Drosophila duplicate genes evolve new functions on the fly

Gene duplication is thought to play a key role in phenotypic innovation. While several processes have been hypothesized to drive the retention and functional evolution of duplicate genes, their genomic contributions have never been determined. We recently developed the first genome-wide method to classify these processes by comparing distances between expression profiles of duplicate genes and their ancestral single-copy orthologs. Application of our approach to spatial gene expression profiles in two Drosophila species revealed that a majority of young duplicate genes possess new functions, and that new functions are acquired rapidly—often within a few million years. Surprisingly, new functions tend to arise in younger copies of duplicate gene pairs. Moreover, we found that young duplicates are often specifically expressed in testes, whereas old duplicates are broadly expressed across several tissues, providing strong support for the hypothetical “out-of-testes” origin of new genes. In this Extra View, I discuss our findings in the context of theoretical predictions about gene duplication, with a particular emphasis on the importance of natural selection in the evolution of novel phenotypes.     

Evolutionary divergence of the APETALA1 and CAULIFLOWER proteins

Abstract APETALA1 (AP1) and CAULIFLOWER (CAL) are a pair of paralogous genes that were generated through the pre‐Brassicaceae whole‐genome duplication event. AP1 and CAL have both partially redundant and unique functions. Previous studies have shown that the K and C regions of their proteins are essential for the functional divergence. However, which differences in these regions are the major contributors and how the differences were accumulated remain unknown. In the present study, we compared the sequences of the two proteins and identified five gaps and 55 amino acid replacements between them. Investigation of genomic sequences further indicated that the differences in the proteins were caused by non‐synonymous substitutions and changes in exon–intron structures. Reconstruction of three‐dimensional structures revealed that the sequence divergence of AP1 and CAL has resulted in differences between the two in terms of the number, length, position and orientation of α‐helices, especially in the K and C regions. Comparisons of sequences and three‐dimensional structures of ancestral proteins with AP1 and CAL suggest that the ancestral AP1 protein experienced fewer changes, whereas the ancestral CAL protein accumulated more changes shortly after gene duplication, relative to their common ancestor. Thereafter, AP1‐like proteins experienced few mutations, whereas CAL‐like proteins were not conserved until the diversification of the Brassicaceae lineage I. This indicates that AP1‐ and CAL‐like proteins evolved asymmetrically after gene duplication. These findings provide new insights into the functional divergence of AP1 and CAL genes.     

Asymmetric Functional Divergence of Young, Dispersed Gene Duplicates in Arabidopsis thaliana

One prediction of the classic Ohno model of gene duplication predicts that new genes form from the asymmetric functional divergence of a newly arisen, redundant duplicate locus. In order to understand the mechanisms which give rise to functional divergence of newly formed dispersed duplicates, we assessed the expression and molecular evolutionary divergence of a suite of 19 highly similar dispersed duplicates in Arabidopsis thaliana. These duplicates have a K _sil equal to or less than 5 % and are specific to the A. thaliana lineage; thus, they predictably represent some of the youngest duplicates in the A. thaliana genome. We found that the majority of young duplicate loci exhibit asymmetric expression patterns, with the daughter locus exhibiting reduced expression across all tissues analyzed relative to the progenitor locus or simply not expressed. Furthermore, daughter loci, on the whole, have significantly more nonsynonymous substitutions than the progenitor loci. We also identified four pairs of loci which exhibit significant (P < 0.05) evolutionary rate asymmetry, three of which exhibit elevated dN/dS in the duplicate copy. We suggest, based on these data, that functional diversification initially takes the form of asymmetric regulatory divergence that can be a direct consequence of the mode of duplication. The reduced and/or absence of expression in the daughter copy relaxes functional constraint on its protein coding sequence leading to the asymmetric accumulation of nonsynonymous mutations. Thus, our data both affirm Ohno’s prediction while explaining the mechanism by which functional divergence initially occurs following duplication for dispersed gene duplicates.     

