Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution

An excess of nonsynonymous substitutions over synonymous ones is an important indicator of positive selection at the molecular level. A lineage that underwent Darwinian selection may have a nonsynonymous/synonymous rate ratio (dN/dS) that is different from those of other lineages or greater than one. In this paper, several codon-based likelihood models that allow for variable dN/dS ratios among lineages were developed. They were then used to construct likelihood ratio tests to examine whether the dN/dS ratio is variable among evolutionary lineages, whether the ratio for a few lineages of interest is different from the background ratio for other lineages in the phylogeny, and whether the dN/dS ratio for the lineages of interest is greater than one. The tests were applied to the lysozyme genes of 24 primate species. The dN/dS ratios were found to differ significantly among lineages, indicating that the evolution of primate lysozymes is episodic, which is incompatible with the neutral theory. Maximum- likelihood estimates of parameters suggested that about nine nonsynonymous and zero synonymous nucleotide substitutions occurred in the lineage leading to hominoids, and the dN/dS ratio for that lineage is significantly greater than one. The corresponding estimates for the lineage ancestral to colobine monkeys were nine and one, and the dN/dS ratio for the lineage is not significantly greater than one, although it is significantly higher than the background ratio. The likelihood analysis thus confirmed most, but not all, conclusions Messier and Stewart reached using reconstructed ancestral sequences to estimate synonymous and nonsynonymous rates for different lineages.      

Uncorrected nucleotide bias in mtDNA can mimic the effects of positive Darwinian selection

The relative rates of nucleotide substitution at synonymous and nonsynonymous sites within protein-coding regions have been widely used to infer the action of natural selection from comparative sequence data. It is known, however, that mutational and repair biases can affect rates of evolution at both synonymous and nonsynonymous sites. More importantly, it is also known that synonymous sites are particularly prone to the effects of nucleotide bias. This means that nucleotide biases may affect the calculated ratio of substitution rates at synonymous and nonsynonymous sites. Using a large data set of animal mitochondrial sequences, we demonstrate that this is, in fact, the case. Highly biased nucleotide sequences are characterized by significantly elevated dN/dS ratios, but only when the nucleotide frequencies are not taken into account. When the analysis is repeated taking the nucleotide frequencies at each codon position into account, such elevated ratios disappear. These results suggest that the recently reported differences in dN/dS ratios between vertebrate and invertebrate mitochondrial sequences could be explained by variations in mitochondrial nucleotide frequencies rather than the effects of positive Darwinian selection.     

Spatial covariation of mutation and nonsynonymous substitution rates in vertebrate mitochondrial genomes

Mitochondrial genomes encode fundamental subunits of the basic energy producing machinery of eukaryotic cells that are under strong functional constraint. Paradoxically, these genes evolve rapidly in general, and there is substantial variation in evolutionary rates among genes within genomes. In order to investigate spatial variation in selection intensity, we conducted tests of neutrality using ratios of synonymous to nonsynonymous substitutions (dN/dS = omega) on numerous protein gene segments from fishes and mammals. Values of omega were very low for nearly all genomic regions. However, values of both omega and dN varied in a clinal pattern with increasing distance from the light-strand origin of replication. Spatial heterogeneity of nonsynonymous substitution rates exhibits a significantly positive correlation with variation in mutation rates that are related to the mode of mitochondrial DNA replication. The finding that nonsynonymous substitution rates are proportional to mutation rates is expected if a majority of substitutions are selectively neutral or slightly deleterious. Spatial patterns of among-gene variation in nonsynonymous rates were highly similar between fishes and mammals, suggesting that forces governing mitochondrial gene evolution have remained relatively constant over 450 Myr of vertebrate evolution. Conservation of substitution patterns despite major shifts in thermal habit and metabolic demands among taxa implicates a conserved replication mechanism controlling relative mutation rates as a major determinant of mitochondrial protein evolution.     

Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E)

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.     

