Diversifying selection drives the evolution of the type III secretion system pilus of Pseudomonas syringae

The plant pathogenic bacterium Pseudomonas syringae uses a type III secretion system to inject virulence proteins directly into the cytoplasm of its hosts. The P. syringae type III secretion apparatus is encoded, in part, by the HrpZ operon, which carries the hrpA gene encoding the pilin subunit of the pilus, various components of the structural apparatus, and the HrpZ harpin protein that is believed to produce pores in the host cell membrane. The pilus of the type III system comes into direct contact with the host cell and is, therefore, a likely target of the host's pathogen surveillance systems. We sequenced and analyzed 22 HrpZ operons from P. syringae strains spanning the diversity of the species. Selection analyses, including K(a)/K(s) tests and Tajima's D, revealed strong diversifying selection acting on the hrpA gene. This form of selection enables pathogens to maintain genetic diversity within their populations and is often driven by selection imposed by host defense systems. The HrpZ operon also revealed a single significant recombination event that dramatically changed the evolutionary relationships among P. syringae strains from 2 quite distinct phylogroups. This recombination event appears to have introduced genetic diversity into a clade of strains that may now be undergoing positive selection. The identification of diversifying selection acting on the Hrp pilus across the whole population sample and positive selection within one P. syringae lineage supports a trench warfare coevolutionary model between P. syringae and its plant hosts.     

Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae

Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems. Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts. Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts. To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P. syringae type III effectors and two effector chaperones. We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer. We assessed the role of diversifying and purifying selection in the evolution of these gene families. While some P. syringae type III effectors were acquired recently, others have evolved predominantly by descent. The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function. However, members of 7 families had domains subject to diversifying selection.     

Concerted evolution in the repeats of an immunomodulating cell surface protein, SOWgp, of the human pathogenic fungi Coccidioides immitis and C. posadasii

Genome dynamics that allow pathogens to escape host immune responses are fundamental to our understanding of host-pathogen interactions. Here we present the first population-based study of the process of concerted evolution in the repetitive domain of a protein-coding gene. This gene, SOWgp, encodes the immunodominant protein in the parasitic phase of the human pathogenic fungi Coccidioides immitis and C. posadasii. We sequenced the entire gene from strains representing the geographic ranges of the two Coccidioides species. By using phylogenetic and genetic distance analyses we discovered that the repetitive part of SOWgp evolves by concerted evolution, predominantly by the mechanism of unequal crossing over. We implemented a mathematical model originally developed for multigene families to estimate the rate of homogenization and recombination of the repetitive array, and the results indicate that the pattern of concerted evolution is a result of homogenization of repeat units proceeding at a rate close to the nucleotide point mutation rate. The release of the SOWgp molecules by the pathogen during proliferation may mislead the host: we speculate that the pathogen benefits from concerted evolution of repeated domains in SOWgp by an enhanced ability to misdirect the host's immune system.     

Diversifying selection of the tumor-growth promoter angiogenin in primate evolution

Diversifying selection drives the rapid differentiation of gene sequences and is one of the main forces behind adaptive evolution. Most genes known to be shaped by diversifying selection are those involved in host-pathogen or male-female interactions characterized as molecular "arms races." Here we report the unexpected detection of diversifying selection in the evolution of a tumor-growth promoter, angiogenin (ANG). A comparison among 11 primate species demonstrates that ANG has a significantly higher rate of nucleotide substitution at nonsynonymous sites than at synonymous sites, a hallmark of positive selection acting at the molecular level. Furthermore, we observed significant charge diversity at the molecular surface, suggesting the presence of selective pressures in the microenvironment of ANG, including its binding molecules. A population survey of ANG in chimpanzees, however, reveals no polymorphism, which may have resulted from a recent selective sweep of a charge-altering substitution in chimpanzee evolution. Functional assays of recombinant ANGs from the human and owl monkey indicate that primate ANGs retain angiogenic activity despite rapid evolution. Our study, together with findings of similar selection in the primate breast cancer suppressor gene, BRCA1, reveals an intriguing phenomenon of unusual selective pressures on, and adaptive evolution of, cancer-related genes in primate evolution.     

Molecular Evolution of the Hepatitis Delta Virus Antigen Gene: Recombination or Positive Selection?