Predominant gain of promoter TATA box after gene duplication associated with stress responses

TATA box, the core promoter element, exists in a broad range of eukaryotes, and the expression of TATA-containing genes usually responds to various environmental stresses. Hence, the evolution of TATA-box in duplicate genes may provide some clues for the interrelationship among environmental stress, expression differentiation, and duplicate gene preservation. In the present study, we observed that the TATA box is significantly overrepresented in duplicate genes compared with singletons in human, worm, Arabidopsis, and yeast genomes. We then conducted an extensive functional genomic analysis to investigate the evolution of TATA box along over 700 yeast gene family phylogenies. After reconstructing the ancestral TATA-box states (presence or absence), we found that significantly higher numbers of TATA box gain events than loss events had occurred after yeast gene duplications-the overall gain-loss ratio is about 3-4 to 1. Interestingly, these TATA-gain duplicate genes on average have experienced greater expression divergence from the ancestral expression states than their most closely related TATA-less duplicate partners, but only under environmental stress conditions (asymmetric evolution); indeed, under normal physiological conditions, they have similar expression divergence (symmetric evolution). Moreover, we showed that TATA-gain duplicates are enriched in stress-associated functional categories but that is not the case for TATA-ancestral duplicates (those inherited from their ancestors prior to duplication). Together, we conclude that after the gene duplication, gain of the TATA box in duplicate promoters may have played an important role in yeast duplicate preservation by accelerating expression divergence that may facilitate the adaptive evolution of the organism in response to environmental changes.     

Positive selection and subfunctionalization of duplicated CCT chaperonin subunits

To reach a functional and energetically stable conformation, many proteins need molecular helpers called chaperonins. Among the group II chaperonins, CCT proteins provide crucial machinery for the stabilization and proper folding of several proteins in the cytosol of eukaryotic cells through interactions that are subunit-specific and geometry-dependent. CCT proteins are made up of eight different subunits, all with similar sequences, positioned in a precise arrangement. Each subunit has been proposed to have a specialized function during the binding and folding of the CCT protein substrate. Here, we demonstrate that functional divergence occurred after several CCT duplication events due to the fixation of amino acid substitutions by positive selection. Sites critical for ATP binding and substrate binding were found to have undergone positive selection and functional divergence predominantly in subunits that bind tubulin but not actin. Furthermore, we show clear functional divergence between CCT subunits that bind the C-terminal domains of actin and tubulin and those that bind the N-terminal domains. Phylogenetic analyses could not resolve the deep relationships between most subunits, except for the groups alpha/beta/eta and delta/epsilon, suggesting several almost simultaneous ancient duplication events. Together, the results support the idea that, in contrast to homo-oligomeric chaperonins such as GroEL, the high divergence level between CCT subunits is the result of positive selection after each duplication event to provide a specialized role for each CCT subunit in the different steps of protein folding.     

Functional divergence of the duplicated AtKIN14a and AtKIN14b genes: critical roles in Arabidopsis meiosis and gametophyte development

Gene duplication is important for gene family evolution, allowing for functional divergence and innovation. In flowering plants, duplicated genes are widely observed, and functional redundancy of closely related duplicates has been reported, but few cases of functional divergence of close duplicates have been described. Here, we show that the Arabidopsis AtKIN14a and AtKIN14b genes encoding highly similar kinesins are two of the most closely related Arabidopsis paralogs, which were formed by a duplication event that occurred after the split of Arabidopsis and poplar. In addition, AtKIN14a and AtKIN14b exhibit varying degrees of coding sequence divergence. Further genetic studies of plants carrying atkin14a and/or atkin14b mutations indicate that, although these two genes have similar functions, there is clear evidence for functional divergence. Although both genes are important for male and female meiosis, AtKIN14a plays a more critical role in male meiosis than AtKIN14b . Moreover, either one of these two genes is necessary and sufficient for gametophyte development, indicating that they are redundant for this function. Therefore, AtKIN14a and AtKIN14b together play important roles in controlling plant reproductive development. Our results suggest that the AtKIN14a and AtKIN14b genes have retained similar functions in gametophyte development and female meiosis, but have evolved partially distinct functions in male meiosis, with AtKIN14a playing a more substantive role.     

