Enhanced Synonymous Site Divergence in Positively Selected VertebrateAntimicrobial Peptide Genes

Nonrandom patterns associated with adaptively evolving genes can shed light on how selection and mutation produce rapid changes in sequences. I examine such patterns in two independent families of antimicrobial peptide genes: those in frogs, which are known to have evolved under positive selection, and those in flatfishes, which I show have also evolved under positive selection. I address two recently proposed hypotheses about the molecular evolution of antimicrobial peptide genes. The first is that the mature peptide region is replicated by an error-prone polymerase that increases the mutation rate and the transversion/transition ratio compared to the signal sequence of the same genes. The second is that mature peptides evolve in a coordinated fashion with their propieces, such that a change in net charge in one molecular region prompts an opposite change in charge in the other region. I test these hypotheses using alternative methods that minimize alignment errors, correct for phylogenetic nonindependence, reduce sequence saturation, and account for differing selection pressures on different regions of the gene. In both gene families I show that divergence at both synonymous and nonsynonymous sites within the mature peptide region is enhanced. However, in neither gene family is there evidence of an increased mutational transversion/transition ratio or coordinated evolution. My observations are consistent with either an elevated mutation rate in an adaptively evolving gene region or widespread selection on “silent” sites. These hypotheses challenge the assumption that mutations are random and can be measured by the synonymous substitution rate. [Reviewing Editor: Dr. Willie J. Swanson]     

Adaptive protein evolution and regulatory divergence in Drosophila

Two recent studies demonstrated a positive correlation between divergence in gene expression and protein sequence in Drosophila. This correlation could be driven by positive selection or variation in functional constraint. To distinguish between these alternatives, we compared patterns of molecular evolution for 1,862 genes with two previously reported estimates of expression divergence in Drosophila. We found a slight negative trend (nonsignificant) between positive selection on protein sequence and divergence in expression levels between Drosophila melanogaster and Drosophila simulans. Conversely, shifts in expression patterns during Drosophila development showed a positive association with adaptive protein evolution, though as before the relationship was weak and not significant. Overall, we found no strong evidence for an increase in the incidence of positive selection on protein-coding regions in genes with divergent expression in Drosophila, suggesting that the previously reported positive association between protein and regulatory divergence primarily reflects variation in functional constraint.     

Protein evolution and codon usage bias on the neo-sex chromosomes of Drosophila miranda

The neo-sex chromosomes of Drosophila miranda constitute an ideal system to study the effects of recombination on patterns of genome evolution. Due to a fusion of an autosome with the Y chromosome, one homolog is transmitted clonally. Here, I compare patterns of molecular evolution of 18 protein-coding genes located on the recombining neo-X and their homologs on the nonrecombining neo-Y chromosome. The rate of protein evolution has significantly increased on the neo-Y lineage since its formation. Amino acid substitutions are accumulating uniformly among neo-Y-linked genes, as expected if all loci on the neo-Y chromosome suffer from a reduced effectiveness of natural selection. In contrast, there is significant heterogeneity in the rate of protein evolution among neo-X-linked genes, with most loci being under strong purifying selection and two genes showing evidence for adaptive evolution. This observation agrees with theory predicting that linkage limits adaptive protein evolution. Both the neo-X and the neo-Y chromosome show an excess of unpreferred codon substitutions over preferred ones and no difference in this pattern was observed between the chromosomes. This suggests that there has been little or no selection maintaining codon bias in the D. miranda lineage. A change in mutational bias toward AT substitutions also contributes to the decline in codon bias. The contrast in patterns of molecular evolution between amino acid mutations and synonymous mutations on the neo-sex-linked genes can be understood in terms of chromosome-specific differences in effective population size and the distribution of selective effects of mutations.     

