An attractive and long-standing hypothesis regarding the evolution of genes after duplication posits that the duplication event creates new evolutionary possibilities by releasing a copy of the gene from constraint. Apparent support was found in numerous analyses, particularly, the observation of higher rates of evolution in duplicated as compared with singleton genes. Could it, instead, be that more duplicable genes (owing to mutation, fixation, or retention biases) are intrinsically faster evolving? To uncouple the measurement of rates of evolution from the determination of duplicate or singleton status, we measure the rates of evolution in singleton genes in outgroup primate lineages but classify these genes as to whether they have duplicated or not in a crown group of great apes. We find that rates of evolution are higher in duplicable genes prior to the duplication event. In part this is owing to a negative correlation between coding sequence length and rate of evolution, coupled with a bias toward smaller genes being more duplicable. The effect is masked by difference in expression rate between duplicable genes and singletons. Additionally, in contradiction to the classical assumption, we find no convincing evidence for an increase in dN/dS after duplication, nor for rate asymmetry between duplicates. We conclude that high rates of evolution of duplicated genes are not solely a consequence of the duplication event, but are rather a predictor of duplicability. These results are consistent with a model in which successful gene duplication events in mammals are skewed toward events of minimal phenotypic impact.

Knocking out a gene from a genome often causes no phenotypic effect. This phenomenon has been explained in part by the existence of duplicate genes. However, it was found that in mouse knockout data duplicate genes are as essential as singleton genes. Here, we study whether it is also true for the knockout data in Arabidopsis. From the knockout data in Arabidopsis thaliana obtained in our study and in the literature, we find that duplicate genes show a significantly lower proportion of knockout effects than singleton genes. Because the persistence of duplicate genes in evolution tends to be dependent on their phenotypic effect, we compared the ages of duplicate genes whose knockout mutants showed less severe phenotypic effects with those with more severe effects. Interestingly, the latter group of genes tends to be more anciently duplicated than the former group of genes. Moreover, using multiple-gene knockout data, we find that functional compensation by duplicate genes for a more severe phenotypic effect tends to be preserved by natural selection for a longer time than that for a less severe effect. Taken together, we conclude that duplicate genes contribute to genetic robustness mainly by preserving compensation for severe phenotypic effects in A. thaliana.

It is now widely recognized that robustness is an inherent property of biological systems [1],[2],[3]. The contribution of close sequence homologs to genetic robustness against null mutations has been previously demonstrated in simple organisms [4],[5]. In this paper we investigate in detail the contribution of gene duplicates to back-up against deleterious human mutations. Our analysis demonstrates that the functional compensation by close homologs may play an important role in human genetic disease. Genes with a 90% sequence identity homolog are about 3 times less likely to harbor known disease mutations compared to genes with remote homologs. Moreover, close duplicates affect the phenotypic consequences of deleterious mutations by making a decrease in life expectancy significantly less likely. We also demonstrate that similarity of expression profiles across tissues significantly increases the likelihood of functional compensation by homologs.

Gene duplication generates extra gene copies in which mutations can accumulate without risking the function of pre-existing genes. Such mutations modify duplicates and contribute to evolutionary novelties. However, the vast majority of duplicates appear to be short-lived and experience duplicate silencing within a few million years. Little is known about the molecular mechanisms leading to these alternative fates. Here we delineate differing molecular trajectories of a relatively recent duplication event between humans and chimpanzees by investigating molecular properties of a single duplicate: DNA sequences, gene expression and promoter activities. The inverted duplication of the Glutathione S-transferase Theta 2 (GSTT2) gene had occurred at least 7 million years ago in the common ancestor of African great apes and is preserved in chimpanzees (Pan troglodytes), whereas a deletion polymorphism is prevalent in humans. The alternative fates are associated with expression divergence between these species, and reduced expression in humans is regulated by silencing mutations that have been propagated between duplicates by gene conversion. In contrast, selective constraint preserved duplicate divergence in chimpanzees. The difference in evolutionary processes left a unique DNA footprint in which dying duplicates are significantly more similar to each other (99.4%) than preserved ones. Such molecular trajectories could provide insights for the mechanisms underlying duplicate life and death in extant genomes.