Asymmetric evolution of duplicate genes encoding the CCAAT-binding factor NF-Y in plant genomes

NF-Y is a ubiquitous CCAAT-binding factor composed of NF-YA, NF-YB and NF-YC. Multiple genes encoding NF-Y subunits have been identified in plant genomes. It remains unclear whether the duplicate genes underwent different evolutionary patterns. Likelihood-ratio tests were used to examine whether the amino acid substitution rates are the same between duplicate genes. The influences of selection on evolution were evaluated by comparing the conservative and radical amino acid substitution rates, as well as maximum-likelihood analysis. Some NF-YB and NF-YC duplicates showed significant evidence of asymmetric evolution but not the NF-YA duplicates. Most amino acid replacements in the NF-YB and NF-YC duplicates result in changes in hydropathy, polar requirement and polarity. The physicochemical changes in the sequences of NF-YB seem to be coupled to asymmetric divergence in gene function. Plant NF-Y genes have evolved in different patterns. Relaxed selective constraints following gene duplication are most likely responsible for the unequal evolutionary rates and distinct divergence patterns of duplicate NF-Y genes. Positive selection may have promoted amino acid hydropathy changes in the NF-YC duplicates.     

Multiple recombining loci encode MaSp1, the primary constituent of dragline silk, in widow spiders (Latrodectus: Theridiidae)

Spiders spin a functionally diverse array of silk fibers, each composed of one or more unique proteins. Most of these proteins, in turn, are encoded by members of a single gene family thought to have arisen through duplication and divergence of an ancestral silk gene. Because of its remarkable mechanical properties, orb weaver dragline silk, a composite of 2 proteins (MaSp1 and MaSp2), is the best studied. Here, we demonstrate that multiple loci encode MaSp1 in widow spiders (Latrodectus). Because these copies may be the result of more recent duplication events than those leading to the currently recognized silk gene paralogs, they offer insight into the early evolutionary fate of silk gene duplicates. In addition to 3 presumed functional MaSp1 loci in Latrodectus hesperus (Western black widow) and Latrodectus geometricus (brown widow) genomes, we find a MaSp1 pseudogene in L. hesperus, demonstrating the potential for unrecognized extinction of silk gene paralogs. We also document recombination events among L. hesperus MaSp1 loci and between Latrodectus MaSp1 loci and MaSp2. This result supports the hypothesis that concerted evolution occurs not only within an individual silk gene but also among silk gene paralogs. One of the L. geometricus MaSp1 copies encodes a protein that has diverged in amino acid composition and potentially converged on the secondary structure of MaSp2. Based on the presence of multiple MaSp1 loci and the phylogenetic distribution of MaSp1 versus MaSp2, we propose that MaSp2 is derived from an ancestral MaSp1 duplicate. Finally, divergence has occurred in the upstream flanking sequences of the L. hesperus MaSp1 loci, the region most likely to contain regulatory motifs, providing ample opportunity for differential expression. However, the benefits associated with increased protein production may be the primary mechanism maintaining multiple functional MaSp1 copies in widow genomes.     

Divergence pattern of duplicate genes in protein-protein interactions follows the power law

The impact of the biological network structures on the divergence between the two copies of one duplicate gene pair involved in the networks has not been documented on a genome scale. Having analyzed the most recently updated Database of Interacting Proteins (DIP) by incorporating the information for duplicate genes of the same age in yeast, we find that there was a highly significantly positive correlation between the level of connectivity of ancient genes and the number of shared partners of their duplicates in the protein-protein interaction networks. This suggests that duplicate genes with a low ancestral connectivity tend to provide raw materials for functional novelty, whereas those duplicate genes with a high ancestral connectivity tend to create functional redundancy for a genome during the same evolutionary period. Moreover, the difference in the number of partners between two copies of a duplicate pair was found to follow a power-law distribution. This suggests that loss and gain of interacting partners for most duplicate genes with a lower level of ancestral connectivity is largely symmetrical, whereas the "hub duplicate genes" with a higher level of ancient connectivity display an asymmetrical divergence pattern in protein-protein interactions. Thus, it is clear that the protein-protein interaction network structures affect the divergence pattern of duplicate genes. Our findings also provide insights into the origin and development of biological networks.     