Positive and negative selection on mammalian Y chromosomes

Y chromosomes are genetically degenerate in most organisms studied. The loss of genes from Y chromosomes is thought to be due to the inefficiency of purifying selection in nonrecombining regions, which leads to the accumulation of deleterious mutations via the processes of hitchhiking, background selection, and Muller's ratchet. As the severity of these processes depends on the number of functional genes linked together on the nonrecombining Y, it is not clear whether these processes are still at work on the old, gene-poor mammalian Y chromosomes. If purifying selection is indeed less efficient in the Y-linked, compared to the X-linked genes, deleterious nonsynonymous substitutions are expected to accumulate faster on the Y chromosome. However, positive selection on Y-linked genes could also increase the rate of amino acid-changing substitutions. Thus, the previous reports of an elevated nonsynonymous substitution rate in Y-linked genes are still open to interpretation. Here, we report evidence for positive selection in two out of three studied mammalian Y-linked genes, suggesting that adaptive Darwinian evolution may be common on mammalian Y chromosomes. Taking positive selection into account, we demonstrate that purifying selection is less efficient in mammalian Y-linked genes compared to their X-linked homologues, suggesting that these genes continue to degenerate.     

Adaptive evolution after duplication of penaeidin antimicrobial peptides

Penaeidin antimicrobial peptides in penaeid shrimps are an important component of their innate immune system that provides immunity against infection caused by several gram-positive bacteria and filamentous fungal species. Despite the knowledge on the identification and characterization of these peptides in penaeid shrimps, little is known about the evolutionary pattern of these peptides and the underlying genetic mechanisms that maintain high sequence diversities in the penaeidin gene family. Based on the phylogenetic analyses and maximum likelihood-based codon substitution analyses, here we present the convincing evidence that multiple copies of penaeidins have evolved by gene duplication, and positive Darwinian selection (adaptive evolution) is the likely cause of accelerated rate of amino acid substitutions among these duplicated genes. While the average ratio of non-synonymous to synonymous substitutions (omega) for the entire coding region of both active domains is 0.9805, few codon sites showed significantly higher omega (3.73). The likelihood ratio tests that compare models incorporating positive selection (omega>1) at certain codon sites with models not incorporating positive selection (omega<1), failed to reject (p=0) the evidence of positive Darwinian selection. The rapid adaptive evolution of this gene family might be directed by the pathogens and the faster rate of amino acid substitutions in the N-terminal proline-rich and C-terminal cysteine-rich domains could be due to their direct involvement in the protection against pathogens. When the host expose to different habitats/environment an accelerated rate of amino acid substitutions in both the active domains may also be expected.     

Selection for Antimicrobial Peptide Diversity in Frogs Leads to Gene Duplication and Low Allelic Variation

Antimicrobial peptides are highly diverse pathogen-killing molecules. In many taxa, their evolution is characterized by positive selection and frequent gene duplication. It has been proposed that genes encoding antimicrobial peptides might be subject to balancing selection and/or an enhanced mutation rate, but these hypotheses have not been well evaluated because allelic variation has rarely been studied at antimicrobial peptide loci. We present an evolutionary analysis of novel antimicrobial peptide genes from leopard frogs, Rana. Our results demonstrate that a single genome contains multiple homologous copies, among which there is an excess of nonsynonymous nucleotide site divergence relative to that expected from synonymous site divergence. Thus, we confirm the trends of recurrent duplication and positive selection. Allelic variation is quite low relative to interspecies divergence, indicating a recent positive selective sweep with no evidence of balancing selection. Repeated gene duplication, rather than a balanced maintenance of divergent allelic variants at individual loci, appears to be how frogs have responded to selection for a diverse suite of antimicrobial peptides. Our data also support a pattern of enhanced synonymous site substitution in the mature peptide region of the gene, but we cannot conclude that this is due to an elevated mutation rate.     

Sexually dimorphic gene expression and transcriptome evolution provide mixed evidence for a fast‐Z effect in Heliconius

Sex chromosomes have different evolutionary properties compared to autosomes due to their hemizygous nature. In particular, recessive mutations are more readily exposed to selection, which can lead to faster rates of molecular evolution. Here, we report patterns of gene expression and molecular evolution for a group of butterflies. First, we improve the completeness of the Heliconius melpomene reference annotation, a neotropical butterfly with a ZW sex determination system. Then, we analyse RNA from male and female whole abdomens and sequence female ovary and gut tissue to identify sex‐ and tissue‐specific gene expression profiles in H. melpomene. Using these expression profiles, we compare (a) sequence divergence and polymorphism; (b) the strength of positive and negative selection; and (c) rates of adaptive evolution, for Z and autosomal genes between two species of Heliconius butterflies, H. melpomene and H. erato. We show that the rate of adaptive substitutions is higher for Z than autosomal genes, but contrary to expectation, it is also higher for male‐biased than female‐biased genes. Additionally, we find no significant increase in the rate of adaptive evolution or purifying selection on genes expressed in ovary tissue, a heterogametic‐specific tissue. Our results contribute to a growing body of literature from other ZW systems that also provide mixed evidence for a fast‐Z effect where hemizygosity influences the rate of adaptive substitutions.     

