High rate of recent intron gain and loss in simultaneously duplicated Arabidopsis genes

We examined the gene structure of a set of 2563 Arabidopsis thaliana paralogous pairs that were duplicated simultaneously 20-60 MYA by tetraploidy. Out of a total of 23,164 introns in these genes, we found that 10,004 pairs have been conserved and 578 introns have been inserted or deleted in the time since the duplication event. This intron insertion/deletion rate of 2.7 x 10(-3) to 9.1 x 10(-4) per site per million years is high in comparison to previous studies. At least 56 introns were gained and 39 lost based on parsimony analysis of the phylogenetic distribution of these introns. We found weak evidence that genes undergoing intron gain and loss are biased with respect to gene ontology terms. Gene pairs that experienced at least 2 intron insertions or deletions show evidence of enrichment for membrane location and transport and transporter activity function. We do not find any relationship of intron flux to expression level or G + C content of the gene. Detection of a bias in the location of intron gains and losses within a gene depends on the method of measurement: an intragene method indicates that events (specifically intron losses) are biased toward the 3' end of the gene. Despite the relatively recent acquisition of these introns, we found only one case where we could identify the mechanism of intron origin--the TOUCH3 gene has experienced 2 tandem, partial, internal gene duplications that duplicated a preexisting intron and also created a novel, alternatively spliced intron that makes use of a duplicated pair of cryptic splice sites.     

Very little intron gain in Entamoeba histolytica genes laterally transferred from prokaryotes

The evolution of spliceosomal introns remains intensely debated. We studied 96 Entamoeba histolytica genes previously identified as having been laterally transferred from prokaryotes, which were presumably intronless at the time of transfer. Ninety out of the 96 are also present in the reptile parasite Entamoeba invadens, indicating lateral transfer before the species' divergence approximately 50 MYA. We find only 2 introns, both shared with E. invadens. Thus, no intron gains have occurred in approximately 50 Myr, implying a very low rate of intron gain of less than one gain per gene per approximately 4.5 billion years. Nine other predicted introns are due to annotation errors reflecting apparent mistakes in the E. histolytica genome assembly. These results underscore the massive differences in intron gain rates through evolution.     

Widespread intron loss suggests retrotransposon activity in ancient apicomplexans

Several facets of spliceosomal intron in apicomplexans remain mysterious. First, intron numbers vary across species by 2 orders of magnitude, indicating massive intron loss and/or gain. Second, previous studies have shown very different evolutionary patterns over different timescales, with 100-fold higher rates of intron loss/gain between genera than within genera. Third, the timing and dynamics of nearly complete intron loss in Cryptosporidium species, as well as reasons for retention of the few remaining introns, remain unknown. We compared intron positions in 785 orthologous genes between 3 moderate to intron-rich apicomplexan species. We estimate that the Theileria-Plasmodium ancestor had 4.5 times as many introns as modern Plasmodium species and 38% more than modern Theileria species, and that subsequent intron losses have outnumbered intron gains by 5.8 to 1 in Theileria and by some 56 to 1 in Plasmodium. Several patterns suggest that these intron losses occurred by recombination with reverse-transcribed mRNAs. Intriguingly, this finding suggests significant retrotransposon activity in the lineages leading to both Theileria and Plasmodium, in contrast to the dearth of known retrotransposons and intron loss within modern species from both genera. We also compared genomes from Cryptosporidium parvum and C. hominis and found no evidence of ongoing intron loss, nor of intron gain. By contrast, Cryptosporidium introns are less evolutionary conserved with Toxoplasma than are introns from other apicomplexans; thus the few remaining introns are not simply indispensable ancestral introns.     

Faster evolution of Z-linked duplicate genes in chicken

It has been shown that duplicate genes on the X chromosome evolve much faster than duplicate genes on autosomes in Drosophila melanogaster.However,whether this phenomenon is general and can be applied to other species is not known.Here we examined this issue in chicken that have heterogametic females(females have ZW sex chromosome).We compared sequence divergence of duplicate genes on the Z chromosome with those on autosomes.We found that duplications on the Z chromosome indeed evolved faster than those on autosomes and show distinct patterns of molecular evolution from autosomal duplications.Examination of the expression of duplicate genes revealed an enrichment of duplications on the Z chromosome having male-biased expression and an enrichment of duplications on the autosomes having female-biased expression.These results suggest an evolutionary trend of the recruitment of duplicate genes towards reproduction-specific function.The faster evolution of duplications on Z than on the autosomes is most likely contributed by the selective forces driving the fixation of adaptive mutations on Z.Therefore,the common phenomena observed in both flies and chicken suggest that duplicate genes on sex chromosomes have distinct dynamics and are more influenced by natural selection than antosomal duplications,regardless of the kind of sex determination systems.     