Phylogeny, rate variation, and genome size evolution of Pelargonium (Geraniaceae)

The phylogeny of 58 Pelargonium species was estimated using five plastid markers (rbcL, matK, ndhF, rpoC1, trnL-F) and one mitochondrial gene (nad5). The results confirmed the monophyly of three major clades and four subclades within Pelargonium but also indicate the need to revise some sectional classifications. This phylogeny was used to examine karyotype evolution in the genus: plotting chromosome sizes, numbers and 2C-values indicates that genome size is significantly correlated with chromosome size but not number. Accelerated rates of nucleotide substitution have been previously detected in both plastid and mitochondrial genes in Pelargonium, but sparse taxon sampling did not enable identification of the phylogenetic distribution of these elevated rates. Using the multigene phylogeny as a constraint, we investigated lineage- and locus-specific heterogeneity of substitution rates in Pelargonium for an expanded number of taxa and demonstrated that both plastid and mitochondrial genes have had accelerated substitution rates but with markedly disparate patterns. In the plastid, the exons of rpoC1 have significantly accelerated substitution rates compared to its intron and the acceleration was mainly due to nonsynonymous substitutions. In contrast, the mitochondrial gene, nad5, experienced substantial acceleration of synonymous substitution rates in three internal branches of Pelargonium, but this acceleration ceased in all terminal branches. Several lineages also have dN/dS ratios significantly greater than one for rpoC1, indicating that positive selection is acting on this gene, whereas the accelerated synonymous substitutions in the mitochondrial gene are the result of elevated mutation rates.     

Lineage‐specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana

Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the ‘edges’ of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage‐specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage‐specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage‐specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.     

The evolution of mitochondrial genomes in subterranean caviomorph rodents: adaptation against a background of purifying selection

South American tuco-tucos (Ctenomys) and the related coruro (Spalacopus) are two rodent lineages that have independently colonised the subterranean niche. The energetically demanding lifestyles of these species, coupled with the hypoxic atmospheres characteristic of subterranean environments, may have altered the selective regimes on genes encoding proteins related to cellular respiration. Here, we examined the molecular evolution of 13 protein-coding genes in the mitochondrial genome of seven caviomorph rodents, including these two subterranean genera and their above-ground relatives. Using maximum-likelihood and Bayesian approaches, we estimated rates of synonymous (dS) and nonsynonymous (dN) substitutions. We found a significantly higher ω ratio (dN/dS) in subterranean groups as compared to their non-subterranean counterparts in 11 of 13 genes, although no ω ratio was larger than 1. Additionally, we applied a method based on quantitative physicochemical properties to test for positive selection. Amino acid changes implicated in radical structural or functional shifts in the protein property were found to be ubiquitous across the phylogeny, but concentrated in the subterranean lineages. Convergent changes were also found between the subterranean genera used in this study and other mammals adapted to hypoxia. The results of this study suggest a link between niche shifts and weak directional (or episodic) selection at the molecular level against a background of purifying selection.     

Apparent Selection Intensity for the Cytochrome Oxidase Subunit I Gene Varies with Mode of Reproduction in Echinoderms

When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.     

Differential rates of evolution for the ZFY-related zinc finger genes,Zfy, Zfx,and Zfa in the mouse genus Mus

A comparative study of the last exon of the zinc finger genes Zfx, Zfy, and Zfa from species of mice in the genus Mus was conducted to assess the extent of gene-specific and chromosome-specific effects on the evolutionary patterns among related X-, Y-, and autosomal-linked genes. Phylogenetic analyses of 29 sequences from Zfx, Zfa, and Zfy from 10 taxa were performed to infer relatedness among the zinc finger loci, and codon-based maximum likelihood analyses were conducted to assess evolutionary pattern among genes. Five models of nucleotide sequence evolution were applied and compared using a likelihood ratio test. Estimates of nonsynonymous to synonymous changes (dN/dS) for these genes suggest that amino acid substitutions are occurring at a more rapid rate across the autosomal- and Y-specific lineages compared to the X-specific lineage, with the Y-specific lineage showing the highest rate under certain models. The data suggest the action of gene-specific effects on evolutionary pattern. In particular, Zfa and Zfy genes, both with presumed restricted expression, appear less functionally constrained relative to ubiquitously expressed Zfx. Slightly elevated dN/dS for Zfy genes in comparison to Zfa also suggest Y-specific effects.     

