Significance of Population Size on the Fixation of Nonsynonymous Mutations in Genes Under Varying Levels of Selection Pressure

Previous studies observed a higher ratio of divergences at nonsynonymous and synonymous sites (ω = d_N/d_S) in species with a small population size compared to that estimated for those with a large population size. Here we examined the theoretical relationship between ω, effective population size (N_e), and selection coefficient (s). Our analysis revealed that when purifying selection is high, ω of species with small N_e is much higher than that of species with large N_e. However the difference between the two ω reduces with the decline in selection pressure (s → 0). We examined this relationship using primate and rodent genes and found that the ω estimated for highly constrained genes of primates was up to 2.9 times higher than that obtained for their orthologous rodent genes. Conversely, for genes under weak purifying selection the ω of primates was only 17% higher than that of rodents. When tissue specificity was used as a proxy for selection pressure we found that the ω of broadly expressed genes of primates was up to 2.1-fold higher than that of their rodent counterparts and this difference was only 27% for tissue specific genes. Since most of the nonsynonymous mutations in constrained or broadly expressed genes are deleterious, fixation of these mutations is influenced by N_e. This results in a higher ω of these genes in primates compared to those from rodents. Conversely, the majority of nonsynonymous mutations in less-constrained or tissue-specific genes are neutral or nearly neutral and therefore fixation of them is largely independent of N_e, which leads to the similarity of ω in primates and rodents.     

Why Do More Divergent Sequences Produce Smaller Nonsynonymous/Synonymous Rate Ratios in Pairwise Sequence Comparisons?

Several studies have reported a negative correlation between estimates of the nonsynonymous to synonymous rate ratio (ω = d_N/d_S) and the sequence distance d in pairwise comparisons of the same gene from different species. That is, more divergent sequences produce smaller estimates of ω. Explanations for this negative correlation have included segregating nonsynonymous polymorphisms in closely related species and nonlinear dynamics of the ratio of two random variables. Here we study the statistical properties of the maximum-likelihood estimates of ω and d in pairwise alignments and explore the possibility that the negative correlation can be entirely explained by those properties. We show that the ω estimate is positively biased for small d and that the bias decreases with the increase of d. We also show that the estimates of ω and d are negatively correlated when ω < 1 and positively correlated when ω > 1. However, the bias in estimates of ω and the correlation between estimates of ω and d are not enough to explain the much stronger correlation observed in real data sets. We then explore the behavior of the estimates when the model is misspecified and suggest that the observed correlation may be due to protein-level selection that causes very different amino acids to be favored in different domains of the protein. Widely used models fail to account for such among-site heterogeneity and cause underestimation of the nonsynonymous rate and ω, with the bias being much stronger for distant sequences. We point out that tests of positive selection based on the ω ratio are invariant to the parameterization of the model and thus unaffected by bias in the ω estimates or the correlation between estimates of ω and d.     

The Genomic Selfing Syndrome Accompanies the Evolutionary Breakdown of Heterostyly

The evolutionary transition from outcrossing to selfing can have important genomic consequences. Decreased effective population size and the reduced efficacy of selection are predicted to play an important role in the molecular evolution of the genomes of selfing species. We investigated evidence for molecular signatures of the genomic selfing syndrome using 66 species of Primula including distylous (outcrossing) and derived homostylous (selfing) taxa. We complemented our comparative analysis with a microevolutionary study of P. chungensis, which is polymorphic for mating system and consists of both distylous and homostylous populations. We generated chloroplast and nuclear genomic data sets for distylous, homostylous, and distylous–homostylous species and identified patterns of nonsynonymous to synonymous divergence (d_N/d_S) and polymorphism (π_N/π_S) in species or lineages with contrasting mating systems. Our analysis of coding sequence divergence and polymorphism detected strongly reduced genetic diversity and heterozygosity, decreased efficacy of purifying selection, purging of large-effect deleterious mutations, and lower rates of adaptive evolution in samples from homostylous compared with distylous populations, consistent with theoretical expectations of the genomic selfing syndrome. Our results demonstrate that self-fertilization is a major driver of molecular evolutionary processes with genomic signatures of selfing evident in both old and relatively young homostylous populations.     