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.     

On the incidence of intron loss and gain in paralogous gene families

Understanding gene duplication and gene structure evolution are fundamental goals of molecular evolutionary biology. A previous study by Babenko et al. (2004. Prevalence of intron gain over intron loss in the evolution of paralogous gene families. Nucleic Acids Res. 32:3724-3733) employed Dollo parsimony to infer spliceosomal intron losses and gains in paralogous gene families and concluded that there was a general excess of gains over losses. This result contrasts with patterns in orthologous genes, in which most lineages show an excess of intron losses over gains, suggesting the possibility of fundamentally different modes of intron evolution between orthologous and paralogous genes. We further studied the data and found a low level of intron position conservation with outgroups, and this led to problems with using Dollo parsimony to analyze the data. Statistical reanalysis of the data suggests, instead, that intron losses have outnumbered intron gains in paralogous gene families.     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.     

Smoke without fire: most reported cases of intron gain in nematodes instead reflect intron losses

Identification of recently gained spliceosomal introns would provide crucial evidence in the continuing debate concerning the age and evolutionary significance of introns. A previously published genomic analysis reported to have identified 122 introns that had been gained since the divergence of the nematodes Caenorhabidits elegans and Caenorhabditis briggsae approximately 100 MYA. However, using newly available genomic sequence from additional Caenorhabditis species, we show that 74% (60/81) of the reported gains in C. elegans are present in a C. briggsae relative. This pattern indicates that these introns represent losses in C. briggsae, not gains in C. elegans. In addition, 61% (25/41) of the reported gains in C. briggsae are present in the more distant C. briggsae relative, in a pattern suggesting that additional reported gains in C. elegans and/or C. briggsae may in fact represent unrecognized losses. These results underscore the dominance of intron loss over intron gain in recent eukaryotic evolution, the pitfalls associated with parsimony in inferring intron gains, and the importance of genomic sequencing of clusters of closely related species for drawing accurate inferences about genome evolution.     

Two patterns of genome organization in mammals: the chromosomal distribution of duplicate genes in human and mouse

Gene duplication occurs repeatedly in the evolution of genomes, and the rearrangement of genomic segments has also occurred repeatedly over the evolution of eukaryotes. We studied the interaction of these two factors in mammalian evolution by comparing the chromosomal distribution of multigene families in human and mouse. In both species, gene families tended to be confined to a single chromosome to a greater extent than expected by chance. The average number of families shared between chromosomes was nearly 60% higher in mouse than in human, and human chromosomes rarely shared large numbers of gene families with more than one or two other chromosomes, whereas mouse chromosomes frequently did so. A higher proportion of duplicate gene pairs on the same chromosome originated from recent duplications in human than in mouse, whereas a higher proportion of duplicate gene pairs on separate chromosomes arose from ancient duplications in human than in mouse. These observations are most easily explained by the hypotheses that (1) most gene duplications arise in tandem and are subsequently separated by segmental rearrangement events, and (2) that the process of segmental rearrangement has occurred at a higher rate in the lineage of mouse than in that of human.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

The evolution of spliceosomal introns in alveolates

Many issues concerning the evolution of spliceosomal introns remain poorly understood. In this respect, the reconstruction of the evolution of introns in deep branching species such as alveolates is of special significance. In this study, we inferred the intron evolution in alveolates using 3,368 intron positions in 162 orthologs from 10 species (9 alveolates and 1 outgroup, Homo sapiens). We found that although very few intron gains and losses have occurred in Theileria and Plasmodium recently, many intron gains and losses have occurred in the evolution of alveolates. Thus, the rates of intron gain and loss in alveolates have varied greatly across time and lineage. Our results seem to support the notion that massive intron gains and losses have occurred during short episodes, perhaps coinciding with major evolutionary events.     