Selection on an antimicrobial peptide defensin in ants

Ants live in crowded nests with interacting individuals, which makes them particularly prone to infectious diseases. The question is, how do ants cope with the increased risk of pathogen transmission due to sociality? We have studied the molecular evolution of defensin, a gene encoding an antimicrobial protein, in ants. Defensin sequences from several ant species were analyzed with maximum likelihood models of codon substitution to infer selection. Positive selection was detected in the mature region of defensin, whereas the signal and pro regions seem to be evolving neutrally. We also found a significantly higher rate of nonsynonymous substitutions in some phylogenetic lineages, as well as dN/dS >1, suggesting varying selection pressures in different lineages. Earlier studies on the molecular evolution of insect antimicrobial peptide genes have focused on termites and dipteran species, and detected positive selection only in duplicated termicin genes in termites. These findings, together with our present results, provide an indication that the immune systems of social insects (ants and termites) and dipteran insects may have responded differently to the selection pressure caused by microbial pathogens.     

Evidence of positively selected sites in mammalian alpha-defensins

Alpha-defensins are a family of mammalian antimicrobial peptides that exhibit variable activity against a panel of microbes, including bacteria, fungi, and enveloped viruses. We have employed a maximum-likelihood approach to detect evidence of positive selection (adaptive evolution) in the evolution of these important molecules of the innate immune response. We have identified 14 amino acid sites that are predicted to be subject to positive selection. Furthermore, we show that all these sites are located in the mature antimicrobial peptide and not in the prepropeptide region of the molecule, implying that they are of functional importance. These results suggest that mammalian alpha-defensins have been under selective pressure to evolve in response to potentially infectious challenges by fast-evolving microbes.     

Genomic evidence for a large-Z effect

The 'large-X effect' suggests that sex chromosomes play a disproportionate role in adaptive evolution. Theoretical work indicates that this effect may be most pronounced in genetic systems with female heterogamety under both good-genes and Fisher's runaway models of sexual selection (males ZZ, females ZW). Here, I use a comparative genomic approach (alignments of several thousands of chicken-zebra finch-human-mouse-opossum orthologues) to show that avian Z-linked genes are highly overrepresented among those bird-mammalian orthologues that show evidence of accelerated rate of functional evolution in birds relative to mammals; the data suggest a twofold excess of such genes on the Z chromosome. A reciprocal analysis of genes accelerated in mammals found no evidence for an excess of X-linkage. This would be compatible with theoretical expectations for differential selection on sex-linked genes under male and female heterogamety, although the power in this case was not sufficient to statistically show that 'large-Z' was more pronounced than 'large-X'. Accelerated Z-linked genes include a variety of functional categories and are characterized by higher non-synonymous to synonymous substitution rate ratios than both accelerated autosomal and non-accelerated genes. This points at a genomic 'large-Z effect', which is widespread and of general significance for adaptive divergence in birds.     

The dermaseptin superfamily: A gene-based combinatorial library of antimicrobial peptides

Skin secretions of hylid frogs show amazing levels of interspecific and intraspecific diversity and are comprised of a cocktail of genetically-related, but markedly diverse antimicrobial peptides that are grouped into a superfamily, termed the dermaseptins, comprising several families: dermaseptins (sensu stricto), phylloseptins, plasticins, dermatoxins, phylloxins, hyposins, caerins, and aureins. Dermaseptin gene superfamily evolution is characterized by repeated gene duplications and focal hypermutations of the mature peptide coding sequence, followed by positive (diversifying) selection. We review here molecular mechanisms leading to these vast combinatorial peptide libraries, and structural and functional properties of antimicrobial peptides of the dermaseptin and plasticin families, as well as those of dermaseptin S9, an amyloidogenic peptide with antimicrobial and chemoattractant activities.     