We present the statistical analysis of diversifying selective pressures on the hepatitis D antigen gene (HDAg). Thirty-three distinct HDAg sequences from subtypes I, II, and III were tested for positive selection using maximum likelihood methods based on models of codon substitution that allow variable selective pressures across sites. Such methods have been shown to be sufficiently accurate and successful in detecting positive selection in a variety of viral and nonviral protein-coding genes. About 11% of codon sites in HDAg were estimated to be under diversifying selection. Remarkably, most of the residues predicted to evolve under positive selection were located in the immunogenic domain and the N-terminus region with reported antigenic activity. These sites are potential targets of the hosts immune response. Identification of residues mutating to escape immune recognition may help to distinguish the most virulent strains and aid vaccine design. Possible interplay between positive selection and recombination on the gene is discussed but no significant evidence for recombination was found.This article contains online supplementary material.Reviewing Editor: Dr. Nicolas Galtier     

Genetic diversity of MHC class I loci in six non-model frogs is shaped by positive selection and gene duplication

Comparative studies of major histocompatibility complex (MHC) genes across vertebrate species can reveal the evolutionary processes that shape the structure and function of immune regulatory proteins. In this study, we characterized MHC class I sequences from six frog species representing three anuran families (Hylidae, Centrolenidae and Ranidae). Using cDNA from our focal species, we amplified a total of 79 unique sequences spanning exons 2-4 that encode the extracellular domains of the functional alpha chain protein. We compared intra- and interspecific nucleotide and amino-acid divergence, tested for recombination, and identified codon sites under selection by estimating the rate of non-synonymous to synonymous substitutions with multiple codon-based maximum likelihood methods. We determined that positive (diversifying) selection was acting on specific amino-acid sites located within the domains that bind pathogen-derived peptides. We also found significant signals of recombination across the physical distance of the genes. Finally, we determined that all the six species expressed two or three putative classical class I loci, in contrast to the single locus condition of Xenopus laevis. Our results suggest that MHC evolution in anurans is a dynamic process and that variation in numbers of loci and genetic diversity can exist among taxa. Thus, the accumulation of genetic data for more species will be useful in further characterizing the relative importance of processes such as selection, recombination and gene duplication in shaping MHC loci among amphibian lineages.     

Rapid Evolution by Positive Selection and Gene Gain and Loss: PLA<Subscript>2</Subscript> Venom Genes in Closely Related <Emphasis Type="Italic">Sistrurus</Emphasis> Rattlesnakes with Divergent Diets

Rapid evolution of snake venom genes by positive selection has been reported previously but key features of this process such as the targets of selection, rates of gene turnover, and functional diversity of toxins generated remain unclear. This is especially true for closely related species with divergent diets. We describe the evolution of PLA₂ gene sequences isolated from genomic DNA from four taxa of Sistrurus rattlesnakes which feed on different prey. We identified four to seven distinct PLA₂ sequences in each taxon and phylogenetic analyses suggest that these sequences represent a rapidly evolving gene family consisting of both paralogous and homologous loci with high rates of gene gain and loss. Strong positive selection was implicated as a driving force in the evolution of these protein coding sequences. Exons coding for amino acids that make up mature proteins have levels of variation two to three times greater than those of the surrounding noncoding intronic sequences. Maximum likelihood models of coding sequence evolution reveal that a high proportion (∼30%) of all codons in the mature protein fall into a class of codons with an estimated d _N /d _S (ω) ratio of at least 2.8. An analysis of selection on individual codons identified nine residues as being under strong (p < 0.01) positive selection, with a disproportionately high proportion of these residues found in two functional regions of the PLA₂ protein (surface residues and putative anticoagulant region). This is direct evidence that diversifying selection has led to high levels of functional diversity due to structural differences in proteins among these snakes. Overall, our results demonstrate that both gene gain and loss and protein sequence evolution via positive selection are important evolutionary forces driving adaptive divergence in venom proteins in closely related species of venomous snakes.     

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.     

Molecular Evolution of Daphnia Immunity Genes: Polymorphism in a Gram-Negative Binding Protein Gene and an α-2-Macroglobulin Gene

Studies of DNA polymorphism have shown that some immune system genes of mammals and plants are exceptionally diverse, indicating that coevolution between these taxa and their parasites mediates positive selective sweeps and/or balancing selection. The genes of the arthropod immune system remain comparatively unstudied. We isolated two putative immune system genes from the cladoceran crustacean Daphnia and examined DNA sequence diversity. For one gene, encoding a putative gram-negative binding protein, we found evidence of only purifying selection, indicating that this gene is under strong functional constraint and that selection acts to eliminate amino acid variation. For another gene, encoding a putative -2-macroglobulin, we found evidence of positive selection, indicating the possible involvement of this gene in a host–parasite arms race. We discuss the assumed function of these genes and offer speculation regarding which components of the arthropod immune system might experience diversifying adaptive evolution.[Reviewing Editor: Dr. Martin Kreitman]     

Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes

Molecular characterization of Toxoplasma gondii isolates is central for understanding differences in disease transmission and manifestations. Only 3 subgroups (lineages) have been discerned with subtle within-lineage variation, permitting low-resolution classification of isolates. Because proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of housekeeping genes, which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of 5 housekeeping genes with 2 antigen-coding genes. Introns of housekeeping genes were slightly more polymorphic than coding and noncoding regions of antigen-coding genes and only the former showed intralineage variation. Intragenic linkage disequilibrium was complete, but intergenic linkage, although highly significant, was incomplete, suggesting that genes are partially uncoupled. Six of 7 substitutions found within the region coding for the tachyzoite surface antigen, SAG2, were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates on the basis of housekeeping and antigen-coding genes was consistent, but the phylogenetic relationships among the resulting groups was inconsistent. A cougar isolate typed as lineage II using a restriction fragment length polymorphism assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of housekeeping genes are preferred markers for phylogenetic study, and that multilocus genotyping is preferred for typing parasites, especially from feral or unstudied environments.     

