Rate Of Nonsynonymous Nucleotide Substitution Research Articles

Genome reduction occurred in early Prochlorococcus with an unusually low effective population size.

In the oligotrophic sunlit ocean, the most abundant free-living planktonic bacterial lineages evolve convergently through genome reduction. The cyanobacterium Prochlorococcus responsible for 10% global oxygen production is a prominent example. The dominant theory known as "genome streamlining" posits that they have extremely large effective population sizes (Ne) such that selection for metabolic efficiency acts to drive genome reduction. Because genome reduction largely took place anciently, this theory builds on the assumption that their ancestors' Ne was similarly large. Constraining Ne for ancient ancestors is challenging because experimental measurements of extinct organisms are impossible and alternatively reconstructing ancestral Ne with phylogenetic models gives large uncertainties. Here, we develop a new strategy that leverages agent-based modeling to simulate the changes in the genome-wide ratio of radical to conservative nonsynonymous nucleotide substitution rate (dR/dC) in a possible range of Ne in ancestral populations. This proxy shows expected increases with decreases of Ne only when Ne falls to about 10k - 100k or lower, magnitudes characteristic of Ne of obligate endosymbiont species where drift drives genome reduction. Our simulations therefore strongly support a scenario where the primary force of Prochlorococcus genome reduction is drift rather than selection.

Structural organization and sequence diversity of the complete nucleotide sequence encoding the Plasmodium malariae merozoite surface protein-1

The merozoite surface protein-1 (MSP1) is a prime candidate for an asexual blood stage vaccine against malaria. However, polymorphism in this antigen could compromise the vaccine’s efficacy. Although the extent of sequence variation in MSP1 has been analyzed from various Plasmodium species, little is known about structural organization and diversity of this locus in Plasmodium malariae (PmMSP1). Herein, we have shown that PmMSP1 contained five conserved and four variable blocks based on analysis of the complete coding sequences. Variable blocks were characterized by short insertion and deletion variants (block II), polymorphic nonrepeat sequences (block IV), complex repeat structure with size variation (block VI) and degenerate octapeptide repeats (block VIII). Like other malarial MSP1s, evidences of intragenic recombination have been found in PmMSP1. The rate of nonsynonymous nucleotide substitutions significantly exceeded that of synonymous nucleotide substitutions in block IV, suggesting positive selection in this region. Codon-based analysis of deviation from neutrality has identified a codon under purifying selection located in close proximity to the homologous region of the 38 kDa/42 kDa cleavage site of P. falciparum MSP1. A number of predicted linear B-cell epitopes were identified across both conserved and variable blocks of the protein. However, polymorphism in repeat-containing blocks resulted in alteration of the predicted linear B-cell epitope scores across variants. Although a number of predicted HLA-class II-binding peptides were identified in PmMSP1, all variants of block IV seemed not to be recognized by common HLA-class II alleles among Thai population, suggesting that diversity in this positive selection region could probably affect host immune recognition. The data on structural diversity in PmMSP1 could be useful for further studies such as vaccine development and strain characterization of this neglected malaria parasite.

Assessing a Role of Genetic Drift for Deep-Time Evolutionary Events.

Effective population size (Ne) determines the amount of genetic diversity and the fate of genetic variants in a species and thus is an essential parameter in evolutionary genetics. There are standard approaches to determine the Ne of evolving species. For example, the long-term Ne of an extant species is calculated based on its unbiased global mutation rate and the neutral genetic diversity of the species. However, approaches for inferring Ne of ancestral lineages are less known. Here, we introduce an evolutionary genetic statistic and an analytical procedure to assess the efficiency of natural selection for deep nodes by calculating rates of nonsynonymous nucleotide substitutions leading to radical (dR) and conservative (dC) amino acid replacements, respectively. Given that radical variants are more likely to be deleterious than conservative ones, an elevated dR/dC ratio in gene families across the genome means an accelerated genome-wide accumulation of the more deleterious type of mutations (i.e., radical variants), which indicates that natural selection is less efficient and genetic drift becomes more powerful. Earlier approaches that calculate dR/dC do not consider the impact of nucleotide composition (G+C content) on the dR/dC result, which is partially accounted for in more recent methods. Here, we use these methods to demonstrate that genetic drift may have driven the early evolution of Prochlorococcus, the most abundant carbon-fixing photosynthetic bacteria in the ocean.

Molecular Evolution of rbcL in Orthotrichales (Bryophyta): Site Variation, Adaptive Evolution, and Coevolutionary Patterns of Amino Acid Replacements.