A Survey of the Molecular Evolutionary Dynamics of Twenty-Five Multigene Families from Four Grass Taxa

We surveyed the molecular evolutionary characteristics of 25 plant gene families, with the goal of better understanding general processes in plant gene family evolution. The survey was based on 247 GenBank sequences representing four grass species (maize, rice, wheat, and barley). For each gene family, orthology and paralogy relationships were uncertain. Recognizing this uncertainty, we characterized the molecular evolution of each gene family in four ways. First, we calculated the ratio of nonsynonymous to synonymous substitutions (d _N/d _S) both on branches of gene phylogenies and across codons. Our results indicated that the d _N/d _S ratio was statistically heterogeneous across branches in 17 of 25 (68%) gene families. The vast majority of d _N/d _S estimates were <<1.0, suggestive of selective constraint on amino acid replacements, and no estimates were >1.0, either across phylogenetic lineages or across codons. Second, we tested separately for nonsynonymous and synonymous molecular clocks. Sixty-eight percent of gene families rejected a nonsynonymous molecular clock, and 52% of gene families rejected a synonymous molecular clock. Thus, most gene families in this study deviated from clock-like evolution at either synonymous or nonsynonymous sites. Third, we calculated the effective number of codons and the proportion of G+C synonymous sites for each sequence in each gene family. One or both quantities vary significantly within 18 of 25 gene families. Finally, we tested for gene conversion, and only six gene families provided evidence of gene conversion events. Altogether, evolution for these 25 gene families is marked by selective constraint that varies among gene family members, a lack of molecular clock at both synonymous and nonsynonymous sites, and substantial variation in codon usage. Received: 25 May 2000 / Accepted: 16 October 2000     

Invertebrate Species with Nonpelagic Larvae Have Elevated Levels of Nonsynonymous Substitutions and Reduced Nucleotide Diversities

Under a nearly neutral model in which most amino acid substitutions are slightly deleterious, variation in demography, population structure, and other ecological factors among closely related species can potentially modify the effective population size or the selective regime, leading to differences in the rate of nonsynonymous substitution. Ratios of nonsynonymous to synonymous substitutions (d_N/d_S) between species were analyzed in a sea star genus (Patiriella) and a molluscan genus (Littorina), each with diverse modes of reproduction, including multiple lineages with pelagic and nonpelagic larvae. In both genera, lineages with nonpelagic larvae had significantly higher d_N/d_S ratios than lineages with pelagic larvae. The hypothesis that the elevated d_N/d_S ratios in species with nonpelagic larvae was due to reduced effective population size was tested by comparing nucleotide diversities in three genera of gastropod mollusks (Littorina, Crepidula, and Hydrobia), each with several modes of reproduction. Overall, there was a significant (p < 0.05) reduction in nucleotide diversity in species with nonpelagic larvae compared to species with pelagic larvae.     

How Common Are Intragene Windows with <Emphasis Type="Italic">K</Emphasis><Subscript>A</Subscript> > <Emphasis Type="Italic">K</Emphasis><Subscript>S</Subscript> Owing to Purifying Selection on Synonymous Mutations?