Rapidly evolving genes in human. I. The glycophorins and their possible role in evading malaria parasites

In an attempt to identify all fast-evolving genes between human and other primates, we found three glycophorins, GPA, GPB, and GPE, to have the highest rate of nonsynonymous substitutions among the 280 genes surveyed. The Ka/Ks ratios are generally greater than 3 for GPA, GPB, and GPE in human, chimpanzee, and gorilla, indicating positive selection. The uniformly high substitution rate across loci can be explained by the frequent sequence exchanges among genes. GPA is the receptor for the binding ligand EBA-175 of the malaria parasite, Plasmodium falciparum. The levels of nonsynonymous divergence and polymorphism of EBA-175 are also the highest in the genome of P. falciparum. We hypothesize that GPA has been evolving rapidly to evade malaria parasites. Both the high rate of nonsynonymous substitutions and the frequent interlocus conversions may be means of evasion. The support for the evasion hypothesis is still indirect, but, unlike other hypotheses, it can be tested specifically and systematically.     

Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum

Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5 non-coding leader, the 11 introns, and 3 non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3 non-coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3 tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between the GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these twin GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed.     

Evolution of vitamin B6 (pyridoxine) metabolism by gain and loss of genes

Vitamin B(6) (VB6) functions as a cofactor of many diverse enzymes in amino acid metabolism. Three metabolic pathways for pyridoxal 5'-phosphate (PLP; the active form of VB6) are known: the de novo pathway, the salvage pathway, and the fungal type pathway. Most unicellular organisms and plants biosynthesize VB6 using one or two of these three biosynthetic pathways. However, animals such as insects and mammals do not possess any of the pathways and, thus, need to intake VB6 in their diet to survive. It is conceivable that breakdowns of these pathways occurred in the evolutionary lineages of insects and mammals, and one of the major reasons for this would be the loss of pertinent genes. We studied the evolution of VB6 biosynthesis from the view of the gain and loss of 10 pertinent genes in 122 species whose genome sequences were completely determined. The results revealed that each gene in the pathways was lost more than once in the entire evolutionary lineages of the 122 species. We also found the following three points regarding the evolution of PLP biosynthesis: (1) the breakdown of the PLP biosynthetic pathways occurred independently at least three times in animal lineages, (2) the de novo pathway was formed by the generation of pdxB in gamma-proteobacteria, and (3) the order of the gene loss in VB6 metabolism was conserved among different evolutionary lineages. These results suggest that the evolution of VB6 metabolism was subject to gains and frequent losses of related genes in the 122 species examined. This dynamic nature of the evolutionary changes must have been responsible for the breakdowns of the pathways, resulting in profound differentiation of heterotrophy among the species.     

Evaluation of whether accelerated protein evolution in chordates has occurred before, after, or simultaneously with gene duplication

Gene duplication and loss are predicted to be at least of the order of the substitution rate and are key contributors to the development of novel gene function and overall genome evolution. Although it has been established that proteins evolve more rapidly after gene duplication, we were interested in testing to what extent this reflects causation or association. Therefore, we investigated the rate of evolution prior to gene duplication in chordates. Two patterns emerged; firstly, branches, which are both preceded by a duplication and followed by a duplication, display an elevated rate of amino acid replacement. This is reflected in the ratio of nonsynonymous to synonymous substitution (mean nonsynonymous to synonymous nucleotide substitution rate ratio [Ka:Ks]) of 0.44 compared with branches preceded by and followed by a speciation (mean Ka:Ks of 0.23). The observed patterns suggest that there can be simultaneous alteration in the selection pressures on both gene duplication and amino acid replacement, which may be consistent with co-occurring increases in positive selection, or alternatively with concurrent relaxation of purifying selection. The pattern is largely, but perhaps not completely, explained by the existence of certain families that have elevated rates of both gene duplication and amino acid replacement. Secondly, we observed accelerated amino acid replacement prior to duplication (mean Ka:Ks for postspeciation preduplication branches was 0.27). In some cases, this could reflect adaptive changes in protein function precipitating a gene duplication event. In conclusion, the circumstances surrounding the birth of new proteins may frequently involve a simultaneous change in selection pressures on both gene-copy number and amino acid replacement. More precise modeling of the relative importance of preduplication, postduplication, and simultaneous amino acid replacement will require larger and denser genomic data sets from multiple species, allowing simultaneous estimation of lineage-specific fluctuations in mutation rates and adaptive constraints.     