Molecular evolution of the large subunit of the RuBisCO enzyme is understudied in early diverging land plants. These groups show morphological and eco-physiological adaptations to the uneven and intermittent distribution of water in the terrestrial environment. This might have prompted a continuous fine-tuning of RuBisCO under a selective pressure modifying the species-specific optima for photosynthesis in contrasting microdistributions and environmental niches. To gain a better insight into the molecular evolution of RuBisCO large subunits, the aim of this study was to assess the pattern of evolutionary change in the amino acid residues in a monophyletic group of Bryophyta (Orthotrichaceae). Tests for positive, neutral, or purifying selection at the amino acid level were assessed by comparing rates (ω) of non-synonymous (dN) and synonymous (dS) nucleotide substitutions along a Maximum Likelihood phylogenetic tree. Molecular adaptation tests using likelihood ratio tests, reconstruction of ancestral amino acid sites, and intra-protein coevolution analyses were performed. Variable amino acid sites (39) were unevenly distributed across the LSU. The residues are located on rbcL sites that are highly variable in higher plants and close to key regions implying dimer-dimer (L2L2), RuBisCO-activase interactions, and conformational functions during catalysis. Ten rbcL sites (32, 33, 91, 230, 247, 251, 255, 424, 449 and 475) have been identified by the Bayesian Empirical Bayes inference to be under positive selection and under adaptive evolution under the M8 model. The pattern of amino acid variation suggests that it is not lineage specific, but rather representative of a case of convergent evolution, suggesting recurrent changes that potentially favor the same amino acid substitutions that are likely optimized the RuBisCO activity.

Genome-wide analysis of cotton C2H2-zinc finger transcription factor family and their expression analysis during fiber development

BackgroundC2H2-zinc finger protein family is commonly found in the plant, and it is known as the key actors in the regulation of transcription and vital component of chromatin structure. A large number of the C2H2-zinc finger gene members have not been well characterized based on their functions and structure in cotton. However, in other plants, only a few C2H2-zinc finger genes have been studied.ResultsIn this work, we performed a comprehensive analysis and identified 386, 196 and 195 C2H2-zinc finger genes in Gossypium hirsutum (upland cotton), Gossypium arboreum and Gossypium raimondii, respectively. Phylogenetic tree analysis of the C2H2-zinc finger proteins encoding the C2H2-zinc finger genes were classified into seven (7) subgroups. Moreover, the C2H2-zinc finger gene members were distributed in all cotton chromosomes though with asymmetrical distribution patterns. All the orthologous genes were detected between tetraploid and the diploid cotton, with 154 orthologous genes pair detected between upland cotton and Gossypium arboreum while 165 orthologous genes were found between upland cotton and Gossypium raimondii. Synonymous (Ks) and non-synonymous (Ka) nucleotide substitution rates (Ka/Ks) analysis indicated that the cotton C2H2-zinc finger genes were highly influenced mainly by negative selection, which maintained their protein levels after the duplication events. RNA-seq data and RT-qPCR validation of the RNA seq result revealed differential expression pattern of some the C2H2-zinc finger genes at different stages of cotton fiber development, an indication that the C2H2-zinc finger genes play an important role in initiating and regulating fiber development in cotton.ConclusionsThis study provides a strong foundation for future practical genome research on C2H2-zinc finger genes in upland cotton. The expression levels of C2H2-zinc finger genes family is a pointer of their involvement in various biochemical and physiological functions which are directly related to cotton fiber development during initiation and elongation stages. This work not only provides a basis for determining the nominal role of the C2H2-zinc finger genes in fiber development but also provide valuable information for characterization of potential candidate genes involved in regulation of cotton fiber development.

Molecular evolution of mammalian genes with epistatic interactions in fertilization

BackgroundGenes that encode proteins associated with sperm competition, fertilization, and sexual conflicts of interest are often among the most rapidly evolving parts of animal genomes. One family of sperm-expressed genes (Zp3r, C4bpa) in the mammalian gene cluster called the regulator of complement activation (RCA) encodes proteins that bind eggs and mediate reproductive success, and are therefore expected to show high relative rates of nonsynonymous nucleotide substitution in response to sexual selection in comparison to other genes not involved in gamete binding at fertilization. We tested that working hypothesis by using phylogenetic models of codon evolution to identify episodes of diversifying positive selection. We used a comparative approach to quantify the evidence for episodic diversifying selection acting on RCA genes with known functions in fertilization (and sensitivity to sexual selection), and contrast them with other RCA genes in the same gene family that function in innate immunity (and are not sensitive to sexual selection).ResultsWe expected but did not find evidence for more episodes of positive selection on Zp3r in Glires (the rodents and lagomorphs) or on C4BPA in Primates, in comparison to other paralogous RCA genes in the same taxon, or in comparison to the same orthologous RCA gene in the other taxon. That result was not unique to RCA genes: we also found little evidence for more episodes of diversifying selection on genes that encode selective sperm-binding molecules in the egg coat or zona pellucida (Zp2, Zp3) in comparison to members of the same gene family that encode structural elements of the egg coat (Zp1, Zp4). Similarly, we found little evidence for episodic diversifying selection acting on two other recently discovered genes (Juno, Izumo1) that encode essential molecules for sperm–egg fusion.ConclusionsThese negative results help to illustrate the importance of a comparative context for this type of codon model analysis. The results may also point to other phylogenetic contexts in which the effects of selection acting on these fertilization proteins might be more readily discovered and documented in mammals and other taxa.