One method for diagnosing the mode of sequence evolution considers the ratio of nonsynonymous substitutions per nonsynonymous site (K _A) to the corresponding figure for synonymous substitutions (K _S). A ratio (K _A/K _S) greater than unity is taken as evidence for positive selection. This, however, need not necessarily be the case. Notably, there is one instance of a high intragenic K _A/K _S peak, revealed by sliding window analysis and observed in two pairwise comparisons, better accounted for by localised purifying selection on synonymous mutations that affect splicing. Is this example exceptional? To address this we isolate intragenic domains with K _A/K _S > 1 from more than 1000 long mouse-rat orthologues. Approximately one K _A/K _S > 1 peak is found per 12–15 kb of coding sequence. Surprisingly, low synonymous substitution rates underpin more incidences than do high nonsynonymous rates. Several reasons, however, prevent us from supposing that the low synonymous rates reflect purifying selection on synonymous mutations. First, for many peaks, the null that the peak is no higher than expected given the underlying rates of evolution, cannot be rejected. Second, of 18 statistically significant incidences with unusually low K _S values, only 3 are repeatable across independent comparisons. At least two of these are within alternatively spliced exons. We conclude that repeatable statistically significant intragenic domains of low intragenic K _S are rare. As so few K _A/K _S peaks reflect increased rates of protein evolution and so few hold statistical support, we additionally conclude that sliding window analysis to infer domains of positive selection is highly error-prone.     

The evolutionary landscape of the Mycobacterium tuberculosis genome

Mycobacterium tuberculosis is one of the most deadly human pathogens. The major mechanism for the adaptations of M. tuberculosis is nucleotide substitution. Previous studies have relied on the nonsynonymous-to-synonymous substitution rate (d_N/d_S) ratio as a measurement of selective constraint based on the assumed selective neutrality of synonymous substitutions. However, this assumption has been shown to be untrue in many cases. In this study, we used the substitution rate in intergenic regions (d_i) of the M. tuberculosis genome as the neutral reference, and conducted a genome-wide profiling for d_i, d_S, and the rate of insertions/deletions (indel rate) as compared with the genome of M. canettii using a 50 kb sliding window. We demonstrate significant variations in all of the three evolutionary measurements across the M. tuberculosis genome, even for regions in close vicinity. Furthermore, we identified a total of 233 genes with their d_S deviating significantly from d_i within the same window. Interestingly, d_S also varies significantly in some of the windows, indicating drastic changes in mutation rate and/or selection pressure within relatively short distances in the M. tuberculosis genome. Importantly, our results indicate that selection on synonymous substitutions is common in the M. tuberculosis genome. Therefore, the d_N/d_S ratio test must be applied carefully for measuring selection pressure on M. tuberculosis genes.     

ASPM and the Evolution of Cerebral Cortical Size in a Community of New World Monkeys

The ASPM (abnormal spindle-like microcephaly associated) gene has been proposed as a major determinant of cerebral cortical size among primates, including humans. Yet the specific functions of ASPM and its connection to human intelligence remain controversial. This debate is limited in part by a taxonomic focus on Old World monkeys and apes. Here we expand the comparative context of ASPM sequence analyses with a study of New World monkeys, a radiation of primates in which enlarged brain size has evolved in parallel in spider monkeys (genus Ateles) and capuchins (genus Cebus). The primate community of Costa Rica is perhaps a model system because it allows for independent pairwise comparisons of smaller- and larger-brained species within two taxonomic families. Accordingly, we analyzed the complete sequence of exon 18 of ASPM in Ateles geoffroyi, Alouatta palliata, Cebus capucinus, and Saimiri oerstedii. As the analysis of multiple species in a genus improves phylogenetic reconstruction, we also analyzed eleven published sequences from other New World monkeys. Our exon-wide, lineage-specific analysis of eleven genera and the ratio of rates of nonsynonymous to synonymous substitutions (d_N/d_S) on ASPM revealed no detectable evidence for positive selection in the lineages leading to Ateles or Cebus, as indicated by d_N/d_S ratios of <1.0 (0.6502 and 0.4268, respectively). Our results suggest that a multitude of interacting genes have driven the evolution of larger brains among primates, with different genes involved in this process in different encephalized lineages, or at least with evidence for positive selection not readily apparent for the same genes in all lineages. The primate community of Costa Rica may serve as a model system for future studies that aim to elucidate the molecular mechanisms underlying cognitive capacity and cortical size.     