Molecular evolution of the moonlighting protein SMN in metazoans

The spinal muscular atrophy (SMA) associated protein survival of motor neuron (SMN) is known to be a moonlighting protein: having one primary, ancestral function (presumed to be involvement in U snRNP assembly) along with one or more secondary functions. One hypothesis for the evolution of moonlighting proteins is that regions of a structure under relatively weak negative selection could gain new functions without interfering with the primary function. To test this hypothesis, we investigated sequence conservation and dN/dS, which reflects the selection acting on a coding sequence, in SMN and a related protein, splicing factor 30 (SPF30), which is not currently known to be multifunctional. We found very different patterns of evolution in the two genes, with SPF30 characterized by strong sequence conservation and negative selection in most animal taxa investigated, and SMN with much lower sequence conservation, and much weaker negative selection at many sites. Evidence was found of positive selection acting on some sites in primate genes for SMN. SMN was also found to have been duplicated in a number of species, and with patterns that indicate reduced negative selection following some of these duplications. There were also several animal species lacking an SMN gene.     

Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System

The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context - which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans - is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.     

Evolutionary dynamics of the ABCA chromosome 17q24 cluster genes in vertebrates

ABCA is a subfamily of ATP-binding-cassette (ABC) transporter genes. In this subfamily, it was found that five ABCA genes cluster in a head-to-tail pattern in the human and mouse genomes, but only one was found in fish. To understand better the evolution of this cluster of genes, we screened 11 vertebrate genome sequences and newly identified 28 ABCA cluster genes. Comparative genomic analysis reveals that the ABCA5 gene is relatively evolutionarily conserved. In contrast, the repertoires of the other ABCA genes in this cluster diverge tremendously among species, which is due mainly to postspeciation duplications. In addition, maximum likelihood analysis reveals that positive selection is acting on the paralogous genes ABCA6 and Abca8a, suggesting that these two genes have possibly acquired new functions after duplication. Because most eukaryotic ABC proteins integrate into the cytoplasmic membrane and transport a wide range of substrates across it, we conjecture that newly duplicated ABCA cluster genes are under diversifying selection for the ability to recognize a diverse array of substrates.     

Gene genealogies, cryptic species, and molecular evolution in the human pathogen Coccidioides immitis and relatives (Ascomycota, Onygenales).

Previous genealogical analyses of population structure in Coccidioides immitis revealed the presence of two cryptic and sexual species in this pathogenic fungus but did not clarify their origin and relationships with respect to other taxa. By combining the C. immitis data with those of two of its closest relatives, the free-living saprophytes Auxarthron zuffianum and Uncinocarpus reesii, we show that the C. immitis species complex is monophyletic, indicating a single origin of pathogenicity. Cryptic species also were found in both A. zuffianum and U. reesii, indicating that they can be found in both pathogenic and free-living fungi. Our study, together with a few others, indicates that the current list of known fungal species might be augmented by a factor of at least two. However, at least in the C. immitis, A. zuffianum, and U. reesii complexes, cryptic species represent subdivisions at the tips of deep monophyletic clades and thus well within the existing framework of generic classification. An analysis of silent and expressed divergence and polymorphism values between and within the taxa identified by genealogical concordance did not reveal faster evolution in C. immitis as a consequence of adaptation to the pathogenic habit, nor did it show positive Darwinian evolution in a region of a dioxygenase gene (tcrP gene coding for 4-HPPD) known to cause antigenic responses in humans. Instead, the data suggested relative stasis, indicative of purifying selection against mostly deleterious mutations. Two introns in the same gene fragment were considerably more divergent than exons and were unalignable between species complexes but had very low polymorphism within taxa.     