Independent effects of alternative splicing and structural constraint on the evolution of mammalian coding exons

Alternative splicing (AS) is known to significantly affect exon-level protein evolutionary rates in mammals. Particularly, alternatively spliced exons (ASEs) have a higher nonsynonymous-to-synonymous substitution rate (dN/dS) ratio than constitutively spliced exons (CSEs), possibly because the former are required only occasionally for normal biological functions. Meanwhile, intrinsically disordered regions (IDRs), the protein regions lacking fixed 3D structures, are also reported to have an increased evolutionary rate due to lack of structural constraint. Interestingly, IDRs tend to be located in alternative protein regions. Yet which of these two factors is the major determinant of the increased dN/dS in mammalian ASEs remains unclear. By comparing human-macaque and human-mouse one-to-one orthologous genes, we demonstrate that AS and protein structural disorder have independent effects on mammalian exon evolution. We performed analyses of covariance to demonstrate that the slopes of the (dN/dS-percentage of IDR) regression lines differ significantly between CSEs and ASEs. In other words, the dN/dS ratios of both ASEs and CSEs increase with the proportion of IDR (PIDR), whereas ASEs have higher dN/dS ratios than CSEs when they have similar PIDRs. Since ASEs and IDRs may less frequently overlap with protein domains (which also affect dN/dS), we also examined the correlations between dN/dS ratio and exon type/PIDR by controlling for the density of protein domain. We found that the effects of exon type and PIDR on dN/dS are both independent of domain density. Our results imply that nature can select for different biological features with regard to ASEs and IDRs, even though the two biological features tend to be localized in the same protein regions.     

Evolution of duplicate genes in a tetraploid animal, Xenopus laevis

To understand the evolution of duplicate genes, we compared rates of nucleotide substitution between 17 pairs of nonallelic duplicated genes in the tetraploid frog Xenopus laevis with rates between the orthologous loci of human and rodent. For all duplicated X. laevis genes, the number of synonymous substitutions per site (dS) was greater than the number of nonsynonymous substitutions per site (dN), indicating that these genes are subject to purifying selection. There was also a significant positive correlation (r = 0.915) between dN for the X. laevis genes and dN for the mammalian genes, suggesting that, at the amino acid level, the X. laevis genes and the mammalian genes are under similar constraints. Results of relative-rate tests showed nearly equal rates of nonsynonymous substitution in each copy of the X. laevis genes; apparently there are similar constraints on both copies. No correlation was found between dS for the X. laevis genes and dS for the mammalian genes. There was a significant positive correlation both between members of pairs of duplicated X. laevis genes (r = 0.951) and between human and rodent orthologues (r = 0.854) with respect to third- position G+C content but no such relationship between the X. laevis genes and either of their mammalian orthologues. The results indicate that both copies of a duplicate gene can be subject to purifying selection and thus support the hypothesis of selection against all genotypes containing a null allele at either of two duplicate loci.      

Two mitochondrial genes under episodic positive selection in subterranean octodontoid rodents

Tuco-tucos (Ctenomys) and related coruros (Spalacopus) are South American subterranean rodents. An energetically demanding lifestyle within the hypoxic, underground atmosphere may change the selective regime on oxidative phosphorylation. We examined whether weak and/or episodic positive directional selection affected the evolution of two mitochondrial genes (COX2, CytB), in a background of purifying selection in these lineages. We estimated rates of synonymous (dS) and non-synonymous (dN) substitutions and found: 1) significantly higher dN/dS ratio in subterranean groups relative to non-subterranean related species, and 2) two codons in each gene under episodic selection: 94 and 277 of COX2 and 269 and 307 of CytB.     