Molecular Phylogenies and Evolution of crt Genes in Algae

ABSTRACT

The carotenoids constitute the most widespread class of pigments in nature. Most previous work has concentrated on the identification and characterization of their chemical physical properties and bioavailability. In recent years, significant amounts of research have been conducted in an attempt to analyze the genes and the molecular regulation of the genes involved in the biosynthesis of carotenoids. However, it is important not to lose sight of the early evolution of carotenoid biosynthesis. One of the major obstacles in understanding the evolution of the respective enzymes and their patterns of selection is a lack of a well-supported phylogenic analysis. In the present research, a major long-term objective was to provide a clearer picture of the evolutionary history of genes, together with an evaluation of the patterns of selection in algae. These phylogenies will be important in studies characterizing the evolution of algae. The gene sequences of the enzymes involved in the major steps of the carotenoid biosynthetic pathway in algae (cyanobacteria, rhofophyta, chlorophyta) have been analyzed. Phylogenetic relationships among protein-coding DNA sequences were reconstructed by neighbor-joining (NJ) analysis for the respective carotenoid biosynthetic pathway genes (crt) in algae. The analysis also contains an estimation of the rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N), synonymous nucleotide substitution per synonymous site (d_S), and the ratio of nonsynonmous (d_N/d_S) for the test of selection patterns. The phylogenetic trees show that the taxa of some genera have a closer evolutionary relationship with other genera in some gene sequences, which suggests a common ancient origin and that lateral gene transfer has occurred among unrelated genera. The d_N values of crt genes in the early pathway are relatively low, while those of the following steps are slightly higher, while the d_N values of crt genes in chlorophyta are higher than those in cyanobacteria. Most of the d_N/d_S values exceed 1. The phylogenetic analysis revealed that lateral gene transfer may have taken place across algal genomes and the d_N values suggest that most of the early crt genes are well conserved compared to the later crt genes. Furthermore, d_N values also revealed that the crt genes of chlorophyta are more evolutionary than cyanobacteria. The amino acids' changes are mostly adaptive evolution under the influence of positive diversity selection.     

Domain-Specific Positive Selection Contributes to the Evolution of Arabidopsis Leucine-Rich Repeat Receptor-Like Kinase (LRR RLK) Genes

Leucine-rich repeat receptor-like kinases (LRR RLKs) comprise the largest group within the plant receptor-like kinase (RLK) superfamily, and the Arabidopsis genome alone contains over 200 LRR RLK genes. Although there is clear evidence for diverse roles played by individual LRR RLK genes in Arabidopsis growth and development, the evolutionary mechanism for this functional diversification is currently unclear. In this study, we focused on the LRRII RLK subfamily to investigate the molecular mechanisms that might have led to the functional differentiation of Arabidopsis LRR RLK genes. Phylogenetic analysis of 14 genes in this subfamily revealed three well-supported groups (I, II, and III). RT-PCR analysis did not find many qualitative differences in expression among these 14 genes in various Arabidopsis tissues, suggesting that evolution of regulatory sequences did not play a major role in their functional divergence. We analyzed substitution patterns in the predicted ligand-binding regions of these genes to examine if positive selection has acted to produce novel ligand-binding specificities, using the nonsynonymous/synonymous rate ratio (d _N/d _S) as an indicator of selective pressure. Estimates of d _N/d _S ratios from multiple methods indicate that nonsynonymous substitutions accumulated during divergence of the three lineages. Positive selection is likely to have occurred along the lineages ancestral to groups II and III. We suggest that positive selection on the ligand-binding sites of LRRII RLKs promoted diversification of ligand-binding specificities and thus contributed to the functional differentiation of Arabidopsis LRRII RLK genes during evolution. [Reviewing Editor: Dr. Martin Kreitman]     

Likelihood Analysis of the Chalcone Synthase Genes Suggests the Role of Positive Selection in Morning Glories (<Emphasis Type="Italic">Ipomoea</Emphasis>)

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoides, which are important for the pigmentation of flowers and act as attractants to pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. In morning glories (Ipomoea), five functional CHS genes (A–E) have been described. Phylogenetic analysis of the Ipomoea CHS gene family revealed that CHS A, B, and C experienced accelerated rates of amino acid substitution relative to CHS D and E. To examine whether the CHS genes of the morning glories underwent adaptive evolution, maximum-likelihood models of codon substitution were used to analyze the functional sequences in the Ipomoea CHS gene family. These models used the nonsynonymous/synonymous rate ratio ( = d_N/d_S) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio test suggested significant variation in selection pressure among amino acid sites, with a small proportion of them detected to be under positive selection along the branches ancestral to CHS A, B, and C. Positive Darwinian selection appears to have promoted the divergence of subfamily ABC and subfamily DE and is at least partially responsible for a rate increase following gene duplication.     