Purifying Selective Pressure Suggests the Functionality of a Vitamin B12 Biosynthesis Pathway in a Global Population of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of the deadliest and most challenging pathogens to study in current microbiological research. One of the issues that remains to be resolved is the importance of cobalamin in the metabolism of M. tuberculosis. The functionality of a vitamin B12 biosynthesis pathway in M. tuberculosis is under dispute, and the ability of this pathogen to scavenge vitamin B12 from the host is unknown. Here, we quantified the ratios of nonsynonymous and synonymous nucleotide substitution rates (dN/dS) in the genes involved in vitamin B12 biosynthesis and transport and in genes encoding cobalamin-dependent enzymes in nearly four thousand strains of M. tuberculosis. We showed that purifying selection is the dominant force acting on cobalamin-related genes at the levels of individual codons, genes and groups of genes. We conclude that cobalamin-related genes may not be essential but are adaptive for M. tuberculosis in clinical settings. Furthermore, the cobalamin biosynthesis pathway is likely to be functional in this species.

Excess of non-conservative amino acid changes in marine bacterioplankton lineages with reduced genomes.

Surface ocean waters are dominated by planktonic bacterial lineages with highly reduced genomes. The best examples are the cyanobacterial genus Prochlorococcus, the alphaproteobacterial clade SAR11 and the gammaproteobacterial clade SAR86, which together represent over 50% of the cells in surface oceans. Several studies have identified signatures of selection on these lineages in today's ocean and have postulated selection as the primary force throughout their evolutionary history. However, massive loss of genomic DNA in these lineages often occurred in the distant past, and the selective pressures underlying these ancient events have not been assessed. Here, we probe ancient selective pressures by computing %GC-corrected rates of conservative and radical nonsynonymous nucleotide substitutions. Surprisingly, we found an excess of radical changes in several of these lineages in comparison to their relatives with larger genomes. Furthermore, analyses of allelic genome sequences of several populations within these lineages consistently supported that radical replacements are more likely to be deleterious than conservative changes. Our results suggest coincidence of massive genomic DNA losses and increased power of genetic drift, but we also suggest that additional evidence independent of the nucleotide substitution analyses is needed to support a primary role of genetic drift driving ancient genome reduction of marine bacterioplankton lineages.

Degeneration of photosynthetic capacity in mixotrophic plants, Chimaphila japonica and Pyrola decorata (Ericaceae)

The evolution of photosynthesis is an important feature of mixotrophic plants. Previous inferences proposed that mixotrophic taxa tend to retain most genes relating to photosynthetic functions but vary in plastid gene content. However, no sequence data are available to test this hypothesis in Ericaceae. To investigate changes in plastid genomes that may result from a transition from autotrophy to mixotrophy, the plastomes of two mixotrophic plants, Pyrola decorata and Chimaphila japonica, were sequenced at Illumina's Genome Analyzer and compared to the published plastome of the autotrophic plant Rhododendron simsii, which also belongs to Ericaceae. The greatest discrepancy between mixotrophic and autotrophic plants was that ndh genes for both P. decorata and C. japonica plastomes have nearly all become pseudogenes. P. decorata and C. japonica also retained all genes directly involved in photosynthesis under strong selection. The calculated rate of nonsynonymous nucleotide substitutions and synonymous substitutions of protein-coding genes (dN/dS) showed that substitution rates in shade plants were apparently higher than those in sunlight plants. The two mixotrophic plastomes were generally very similar to that of non-parasitic plants, although ndh genes were largely pseudogenized. Photosynthesis genes under strong selection were retained in the two mixotrophs, however, with greatly increased substitution rates. Further research is needed to gain a clearer understanding of the evolution of autotrophy and mixotrophy in Ericaceae.

Comparative transcriptome analysis highlights the crucial roles of photosynthetic system in drought stress adaptation in upland rice.