In contrast to S. cerevisiae and C. elegans, analyses based on the current knockout (KO) mouse phenotypes led to the conclusion that duplicate genes had almost no role in mouse genetic robustness. It has been suggested that the bias of mouse KO database toward ancient duplicates may possibly cause this knockout duplicate puzzle, that is, a very similar proportion of essential genes (PE) between duplicate genes and singletons. In this paper, we conducted an extensive and careful analysis for the mouse KO phenotype data and corroborated a strong effect of duplicate genes on mouse genetics robustness. Moreover, the effect of duplicate genes on mouse genetic robustness is duplication-age dependent, which holds after ruling out the potential confounding effect from coding-sequence conservation, protein-protein connectivity, functional bias, or the bias of duplicates generated by whole genome duplication (WGD). Our findings suggest that two factors, the sampling bias toward ancient duplicates and very ancient duplicates with a proportion of essential genes higher than that of singletons, have caused the mouse knockout duplicate puzzle; meanwhile, the effect of genetic buffering may be correlated with sequence conservation as well as protein-protein interactivity.

Following gene duplication, two duplicate genes may experience relaxed functional constraints or acquire different mutations, and may also diverge in function. Whether the two copies will evolve in different patterns remains unclear, however, because previous studies have reached conflicting conclusions. In order to resolve this issue, by providing a general picture, we studied 250 independent pairs of young duplicate genes from the whole human genome.

Gene duplication is the primary source of new genes with novel or altered functions. It is known that duplicates may obtain these new functional roles by evolving divergent expression patterns and/or protein functions after the duplication event. Here, using yeast (Saccharomyces cerevisiae) as a model organism, we investigate a previously little considered mode for the functional diversification of duplicate genes: subcellular adaptation of encoded proteins.

Gene duplication is thought to play a major role in phenotypic evolution. Yet the forces involved in the functional divergence of young duplicate genes remain unclear. Here, we use population-genetic inference to elucidate the role of natural selection in the functional evolution of young duplicate genes in Drosophila melanogaster. We find that negative selection acts on young duplicates with ancestral functions, and positive selection on those with novel functions, suggesting that natural selection may determine whether and how young duplicate genes are retained. Moreover, evidence of natural selection is strongest in protein-coding regions and 3' UTRs of young duplicates, indicating that selection may primarily target encoded proteins and regulatory sequences specific to 3' UTRs. Further analysis reveals that natural selection acts immediately after duplication and weakens over time, possibly explaining the observed bias toward the acquisition of new functions by young, rather than old, duplicate gene copies. Last, we find an enrichment of testis-related functions in young duplicates that underwent recent positive selection, but not in young duplicates that did not undergo recent positive selection, or in old duplicates that either did or did not undergo recent positive selection. Thus, our findings reveal that natural selection is a key player in the functional evolution of young duplicate genes, acts rapidly and in a region-specific manner, and may underlie the origin of novel testis-specific phenotypes in Drosophila.

Gene and genome duplication fosters genetic novelty, but redundant gene copies would undergo mutational decay unless preserved via selective or neutral forces. Molecular mechanisms mediating duplicate preservation remain incompletely understood. Several recent studies showed an association between DNA methylation and expression divergence of duplicated genes and suggested a role of epigenetic mechanism in duplicate retention. Here, we compare genome-wide gene-body CG methylation (BCGM) and duplicate gene expression between a rice mutant null for OsMet1-2(a major CG methytransferase in rice) and its isogenic wild-type. We demonstrate a causal link between BCGM divergence and expression difference of duplicate copies. Interestingly, the higher- and lower-expressing copies of duplicates as separate groups show broadly different responses with respect to direction of expression alteration upon loss of BCGM. A role for BCGM in conditioning expression divergence between copies of duplicates generally holds for duplicates generated by whole genome duplication (WGD) or by small-scale duplication processes. However, differences are evident among these categories, including a higher proportion of WGD duplicates manifesting expression alteration, and differential propensities to lose BCGM by the higher- and lower-expression copies in the mutant. Together, our results support the notion that differential epigenetic marking may facilitate long-term retention of duplicate genes.