Why do paralogs persist? Molecular evolution of CYCLOIDEA and related floral symmetry genes in Antirrhineae (Veronicaceae)

CYCLOIDEA (CYC) and DICHOTOMA (DICH) are paralogous genes that determine adaxial (dorsal) flower identity in the bilaterally symmetric flowers of Antirrhinum majus (snapdragon). We show here that the duplication leading to the existence of both CYC and DICH in Antirrhinum occurred before the radiation of the Antirrhineae (the tribe to which snapdragon belongs). We find no additional gene duplications within Antirrhineae. Using explicit codon-based models of evolution in a likelihood framework, we show that patterns of molecular evolution after the duplication that gave rise to CYC and DICH are consistent with purifying selection acting at both loci, despite their known functional redundancy in snapdragon. However, for specific gene regions, purifying selection is significantly relaxed across DICH lineages, relative to CYC lineages. In addition, we find evidence for relaxed purifying selection along the lineage leading to snapdragon in one of two putative functional domains of DICH. A model of selection accounting for the persistence of paralogous genes in the absence of diversifying selection is presented. This model takes into account differences in the degree of purifying selection acting at the two loci and is consistent with subfunctionalization models of paralogous gene evolution.     

How immunogenetically different are domestic pigs from wild boars: a perspective from single-nucleotide polymorphisms of 19 immunity-related candidate genes

The coexistence of wild boars and domestic pigs across Eurasia makes it feasible to conduct comparative genetic or genomic analyses for addressing how genetically different a domestic species is from its wild ancestor. To test whether there are differences in patterns of genetic variability between wild and domestic pigs at immunity-related genes and to detect outlier loci putatively under selection that may underlie differences in immune responses, here we analyzed 54 single-nucleotide polymorphisms (SNPs) of 19 immunity-related candidate genes on 11 autosomes in three pairs of wild boar and domestic pig populations from China, Iberian Peninsula, and Hungary. Our results showed no statistically significant differences in allele frequency and heterozygosity across SNPs between three pairs of wild and domestic populations. This observation was more likely due to the widespread and long-lasting gene flow between wild boars and domestic pigs across Eurasia. In addition, we detected eight coding SNPs from six genes as outliers being under selection consistently by three outlier tests (BayeScan2.1, FDIST2, and Arlequin3.5). Among four non-synonymous outlier SNPs, one from TLR4 gene was identified as being subject to positive (diversifying) selection and three each from CD36, IFNW1, and IL1B genes were suggested as under balancing selection. All of these four non-synonymous variants were predicted as being benign by PolyPhen-2. Our results were supported by other independent lines of evidence for positive selection or balancing selection acting on these four immune genes (CD36, IFNW1, IL1B, and TLR4). Our study showed an example applying a candidate gene approach to identify functionally important mutations (i.e., outlier loci) in wild and domestic pigs for subsequent functional experiments.     

Evolutionary dynamics of Ralstonia solanacearum

We investigated the genetic diversity, extent of recombination, natural selection, and population divergence of Ralstonia solanacearum samples obtained from sources worldwide. This plant pathogen causes bacterial wilt in many crops and constitutes a serious threat to agricultural production due to its very wide host range and aggressiveness. Five housekeeping genes, dispersed around the chromosome, and three virulence-related genes, located on the megaplasmid, were sequenced from 58 strains belonging to the four major phylogenetic clusters (phylotypes). Whereas genetic variation is high and consistent for all housekeeping loci studied, virulence-related gene sequences are more diverse. Phylogenetic and statistical analyses suggest that this organism is a highly diverse bacterial species containing four major, deeply separated evolutionary lineages (phylotypes I to IV) and a weaker subdivision of phylotype II into two subgroups. Analysis of molecular variations showed that the geographic isolation and spatial distance have been the significant determinants of genetic variation between phylotypes. R. solanacearum displays high clonality for housekeeping genes in all phylotypes (except phylotype III) and significant levels of recombination for the virulence-related egl and hrpB genes, which are limited mainly to phylotype strains III and IV. Finally, genes essential for species survival are under purifying selection, and those directly involved in pathogenesis might be under diversifying selection.     

Adaptive molecular evolution of MC1R gene reveals the evidence for positive diversifying selection in indigenous goat populations

Detecting signatures of selection can provide a new insight into the mechanism of contemporary breeding and artificial selection and further reveal the causal genes associated to the phenotypic variation. However, the signatures of selection on genes entailing for profitable traits between Chinese commercial and indigenous goats have been poorly interpreted. We noticed footprints of positive selection at MC 1R gene containing SNP s genotyped in five Chinese native goat breeds. An experimental distribution of F _ST was built based on approximations of F _ST for each SNP across five breeds. We identified selection using the high F _ST outlier method and found that MC 1R candidate gene show evidence of positive selection. Furthermore, adaptive selection pressure on specific codons was determined using different codon based on maximum‐likelihood methods; signature of positive selection in mammalian MC 1R was explored in individual codons. Evolutionary analyses were inferred under maximum likelihood models, the HyPhy package implemented in the DATAMONKEY Web Server. The results of codon selection displayed positive diversifying selection at the sites were mainly involved in development of genetic variations in coat color in various mammalian species. Positive diversifying selection inferred with recent evolutionary changes in domesticated goat MC 1R provides new insights that the gene evolution may have been modulated by domestication events in goats.