Nonneutral evolution and differential mutation rate of gender-associated mitochondrial DNA lineages in the marine mussel Mytilus.

Mussels have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally, and the F type is transmitted maternally. To test hypotheses of the molecular evolution of both mtDNA genomes, 50 nucleotide sequences were obtained for 396 bp of the COIII gene of European populations of Mytilus edulis and the Atlantic and Mediterranean forms of M. galloprovincialis. Analysis based on the proportion of synonymous and nonsynonymous substitutions indicate that mtDNA is evolving in a non-neutral and complex fashion. Previous studies on American mussels demonstrated that the F genome experiences a higher purifying selection and that the M genome evolves faster. Here we show that these patterns also hold in European populations. However, in contrast to American populations, where an excess of replacement substitution between F and M lineages has been reported, a significant excess of replacement polymorphism within mtDNA lineages is observed in European populations of M. galloprovincialis. European populations also show an excess of replacement polymorphism within the F but not within the M genome with respect to American M. trossulus, as well as a consistent pattern of excess of rare variants in both F and M genomes. These results are consistent with a nearly neutral model of molecular evolution and a recent relaxation of selective constraints on European mtDNA. Levels of diversity are significantly higher for the M than F genome, and the M genome also accumulates synonymous and nonsynonymous substitutions at a higher rate, in contrast with earlier reports where no difference for the synonymous rate was observed. It is suggested that a subtle balance between relaxed selection and a higher mutation rate explains the faster evolutionary rate of the M lineage.     

Evolution of caprine and ovine β-defensin genes

Defensins comprise an important family of anti-microbial peptides. Among vertebrates, numerous defensin genes have been detected, but their evolutionary background is still discussed. We investigated the molecular evolution and variability of β-defensins of Caprini via sequence analyses of defensin introns. Screening of several domestic and wild species of Caprini revealed a total of 13 discrete β-defensin coding sequences, with three of them described before this study. Phylogenetic analyses revealed that the array of newly described defensin genes is of monophyletic origin and has arisen in numerous independent duplication events after separation of the ancestral defensins. As a result of that scenario, recent defensin genes are distributed in a species-specific manner. Values of synonymous and non-synonymous substitutions demonstrated that both modes of evolutionary pressure, positive as well as negative selection, have acted. In addition, conservation of some β-defensin exons is demonstrated. Discrimination of certain β-defensin genes was possible only due to intron-specific differences. Therefore, sequence analyses restricted to the exons would result in underestimation of the number of β-defensin genes. Our study shows that for reconstruction of the phylogenetic history data of defensin introns are more appropriated. Comparisons among the amino acid sequences show moderate substitutions without changing the net charge of the mature peptides. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Selectionism and neutralism in molecular evolution

Charles Darwin proposed that evolution occurs primarily by natural selection, but this view has been controversial from the beginning. Two of the major opposing views have been mutationism and neutralism. Early molecular studies suggested that most amino acid substitutions in proteins are neutral or nearly neutral and the functional change of proteins occurs by a few key amino acid substitutions. This suggestion generated an intense controversy over selectionism and neutralism. This controversy is partially caused by Kimura's definition of neutrality, which was too strict (|2Ns|< or =1). If we define neutral mutations as the mutations that do not change the function of gene products appreciably, many controversies disappear because slightly deleterious and slightly advantageous mutations are engulfed by neutral mutations. The ratio of the rate of nonsynonymous nucleotide substitution to that of synonymous substitution is a useful quantity to study positive Darwinian selection operating at highly variable genetic loci, but it does not necessarily detect adaptively important codons. Previously, multigene families were thought to evolve following the model of concerted evolution, but new evidence indicates that most of them evolve by a birth-and-death process of duplicate genes. It is now clear that most phenotypic characters or genetic systems such as the adaptive immune system in vertebrates are controlled by the interaction of a number of multigene families, which are often evolutionarily related and are subject to birth-and-death evolution. Therefore, it is important to study the mechanisms of gene family interaction for understanding phenotypic evolution. Because gene duplication occurs more or less at random, phenotypic evolution contains some fortuitous elements, though the environmental factors also play an important role. The randomness of phenotypic evolution is qualitatively different from allele frequency changes by random genetic drift. However, there is some similarity between phenotypic and molecular evolution with respect to functional or environmental constraints and evolutionary rate. It appears that mutation (including gene duplication and other DNA changes) is the driving force of evolution at both the genic and the phenotypic levels.     