Molecular Evolution of the Genomic RNA of Apple Stem Grooving Capillovirus

The complete genome of the German isolate AC of Apple stem grooving virus (ASGV) was sequenced. It encodes two overlapping open reading frames (ORFs), similarly to previously described ASGV isolates. Two regions of high variability were detected between the ASGV isolates, variable region 1 (V1, from amino acids (aa) 532 to 570), and variable region 2 (V2, from aa 1,583 to 1,868). The phylogenetic analysis of the V1 and V2 regions suggested that the ASGV diversity was structured by host plant species rather than geographical origin. The dN/dS ratio between nonsynonymous and synonymous nucleotide substitution rates varied greatly along the ASGV genome. Most of ORF1 showed predominant negative selection except for the two regions V1 and V2. V1 showed an elevated dN and an average dS when compared to the ORF1 background but no significant positive selection was detected. The V2 region of ORF1 showed an elevated dN and a low dS when compared to the ORF1 background with an average dN/dS????3.0 indicative of positive selection. However, the V2 area includes overlapping ORFs, making the dN/dS estimate biased. Joint estimates of the selection intensity in the different ORFs by a recent method indicated that this region of ORF1 was in fact evolving close to neutrality. This was convergent with previous results showing that introduction of stop codons in this region of ORF1 did not impair plant infection. These data suggest that the elimination of a stop codon caused the overprinting of a novel coding region over the ancestral ORF.     

Lower prevalence but similar fitness in a parasitic fungus at higher radiation levels near Chernobyl

Nuclear disasters at Chernobyl and Fukushima provide examples of effects of acute ionizing radiation on mutations that can affect the fitness and distribution of species. Here, we investigated the prevalence of Microbotryum lychnidis‐dioicae, a pollinator‐transmitted fungal pathogen of plants causing anther‐smut disease in Chernobyl, its viability, fertility and karyotype variation, and the accumulation of nonsynonymous mutations in its genome. We collected diseased flowers of Silene latifolia from locations ranging by more than two orders of magnitude in background radiation, from 0.05 to 21.03 μGy/h. Disease prevalence decreased significantly with increasing radiation level, possibly due to lower pollinator abundance and altered pollinator behaviour. Viability and fertility, measured as the budding rate of haploid sporidia following meiosis from the diploid teliospores, did not vary with increasing radiation levels and neither did karyotype overall structure and level of chromosomal size heterozygosity. We sequenced the genomes of twelve samples from Chernobyl and of four samples collected from uncontaminated areas and analysed alignments of 6068 predicted genes, corresponding to 1.04 × 10⁷ base pairs. We found no dose‐dependent differences in substitution rates (neither dN, dS, nor dN/dS). Thus, we found no significant evidence of increased deleterious mutation rates at higher levels of background radiation in this plant pathogen. We even found lower levels of nonsynonymous substitution rates in contaminated areas compared to control regions, suggesting that purifying selection was stronger in contaminated than uncontaminated areas. We briefly discuss the possibilities for a mechanistic basis of radio resistance in this nonmelanized fungus.     

