrates' circadian rhythm pathway to find signatures of molecular evolutionary processes such as gene duplication, natural selection, recombination, and functional divergence. The obtained results, then, were used to determine what evolutionary forces have influenced the fates of duplicated genes of each group. We showed in this research that recombination has not been widespread during the evolution of circadian genes and that purifying selection has been the prominent natural pressure operating on circadian genes. We also showed that the evolution of circadian genes has been depended on gene duplication and functional divergence. Finally, we put forward models best describing the evolutionary fates of circadian duplicates.     

The evolutionary demography of duplicate genes

Although gene duplication has generally been viewed as a necessary source of material for the origin of evolutionary novelties, the rates of origin, loss, and preservation of gene duplicates are not well understood. Applying steady-state demographic techniques to the age distributions of duplicate genes censused in seven completely sequenced genomes, we estimate the average rate of duplication of a eukaryotic gene to be on the order of 0.01/gene/million years, which is of the same order of magnitude as the mutation rate per nucleotide site. However, the average half-life of duplicate genes is relatively small, on the order of 4.0 million years. Significant interspecific variation in these rates appears to be responsible for differences in species-specific genome sizes that arise as a consequence of a quasi-equilibrium birth-death process. Most duplicated genes experience a brief period of relaxed selection early in their history and a minority exhibit the signature of directional selection, but those that survive more than a few million years eventually experience strong purifying selection. Thus, although most theoretical work on the gene-duplication process has focused on issues related to adaptive evolution, the origin of a new function appears to be a very rare fate for a duplicate gene. A more significant role of the duplication process may be the generation of microchromosomal rearrangements through reciprocal silencing of alternative copies, which can lead to the passive origin of post-zygotic reproductive barriers in descendant lineages of incipient species.     

Genomic background predicts the fate of duplicated genes: evidence from the yeast genome

Gene duplication with subsequent divergence plays a central role in the acquisition of genes with novel function and complexity during the course of evolution. With reduced functional constraints or through positive selection, these duplicated genes may experience accelerated evolution. Under the model of subfunctionalization, loss of subfunctions leads to complementary acceleration at sites with two copies, and the difference in average rate between the sequences may not be obvious. On the other hand, the classical model of neofunctionalization predicts that the evolutionary rate in one of the two duplicates is accelerated. However, the classical model does not tell which of the duplicates experiences the acceleration in evolutionary rate. Here, we present evidence from the Saccharomyces cerevisiae genome that a duplicate located in a genomic region with a low-recombination rate is likely to evolve faster than a duplicate in an area of high recombination. This observation is consistent with population genetics theory that predicts that purifying selection is less effective in genomic regions of low recombination (Hill-Robertson effect). Together with previous studies, our results suggest the genomic background (e.g., local recombination rate) as a potential force to drive the divergence between nontandemly duplicated genes. This implies the importance of structure and complexity of genomes in the diversification of organisms via gene duplications.