Deleterious Mutations Accumulate Faster in Allopolyploid Than Diploid Cotton (Gossypium) and Unequally between Subgenomes

Whole-genome duplication (polyploidization) is among the most dramatic mutational processes in nature, so understanding how natural selection differs in polyploids relative to diploids is an important goal. Population genetics theory predicts that recessive deleterious mutations accumulate faster in allopolyploids than diploids due to the masking effect of redundant gene copies, but this prediction is hitherto unconfirmed. Here, we use the cotton genus (Gossypium), which contains seven allopolyploids derived from a single polyploidization event 1–2 Million years ago, to investigate deleterious mutation accumulation. We use two methods of identifying deleterious mutations at the nucleotide and amino acid level, along with whole-genome resequencing of 43 individuals spanning six allopolyploid species and their two diploid progenitors, to demonstrate that deleterious mutations accumulate faster in allopolyploids than in their diploid progenitors. We find that, unlike what would be expected under models of demographic changes alone, strongly deleterious mutations show the biggest difference between ploidy levels, and this effect diminishes for moderately and mildly deleterious mutations. We further show that the proportion of nonsynonymous mutations that are deleterious differs between the two coresident subgenomes in the allopolyploids, suggesting that homoeologous masking acts unequally between subgenomes. Our results provide a genome-wide perspective on classic notions of the significance of gene duplication that likely are broadly applicable to allopolyploids, with implications for our understanding of the evolutionary fate of deleterious mutations. Finally, we note that some measures of selection (e.g., dN/dS, π_N/π_S) may be biased when species of different ploidy levels are compared.     

Hotspots of Biased Nucleotide Substitutions in Human Genes

Genes that have experienced accelerated evolutionary rates on the human lineage during recent evolution are candidates for involvement in human-specific adaptations. To determine the forces that cause increased evolutionary rates in certain genes, we analyzed alignments of 10,238 human genes to their orthologues in chimpanzee and macaque. Using a likelihood ratio test, we identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a significant tendency to contain clusters of AT-to-GC (weak-to-strong) biased substitutions. This pattern is also observed in noncoding sequence flanking rapidly evolving exons. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of nonsynonymous substitutions relative to the genomic average. We next analyzed genes with significantly elevated ratios of nonsynonymous to synonymous rates of base substitution (d_N/d_S) along the human lineage, and those with an excess of amino acid replacement substitutions relative to human polymorphism. These genes also show evidence of clusters of weak-to-strong biased substitutions. These findings indicate that a recombination-associated process, such as biased gene conversion (BGC), is driving fixation of GC alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby generating significant results for tests of positive selection.     

Genetic Diversity in Exon 2 of the Major Histocompatibility Complex Class II DQB1 Locus in Nigerian Goats

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions (d _N/d _S) suggests that allelic sequence evolution is driven by balancing selection (P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of ?4.00629 (P_deleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes.     