A Sliding Window-Based Method to Detect Selective Constraints in Protein-Coding Genes and Its Application to RNA Viruses

Here we present a new sliding window-based method specially designed to detect selective constraints in specific regions of a multiple protein-coding sequence alignment. In contrast to previous window-based procedures, our method is based on a nonarbitrary statistical approach to find the appropriate codon-window size to test deviations of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions from the expectation. The probabilities of dN and dS are obtained from simulated data and used to detect significant deviations of dN and dS in a specific window region of the real sequence alignment. The nonsynonymous-to-synonymous rate ratio (w = dN/dS) was used to highlight selective constraints in any window wherein dS or dN was significantly different from the expectation. In these significant windows, w and its variance [V(w)] were calculated and used to test the neutral hypothesis. Computer simulations showed that the method is accurate even for highly divergent sequences. The main advantages of the new method are that it (i) uses a statistically appropriate window size to detect different selective patterns, (ii) is computationally less intensive than maximum likelihood methods, and (iii) detects saturation of synonymous sites, which can give deviations from neutrality. Hence, it allows the analysis of highly divergent sequences and the test of different alternative hypothesis as well. The application of the method to different human immunodeficiency virus type 1 and to foot-and-mouth disease virus genes confirms the action of positive selection on previously described regions as well as on new regions.     

Rapidly evolving mitochondrial genome and directional selection in mitochondrial genes in the parasitic wasp nasonia (hymenoptera: pteromalidae)

We sequenced the nearly complete mtDNA of 3 species of parasitic wasps, Nasonia vitripennis (2 strains), Nasonia giraulti, and Nasonia longicornis, including all 13 protein-coding genes and the 2 rRNAs, and found unusual patterns of mitochondrial evolution. The Nasonia mtDNA has a unique gene order compared with other insect mtDNAs due to multiple rearrangements. The mtDNAs of these wasps also show nucleotide substitution rates over 30 times faster than nuclear protein-coding genes, indicating among the highest substitution rates found in animal mitochondria (normally <10 times faster). A McDonald and Kreitman test shows that the between-species frequency of fixed replacement sites relative to silent sites is significantly higher compared with within-species polymorphisms in 2 mitochondrial genes of Nasonia, atp6 and atp8, indicating directional selection. Consistent with this interpretation, the Ka/Ks (nonsynonymous/synonymous substitution rates) ratios are higher between species than within species. In contrast, cox1 shows a signature of purifying selection for amino acid sequence conservation, although rates of amino acid substitutions are still higher than for comparable insects. The mitochondrial-encoded polypeptides atp6 and atp8 both occur in F0F1ATP synthase of the electron transport chain. Because malfunction in this fundamental protein severely affects fitness, we suggest that the accelerated accumulation of replacements is due to beneficial mutations necessary to compensate mild-deleterious mutations fixed by random genetic drift or Wolbachia sweeps in the fast evolving mitochondria of Nasonia. We further propose that relatively high rates of amino acid substitution in some mitochondrial genes can be driven by a "Compensation-Draft Feedback"; increased fixation of mildly deleterious mutations results in selection for compensatory mutations, which lead to fixation of additional deleterious mutations in nonrecombining mitochondrial genomes, thus accelerating the process of amino acid substitutions.     

Mitochondrial DNA as a tool for reconstructing past life‐history traits in mammals

Reconstructing the ancestral characteristics of species is a major goal in evolutionary and comparative biology. Unfortunately, fossils are not always available and sufficiently informative, and phylogenetic methods based on models of character evolution can be unsatisfactory. Genomic data offer a new opportunity to estimate ancestral character states, through (i) the correlation between DNA evolutionary processes and species life‐history traits and (ii) available reliable methods for ancestral sequence inference. Here, we assess the relevance of mitochondrial DNA – the most popular molecular marker in animals – as a predictor of ancestral life‐history traits in mammals, using the order of Cetartiodactyla as a benchmark. Using the complete set of 13 mitochondrial protein‐coding genes, we show that the lineage‐specific nonsynonymous over synonymous substitution rate ratio (dN/dS) is closely correlated with the species body mass, longevity and age of sexual maturity in Cetartiodactyla and can be used as a marker of ancestral traits provided that the noise introduced by short branches is appropriately dealt with. Based on ancestral dN/dS estimates, we predict that the first cetartiodactyls were relatively small animals (around 20 kg). This finding is in accordance with Cope's rule and the fossil record but could not be recovered via continuous character evolution methods.