Gene duplication is the primary source of new genes, but the mechanisms underlying the functional divergence and retention of duplicate genes are not well understood. Because eukaryotic proteins are localized to subcellular structures and localization can be altered by a single amino acid replacement, it was recently proposed that protein subcellular relocalization (PSR) plays an important role in the functional divergence and retention of duplicate genes. Although numerous examples of distinct subcellular localizations of paralogous proteins have been reported, it is unknown whether PSR occurs more frequently after gene duplication than without duplication. By analyzing experimentally determined and computationally predicted genome-wide protein subcellular localization data of the budding yeast Saccharomyces cerevisiae and two other fungi (Schizosaccharomyces pombe and Kluyveromyces waltii), we show that even singleton genes have an appreciable rate of relocalization in evolution and that duplicate genes do not relocalize more frequently than singletons. These results suggest that subcellular relocalization is unlikely to have been a major mechanism for duplicate gene retention and functional divergence at the genomic scale.

A longstanding puzzle in human genetics is what limits the clinical manifestation of hundreds of hereditary diseases to certain tissues, while their causal genes are expressed throughout the human body. A general conception is that tissue-selective disease phenotypes emerge when masking factors operate in unaffected tissues, but are specifically absent or insufficient in disease-manifesting tissues. Although this conception has critical impact on the understanding of disease manifestation, it was never challenged in a systematic manner across a variety of hereditary diseases and affected tissues. Here, we address this gap in our understanding via rigorous analysis of the susceptibility of over 30 tissues to 112 tissue-selective hereditary diseases. We focused on the roles of paralogs of causal genes, which are presumably capable of compensating for their aberration. We show for the first time at large-scale via quantitative analysis of omics datasets that, preferentially in the disease-manifesting tissues, paralogs are under-expressed relative to causal genes in more than half of the diseases. This was observed for several susceptible tissues and for causal genes with varying number of paralogs, suggesting that imbalanced expression of paralogs increases tissue susceptibility. While for many diseases this imbalance stemmed from up-regulation of the causal gene in the disease-manifesting tissue relative to other tissues, it was often combined with down-regulation of its paralog. Notably in roughly 20% of the cases, this imbalance stemmed only from significant down-regulation of the paralog. Thus, dosage relationships between paralogs appear as important, yet currently under-appreciated, modifiers of disease manifestation.

Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time) is > approximately 0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.

Gene duplication is a widespread phenomenon in genome evolution, and it has been proposed to serve as an engine of evolutionary innovation. In the present study, we performed the first comprehensive analysis of duplicate genes in the bovine genome. A total of 3131 putative duplicated gene pairs were identified, including 712 cattle-specific duplicate gene pairs unevenly distributed across the genome, which are significantly enriched for specific biological functions including immunity, growth, digestion, reproduction, embryonic development, inflammatory response, and defense response to bacterium. Around 97.1% (87.8%) of (cattle-specific) duplicate gene pairs were found to have distinct exon-intron structures. Analysis of gene expression by RNA-Seq and sequence divergence (synonymous or non-synonymous) revealed that expression divergence is correlated with sequence divergence, as has been previously observed in other species. This analysis also led to the identification of a subset of cattle-specific duplicate gene pairs exhibiting very high expression divergence. Interestingly, further investigation revealed a significant relationship between structural and expression divergence while controlling for the effect of synonymous sequence divergence. Together these results provide further insight into duplicate gene sequence and expression divergence in cattle, and their potential contributions to phenotypic divergence.

The evolutionary mechanism, fate and function of duplicate genes in various taxa have been widely studied; however, the mechanism underlying the maintenance and divergence of duplicate genes in Danio rerio remains largely unexplored. Whether and how the divergence of DNA methylation between duplicate pairs is associated with gene expression and evolutionary time are poorly understood. In this study, by analyzing bisulfite sequencing (BS-seq) and RNA-seq datasets from public data, we demonstrated that DNA methylation played a critical role in duplicate gene evolution in zebrafish. Initially, we found promoter methylation of duplicate genes generally decreased with evolutionary time as measured by synonymous substitution rate between paralogous duplicates (Ks). Importantly, promoter methylation of duplicate genes was negatively correlated with gene expression. Interestingly, for 665 duplicate gene pairs, one gene was consistently promoter methylated, while the other was unmethylated across nine different datasets we studied. Moreover, one motif enriched in promoter methylated duplicate genes tended to be bound by the transcription repression factor FOXD3, whereas a motif enriched in the promoter unmethylated sequences interacted with the transcription activator Sp1, indicating a complex interaction between the genomic environment and epigenome. Besides, body-methylated genes showed longer length than body-unmethylated genes. Overall, our results suggest that DNA methylation is highly important in the differential expression and evolution of duplicate genes in zebrafish.