Fast Accumulation of Nonsynonymous Mutations on the Female-Specific W Chromosome in Birds

Following cessation of recombination during sex chromosome evolution, the nonrecombining sex chromosome is affected by a number of degenerative forces, possibly resulting in the fixation of deleterious mutations. This might take place because of weak selection against recessive or partly recessive deleterious mutations due to permanent heterozygosity of nonrecombining chromosomes. Furthermore, population genetic processes, such as selective sweeps, background selection, and Muller’s ratchet, result in a reduction in N_e, which increase the likelihood of fixation of deleterious mutations. Theory thus predicts that nonrecombining genes should show increased levels of nonsynonymous (d_N) to synonymous substitutions (d_S). We tested this in an avian system by estimating the ratio between d_N and d_S in six gametologous gene pairs located on the Z chromosome and the nonrecombining, female-specific W chromosome. In comparisons, we found a significantly higher d_N/d_S ratio for the W-linked than the Z-linked copy in three of the investigated genes. In a concatenated alignment of all six genes, the d_N/d_S ratio was six times higher for W-linked than Z-linked genes. By using human and mouse as outgroup in maximum likelihood analyses, W-linked genes were found to evolve differently compared with their Z-linked gametologues and outgroup sequences. This seems not to be a consequence of functional diversification because d_N/d_S ratios between gametologous gene copies were consistently low. We conclude that deleterious mutations are accumulating at a high rate on the avian W chromosome, probably as a result of the lack of recombination in this female-specific chromosome. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Deborah Charlesworth]     

Helicobacter pylori evolution: lineage- specific adaptations in homologs of eukaryotic Sel1-like genes

Geographic partitioning is postulated to foster divergence of Helicobacter pylori populations as an adaptive response to local differences in predominant host physiology. H. pylori's ability to establish persistent infection despite host inflammatory responses likely involves active management of host defenses using bacterial proteins that may themselves be targets for adaptive evolution. Sequenced H. pylori genomes encode a family of eight or nine secreted proteins containing repeat motifs that are characteristic of the eukaryotic Sel1 regulatory protein, whereas the related Campylobacter and Wolinella genomes each contain only one or two such “Sel1-like repeat” (SLR) genes (“slr genes”). Signatures of positive selection (ratio of nonsynonymous to synonymous mutations, d_N/d_S = ω > 1) were evident in the evolutionary history of H. pylori slr gene family expansion. Sequence analysis of six of these slr genes (hp0160, hp0211, hp0235, hp0519, hp0628, and hp1117) from representative East Asian, European, and African H. pylori strains revealed that all but hp0628 had undergone positive selection, with different amino acids often selected in different regions. Most striking was a divergence of Japanese and Korean alleles of hp0519, with Japanese alleles having undergone particularly strong positive selection (ω_J > 25), whereas alleles of other genes from these populations were intermingled. Homology-based structural modeling localized most residues under positive selection to SLR protein surfaces. Rapid evolution of certain slr genes in specific H. pylori lineages suggests a model of adaptive change driven by selection for fine-tuning of host responses, and facilitated by geographic isolation. Characterization of such local adaptations should help elucidate how H. pylori manages persistent infection, and potentially lead to interventions tailored to diverse human populations.     

BASE: A novel workflow to integrate nonubiquitous genes in comparative genomics analyses for selection

Inferring the selective forces that orthologous genes underwent across different lineages can help us understand the evolutionary processes that have shaped their extant diversity and the phenotypes they underlie. The most widespread metric to estimate the selection regimes of coding genes—across sites and phylogenies—is the ratio of nonsynonymous to synonymous substitutions (dN/dS, also known as ω). Nowadays, modern sequencing technologies and the large amount of already available sequence data allow the retrieval of thousands of orthologous genes across large numbers of species. Nonetheless, the tools available to explore selection regimes are not designed to automatically process all genes, and their practical usage is often restricted to the single‐copy ones which are found across all species considered (i.e., ubiquitous genes). This approach limits the scale of the analysis to a fraction of single‐copy genes, which can be as low as an order of magnitude in respect to those which are not consistently found in all species considered (i.e., nonubiquitous genes). Here, we present a workflow named BASE that—leveraging the CodeML framework—eases the inference and interpretation of gene selection regimes in the context of comparative genomics. Although a number of bioinformatics tools have already been developed to facilitate this kind of analyses, BASE is the first to be specifically designed to allow the integration of nonubiquitous genes in a straightforward and reproducible manner. The workflow—along with all relevant documentation—is available at github.com/for‐giobbe/BASE.