Preferential retention of the slowly evolving gene in pairs of duplicates in angiosperm genomes

Gene duplication provides raw material for functional innovation, but gene duplicability varies considerably. Previous studies have found widespread asymmetrical sequence evolution between paralogs. However, it remains unknown whether the rate of evolution among paralogs affects their propensity of being retained after another round of whole-genome duplication (WGD). In this study, we investigated gene groups that have experienced two successive WGDs to determine which of two older duplicates with different evolutionary rates was more likely to retain both younger duplicates. To uncouple the measurement of evolutionary rates from any assignment of duplicate or singleton status, we measured the evolutionary rates of singleton genes in out-lineages but classified these singleton genes according to whether they are retained or not in a crown group of species. We found that genes that retained younger duplicates in the crown group of genomes were more constrained prior to the younger duplication event than those that failed to leave duplicates. In addition, we also found that the retained clades have more genes in out-lineages. Subsequent analyses showed that genes in the retained clades were expressed more broadly and highly than genes in the singleton clades. We concluded that the set of repeatedly retained genes after two WGDs is biased toward slowly evolving genes in angiosperms, suggesting that the potential of genes for both functional conservation and divergence likely affects their propensity of being retained after WGD in angiosperms.     

Functional divergence of Populus MYB158 and MYB189 gene pair created by whole genome duplication

Whole genome duplication (WGD) providesnew genetic material for genome evolution. After a WGD event, some duplicates are lost, while other duplicates still persist and evolve diverse functions. A particular challenge is to understand how this diversity arises. This study identified two WGD-derived duplicates, MYB158 and MYB189, from Populus tomentosa. Populus MYB158 and MYB189 had expression divergence. Populus tomentosa overexpressing MYB158 or MYB189 had similar phenotypes: creep growth, decreased width of xylem and secondary cell wall thickness. Compared to wild-type, neither myb158 mutant nor myb158 myb189 double mutant showed obvious phenotypic variation in P. tomentosa. Although MYB158 and MYB189 proteins could repress the same structural genes involved in lignin, cellulose, and xylan biosynthesis, the two proteins had their own specific regulatory targets. Populus MYB158 could act as the upstream regulator of secondary cell wall NAC master switch and directly represses the expression of the SND1-B2 gene. Taken together, Populus MYB158 and MYB189 have retained similar functions in negatively regulating secondary cell wall biosynthesis, but have evolved partially distinct functions in direct regulation of NAC master switch, with MYB158 playing a more crucial role. Our findings provide new insights into the evolutionary and functional divergence of WGD-derived duplicate genes.     

The Pattern of Evolution of Smaller-Scale Gene Duplicates in Mammalian Genomes is More Consistent with Neo- than Subfunctionalisation

Gene duplication and the accompanying release of negative selective pressure on the duplicate pair is thought to be the key process that makes functional change in the coding and regulatory regions of genomes possible. However, the nature of these changes remains unresolved. There are a number of models for the fate of gene duplicates, the two most prominent of which are neofunctionalisation and subfunctionalisation, but it is still unclear which is the dominant fate. Using a dataset consisting of smaller-scale (tandem and segmental) duplications identified from the genomes of four fully sequenced mammalian genomes, we characterise two key features of smaller-scale duplicate evolution: the rate of pseudogenisation and the rate of accumulation of replacement substitutions in the coding sequence. We show that the best fitting model for gene duplicate survival is a Weibull function with a downward sloping convex hazard function which implies that the rate of pseudogenisation of a gene declines rapidly with time since duplication. Our analysis of the accumulation of replacement substitutions per replacement site shows that they accumulate on average at 64% of the neutral expectation immediately following duplication and as high as 73% in the human lineage. Although this rate declines with time since duplication, it takes several tens of millions of years before it has declined to half its initial value. We show that the properties of the gene death rate and of the accumulation of replacement substitutions are more consistent with neofunctionalisation (or subfunctionalisation followed by neofunctionalisation) than they are with subfunctionalisation alone or any of the other alternative modes of evolution of smaller-scale duplicates. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

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.     