Gene duplication is a key evolutionary phenomenon, prevalent in all organisms but particularly so in plants, where whole genome duplication (WGD; polyploidy) is a major force in genome evolution. Much effort has been expended in attempting to understand the evolution of duplicate genes, addressing such questions as why some paralog pairs rapidly return to single copy status whereas, in other pairs, both paralogs are retained and may diverge in expression pattern or function. The effect of a gene - its site of expression and thus the initial locus of its function - occurs at the level of a cell comprising a single cell type at a given state of the cell's development. Using Arabidopsis thaliana single cell transcriptomic data we categorized patterns of expression for 11,470 duplicate gene pairs across 36 cell clusters comprising nine cell types and their developmental states. Among these 11,470 pairs, 10,187 (88.8%) had at least one copy expressed in at least one of the 36 cell clusters. Pairs produced by WGD more often had both paralogs expressed in root cells than did pairs produced by small scale duplications. Three quarters of gene pairs expressed in the 36 cell clusters (7,608/10,187) showed extreme expression bias in at least one cluster, including 352 cases of reciprocal bias, a pattern consistent with expression subfunctionalization. More than twice as many pairs showed reciprocal expression bias between cell states than between cell types or between roots and leaves. A group of 33 gene pairs with reciprocal expression bias showed evidence of concerted divergence of gene networks in stele vs. epidermis. Pairs with both paralogs expressed without bias were less likely to have paralogs with divergent mutant phenotypes; such bias-free pairs showed evidence of preservation by maintenance of dosage balance. Overall, we found considerable evidence of shifts in gene expression following duplication, including in >80% of pairs encoding 7,653 genes expressed ubiquitously in all root cell types and states for which we inferred the polarity of change.

Learning about the roles that duplicate genes play in the origins of novel phenotypes requires an understanding of how their functions evolve. A previous method for achieving this goal, CDROM, employs gene expression distances as proxies for functional divergence and then classifies the evolutionary mechanisms retaining duplicate genes from comparisons of these distances in a decision tree framework. However, CDROM does not account for stochastic shifts in gene expression or leverage advances in contemporary statistical learning for performing classification, nor is it capable of predicting the parameters driving duplicate gene evolution. Thus, here we develop CLOUD, a multi-layer neural network built on a model of gene expression evolution that can both classify duplicate gene retention mechanisms and predict their underlying evolutionary parameters. We show that not only is the CLOUD classifier substantially more powerful and accurate than CDROM, but that it also yields accurate parameter predictions, enabling a better understanding of the specific forces driving the evolution and long-term retention of duplicate genes. Further, application of the CLOUD classifier and predictor to empirical data from Drosophila recapitulates many previous findings about gene duplication in this lineage, showing that new functions often emerge rapidly and asymmetrically in younger duplicate gene copies, and that functional divergence is driven by strong natural selection. Hence, CLOUD represents a major advancement in classifying retention mechanisms and predicting evolutionary parameters of duplicate genes, thereby highlighting the utility of incorporating sophisticated statistical learning techniques to address long-standing questions about evolution after gene duplication.

Gene duplication has been proposed to serve as the engine of evolutionary innovation. It is well recognized that eukaryotic genomes contain a large number of duplicated genes that evolve new functions or expression patterns. However, in mollusks, the evolutionary mechanisms underlying the divergence and the functional maintenance of duplicate genes remain little understood. In the present study, we performed a comprehensive analysis of duplicate genes in the protein kinase superfamily using whole genome and transcriptome data for the Pacific oyster. A total of 64 duplicated gene pairs were identified based on a phylogenetic approach and the reciprocal best BLAST method. By analyzing gene expression from RNA-seq data from 69 different developmental and stimuli-induced conditions (nine tissues, 38 developmental stages, eight dry treatments, seven heat treatments, and seven salty treatments), we found that expression patterns were significantly correlated for a number of duplicate gene pairs, suggesting the conservation of regulatory mechanisms following divergence. Our analysis also identified a subset of duplicate gene pairs with very high expression divergence, indicating that these gene pairs may have been subjected to transcriptional subfunctionalization or neofunctionalization after the initial duplication events. Further analysis revealed a significant correlation between expression and sequence divergence (as revealed by synonymous or nonsynonymous substitution rates) under certain conditions. Taken together, these results provide evidence for duplicate gene sequence and expression divergence in the Pacific oyster, accompanying its adaptation to harsh environments. Our results provide new insights into the evolution of duplicate genes and their expression levels in the Pacific oyster.