The effect of tandem substitutions on the correlation between synonymous and nonsynonymous rates in rodents.

Nonsynonymous substitutions in DNA cause amino acid substitutions while synonymous substitutions in DNA leave amino acids unchanged. The cause of the correlation between the substitution rates at nonsynonymous (K(A)) and synonymous (K(S)) sites in mammals is a contentious issue, and one that impacts on many aspects of molecular evolution. Here we use a large set of orthologous mammalian genes to investigate the causes of the K(A)-K(S) correlation in rodents. The strength of the K(A)-K(S) correlation exceeds the neutral theory expectation when substitution rates are estimated using algorithmic methods, but not when substitution rates are estimated by maximum likelihood. Irrespective of this methodological uncertainty the strength of the K(A)-K(S) correlation appears mostly due to tandem substitutions, an excess of which is generated by substitutional nonindependence. Doublet mutations cannot explain the excess of tandem synonymous-nonsynonymous substitutions, and substitution patterns indicate that selection on silent sites is the likely cause. We find no evidence for selection on codon usage. The nature of the relationship between synonymous divergence and base composition is unclear because we find a significant correlation if we use maximum-likelihood methods but not if we use algorithmic methods. Finally, we find that K(S) is reduced at the start of genes, which suggests that selection for RNA structure may affect silent sites in mammalian protein-coding genes.     

Salmonid Opsin Sequences Undergo Positive Selection and Indicate an Alternate Evolutionary Relationship in Oncorhynchus

Positive selection can be demonstrated by statistical analysis when non-synonymous nucleotide substitutions occur more frequently than synonymous substitutions (dN>dS). This pattern of sequence evolution has been observed in the rhodopsin gene of cichlids. Mutations in opsin genes resulting in amino acid (AA) replacement appear to be associated with the evolution of specific color patterns and the evolution of courtship behaviors. Within fish, AA replacements in opsin proteins have improved vision at great depths and have occurred in deep-sea species. Salmonids experience diverse photic environments during their life history. Furthermore, sexual selection has resulted in species-specific male and female coloration during spawning. To look for evidence of positive selection in salmonid opsins, we sequenced the RH1, RH2, LWS, SWS1, and SWS2 genes from six Pacific salmon species as well as the Atlantic salmon. These salmonids include landlocked and migratory species and species that vary in their coloration during spawning. In each opsin gene comparison from all species sampled, traditional dN:dS analysis did not indicate positive selection. However, the more sensitive Creevey–McInerney statistical analysis indicates that RH1 and RH2 experienced positive selection early in the evolution and speciation of salmonids.     

Evolutionary history and complementary selective relaxation of the duplicated PI genes in grasses

Gene duplication plays an important role in the evolution of organisms by allowing functional innovation and the divergence of duplicate genes. Previous studies found two PI-like genes in grass species, suggesting functional divergence between the paralogous copies. Here, we reconstructed the evolutionary history of two PI genes from major lineages of grasses and other monocot species, and demonstrated that two PI genes (PI1 and PI2) arose from a whole genome duplication that occurred in a common ancestor of extant grasses. Molecular evolutionary analyses at the family and tribal levels found strong purifying selection acting on two genes in grasses, consistent with the conserved class B function of the PI genes. Importantly, we detected different patterns of selective relaxation between the duplicated PI genes although no signature of positive selection was found. Likelihood ratio tests revealed that the ω ratio for M domain is significantly higher in PI1 than in PI2 but that for K domain is significantly higher in PI2 than in PI1. These findings imply that complementary selective relaxation occurs in two PI genes after duplication, and provide additional molecular evidence for the subfunctionalization of the duplicated PI genes in grasses.     

The neutral theory in the genomic era

A number of tests have been developed to detect positive selection at the molecular level. These tests are based on DNA polymorphism within and divergence between species. Applications of these tests have revealed a large collection of genes that have evolved under positive selection and some general insights into adaptive evolution. Recently, these tests have been applied on a genomic scale and have provided estimates of the frequency of adaptive substitutions and a critical test of the neutral theory.