Functional divergence after gene duplication and sequence-structure relationship: a case study of G-protein alpha subunits

In this article, we use animal G-protein alpha subunit family as an example to illustrate a comprehensive analytical pipeline for detecting different types of functional divergence of protein families, which is phylogeny-dependent, combined with ancestral sequence inference and available protein structure information. In particular, we focus on (i) Type-I functional divergence, or site-specific rate shift, as typically exemplified by amino acid residue highly conserved in a subset of homologous genes but highly variable in a different subset of homologous genes, and (ii) Type-II functional divergence, or the shift of cluster-specific amino acid property, as exemplified by a radical shift of amino acid property between duplicate genes, which is otherwise evolutionally conserved. We utilized the software DIVERGE2 to carry out these analyses. In the case of G-protein alpha subunit gene family, we have predicted amino acid residues that are related to either Type-I or Type-II functional divergence. The inferred ancestral sequences for these sites are helpful to explore the trends of functional divergence. Finally, these predicted residues are mapped to the protein structures to test whether these residues may have 3D structure or solvent accessibility preference.     

Pervasive indels and their evolutionary dynamics after the fish-specific genome duplication

Insertions and deletions (indels) in protein-coding genes are important sources of genetic variation. Their role in creating new proteins may be especially important after gene duplication. However, little is known about how indels affect the divergence of duplicate genes. We here study thousands of duplicate genes in five fish (teleost) species with completely sequenced genomes. The ancestor of these species has been subject to a fish-specific genome duplication (FSGD) event that occurred approximately 350 Ma. We find that duplicate genes contain at least 25% more indels than single-copy genes. These indels accumulated preferentially in the first 40 my after the FSGD. A lack of widespread asymmetric indel accumulation indicates that both members of a duplicate gene pair typically experience relaxed selection. Strikingly, we observe a 30-80% excess of deletions over insertions that is consistent for indels of various lengths and across the five genomes. We also find that indels preferentially accumulate inside loop regions of protein secondary structure and in regions where amino acids are exposed to solvent. We show that duplicate genes with high indel density also show high DNA sequence divergence. Indel density, but not amino acid divergence, can explain a large proportion of the tertiary structure divergence between proteins encoded by duplicate genes. Our observations are consistent across all five fish species. Taken together, they suggest a general pattern of duplicate gene evolution in which indels are important driving forces of evolutionary change.     

Consequences of Whole-Genome Triplication as Revealed by Comparative Genomic Analyses of the Wild Radish Raphanus raphanistrum and Three Other Brassicaceae Species

Polyploidization events are frequent among flowering plants, and the duplicate genes produced via such events contribute significantly to plant evolution. We sequenced the genome of wild radish (Raphanus raphanistrum), a Brassicaceae species that experienced a whole-genome triplication event prior to diverging from Brassica rapa. Despite substantial gene gains in these two species compared with Arabidopsis thaliana and Arabidopsis lyrata, ∼70% of the orthologous groups experienced gene losses in R. raphanistrum and B. rapa, with most of the losses occurring prior to their divergence. The retained duplicates show substantial divergence in sequence and expression. Based on comparison of A. thaliana and R. raphanistrum ortholog floral expression levels, retained radish duplicates diverged primarily via maintenance of ancestral expression level in one copy and reduction of expression level in others. In addition, retained duplicates differed significantly from genes that reverted to singleton state in function, sequence composition, expression patterns, network connectivity, and rates of evolution. Using these properties, we established a statistical learning model for predicting whether a duplicate would be retained postpolyploidization. Overall, our study provides new insights into the processes of plant duplicate loss, retention, and functional divergence and highlights the need for further understanding factors controlling duplicate gene fate.