The Paramecium aurelia complex is a group of 15 species that share at least three past whole-genome duplications (WGDs). The macronuclear genome sequences of P. biaurelia and P. sexaurelia are presented and compared to the published sequence of P. tetraurelia. Levels of duplicate-gene retention from the recent WGD differ by > 10% across species, with P. sexaurelia losing significantly more genes than P. biaurelia or P. tetraurelia. In addition, historically high rates of gene conversion have homogenized WGD paralogs, probably extending the paralogs' lifetimes. The probability of duplicate retention is positively correlated with GC content and expression level; ribosomal proteins, transcription factors, and intracellular signaling proteins are overrepresented among maintained duplicates. Finally, multiple sources of evidence indicate that P. sexaurelia diverged from the two other lineages immediately following, or perhaps concurrent with, the recent WGD, with approximately half of gene losses between P. tetraurelia and P. sexaurelia representing divergent gene resolutions (i.e., silencing of alternative paralogs), as expected for random duplicate loss between these species. Additionally, though P. biaurelia and P. tetraurelia diverged from each other much later, there are still more than 100 cases of divergent resolution between these two species. Taken together, these results indicate that divergent resolution of duplicate genes between lineages acts to reinforce reproductive isolation between species in the Paramecium aurelia complex.

It has been shown that gene body DNA methylation is associated with gene expression. However, whether and how deviation of gene body DNA methylation between duplicate genes can influence their divergence remains largely unexplored. Here, we aim to elucidate the potential role of gene body DNA methylation in the fate of duplicate genes. We identified paralogous gene pairs from Arabidopsis and rice (Oryza sativa ssp. japonica) genomes and reprocessed their single-base resolution methylome data. We show that methylation in paralogous genes nonlinearly correlates with several gene properties including exon number/gene length, expression level and mutation rate. Further, we demonstrated that divergence of methylation level and pattern in paralogs indeed positively correlate with their sequence and expression divergences. This result held even after controlling for other confounding factors known to influence the divergence of paralogs. We observed that methylation level divergence might be more relevant to the expression divergence of paralogs than methylation pattern divergence. Finally, we explored the mechanisms that might give rise to the divergence of gene body methylation in paralogs. We found that exonic methylation divergence more closely correlates with expression divergence than intronic methylation divergence. We show that genomic environments (e.g., flanked by transposable elements and repetitive sequences) of paralogs generated by various duplication mechanisms are associated with the methylation divergence of paralogs. Overall, our results suggest that the changes in gene body DNA methylation could provide another avenue for duplicate genes to develop differential expression patterns and undergo different evolutionary fates in plant genomes.

Lineage-specific duplicated genes likely contribute to the phenotypic divergence in closely related species. However, neither the frequency of duplication events nor the degree of selection pressures immediately after gene duplication is clear in the speciation process. Here, using Illumina DNA-sequencing reads from Arabidopsis halleri, which has multiple closely related species with high-quality genome assemblies (A. thaliana and A. lyrata), we succeeded in generating orthologous gene groups in Brassicaceae. The duplication frequency of retained genes in the Arabidopsis lineage was ∼10 times higher than the duplication frequency inferred by comparative genomics of Arabidopsis, poplar, rice and moss (Physcomitrella patens). The difference of duplication frequencies can be explained by a rapid decay of anciently duplicated genes. To examine the degree of selection pressure on genes duplicated in either the A. halleri-lyrata or the A. halleri lineage, we examined positive and purifying selection in the A. halleri-lyrata and A. halleri lineages throughout the ratios of nonsynonymous to synonymous substitution rates (KA/KS). Duplicate genes tended to have a higher proportion of positive selection compared with non-duplicated genes. Interestingly, we found that functional divergence of duplicated genes was accelerated several million years after gene duplication compared with immediately after gene duplication.