Gene loss and evolutionary rates following whole-genome duplication in teleost fishes

Teleost fishes provide the first unambiguous support for ancient whole-genome duplication in an animal lineage. Studies in yeast or plants have shown that the effects of such duplications can be mediated by a complex pattern of gene retention and changes in evolutionary pressure. To explore such patterns in fishes, we have determined by phylogenetic analysis the evolutionary origin of 675 Tetraodon duplicated genes assigned to chromosomes, using additional data from other species of actinopterygian fishes. The subset of genes, which was retained in double after the genome duplication, is enriched in development, signaling, behavior, and regulation functional categories. The evolutionary rate of duplicate fish genes appears to be determined by 3 forces: 1) fish proteins evolve faster than mammalian orthologs; 2) the genes kept in double after genome duplication represent the subset under strongest purifying selection; and 3) following duplication, there is an asymmetric acceleration of evolutionary rate in one of the paralogs. These results show that similar mechanisms are at work in fishes as in yeast or plants and provide a framework for future investigation of the consequences of duplication in fishes and other animals.     

Investigation of loss and gain of introns in the compact genomes of pufferfishes (Fugu and Tetraodon)

We have investigated intron evolution in the compact genomes of 2 closely related species of pufferfishes, Fugu rubripes and Tetraodon nigroviridis, that diverged about 32 million years ago (MYA). Analysis of 148,028 aligned intron positions in 13,547 gene pairs using human as an outgroup identified 57 and 24 intron losses in Tetraodon and fugu lineages, respectively, and no gain in either lineage. For comparison, we analyzed 144,545 intron positions in 12,866 orthologous pairs of genes in human and mouse that diverged about 61 MYA using dog as an outgroup and identified 51 intron losses in mouse and 3 losses in human and no gain. The rate of intron loss in Tetraodon is higher than that in fugu, mouse, and human but lower than the previous estimates for other eukaryotes. The introns lost in pufferfishes and mammals are significantly shorter than the mean size of introns in the genome. One intron deleted in fugu and another in Tetraodon have left behind 6 and 3 nucleotides, respectively, suggesting that they were lost due to genomic deletions. Such losses of introns are likely to be the result of a higher rate of DNA deletions experienced by the genomes of pufferfishes compared with mammals. The shorter generation time of Tetraodon compared with fugu, and the rich diversity and higher activity of transposable elements in pufferfishes compared with mammals, may be responsible for the higher rate of intron loss in Tetraodon. Our findings indicate that overall very little intron turnover has occurred in pufferfishes and mammals during recent evolution and that intron gain is an extremely rare event in vertebrate evolution.     

A roadmap of tandemly arrayed genes in the genomes of human, mouse, and rat

Tandemly arrayed genes (TAGs) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. Here we identified all TAGs in the genomes of humans, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG orientations and intergenic distances, and TAG functions. TAGs account for about 14-17% of all genes in the genome and nearly one-third of all duplicated genes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests, whereas others are enriched with TAG deserts. The majority of TAGs are of size 2 for all genomes, similar to the previous findings in Caenorhabditis elegans, Arabidopsis thaliana, and Oryza sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes in the genome with parallel orientation. The analyses of Gene Ontology function indicate that genes with receptor or binding activities are significantly overrepresented by TAGs. Computer simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. Finally, the average proportion of TAGs shows a trend of increase with the increase of family sizes, although the correlation between TAG proportions in individual families and family sizes is not significant.     

Molecular evolution and population genetics of duplicated accessory gland protein genes in Drosophila

To investigate the potential importance of gene duplication in D. melanogaster accessory gland protein (Acp) gene evolution we carried out a computational analysis comparing annotated D. melanogaster Acp genes to the entire D. melanogaster genome. We found that two known Acp genes are actually members of small multigene families. Polymorphism and divergence data from these duplicated genes suggest that in at least four cases, protein divergence between D. melanogaster and D. simulans is a result of directional selection. One putative Acp revealed by our computational analysis shows evidence of a recent selective sweep in a non-African population (but not in an African population). These data support the idea that selection on reproduction-related genes may drive divergence of populations within species, and strengthen the conclusion that Acps may often be under directional selection in Drosophila.