Drought stress is one of the major adverse environmental factors reducing plant growth. With the aim to elucidate the underlying molecular basis of rice response to drought stress, comparative transcriptome analysis was conducted between drought susceptible rice cultivar Zhenshan97 and tolerant cultivar IRAT109 at the seedling stage. 436 genes showed differential expression and mainly enriched in the Gene Ontology (GO) terms of stress defence. A large number of variations exist between these two genotypes including 2564 high-quality insertion and deletions (INDELs) and 70,264 single nucleotide polymorphism (SNPs). 1041 orthologous gene pairs show the ratio of nonsynonymous nucleotide substitution rate to synonymous nucleotide substitutions rate (Ka/Ks) larger than 1.5, indicating the rapid adaptation to different environments during domestication. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of positive selection genes suggested that photosynthesis represents the most significant category. The collocation of positively selected genes with the QTLs of photosynthesis and the different photosynthesis performance of these two cultivars further illuminate the crucial function of photosynthesis in rice adaptation to drought stress. Our results also provide fruitful functional markers and candidate genes for future genetic research and improvement of drought tolerance in rice.

The effect of ecosystem biodiversity on virus genetic diversity depends on virus species: A study of chiltepin-infecting begomoviruses in Mexico.

Current declines in biodiversity put at risk ecosystem services that are fundamental for human welfare. Increasing evidence indicates that one such service is the ability to reduce virus emergence. It has been proposed that the reduction of virus emergence occurs at two levels: through a reduction of virus prevalence/transmission and, as a result of these epidemiological changes, through a limitation of virus genetic diversity. Although the former mechanism has been studied in a few host-virus interactions, very little is known about the association between ecosystem biodiversity and virus genetic diversity. To address this subject, we estimated genetic diversity, synonymous and non-synonymous nucleotide substitution rates, selection pressures, and frequency of recombinants and re-assortants in populations of Pepper golden mosaic virus (PepGMV) and Pepper huasteco yellow vein virus (PHYVV) that infect chiltepin plants in Mexico. We then analyzed how these parameters varied according to the level of habitat anthropization, which is the major cause of biodiversity loss. Our results indicated that genetic diversity of PepGMV (but not of PHYVV) populations increased with the loss of biodiversity at higher levels of habitat anthropization. This was mostly the consequence of higher rates of synonymous nucleotide substitutions, rather than of adaptive selection. The frequency of recombinants and re-assortants was higher in PepGMV populations infecting wild chiltepin than in those infecting cultivated ones, suggesting that genetic exchange is not the main mechanism for generating genetic diversity in PepGMV populations. These findings provide evidence that biodiversity may modulate the genetic diversity of plant viruses, but it may differentially affect even two closely related viruses. Our analyses may contribute to understanding the factors involved in virus emergence.

A simple method for estimating the strength of natural selection on overlapping genes.

Overlapping genes, where one DNA sequence codes for two proteins with different reading frames, are not uncommon in viruses and cellular organisms. Estimating the direction and strength of natural selection acting on overlapping genes is important for understanding their functionality, origin, evolution, maintenance, and potential interaction. However, the standard methods for estimating synonymous (dS) and nonsynonymous (dN) nucleotide substitution rates are inapplicable here because a nucleotide change can be simultaneously synonymous and nonsynonymous when both reading frames involved are considered. We have developed a simple method that can estimate dN/dS and test for the action of natural selection in each relevant reading frame of the overlapping genes. Our method is an extension of the modified Nei-Gojobori method previously developed for nonoverlapping genes. We confirmed the reliability of our method using extensive computer simulation. Applying this method, we studied the longest human sense–antisense overlapping gene pair, LRRC8E and ENSG00000214248. Although LRRC8E (leucine-rich repeat containing eight family, member E) is known to regulate cell size, the function of ENSG00000214248 is unknown. Our analysis revealed purifying selection on ENSG00000214248 and suggested that it originated in the common ancestor of bony vertebrates.

The Implication of Codon Usage Design and Expression Level in Determining the Nature of Selection and Functionality Amongst the Amino Acid Biosynthetic Pathway Coding Sequences of Arthrobacter sp. FB24

Amino acid biosynthesis is an immensely important life process requiring the participation of nearly hundred gene products, differing in terms of codon usage pattern, expressivity and selection pressure. In this study an attempt was made to categorize the gene products according to their expression level, nature of selection and functionality analyzing the amino acid biosynthetic pathway genes of the soil bacterium Arthrobacter, known for its varied metabolic activities and a key agent for industrial production of amino acids. The relationship between the different parameters like expression level, codon usage and selection pressure was calculated to find out the correlation of biasness, expression level, nature of selection and functionality amongst the genes involved in amino acid biosynthetic pathways. We found that genes coding for enzymes and proteins with large number of functions have higher codon usage bias and are subjected mostly to purifying selection. Gene sequences of proteins and enzymes with low functionality showed less codon usage bias and are under positive selection. The multifunctional genes mostly show high expression level and it is highly correlated with codon usage bias. The results of this study demonstrate that functionality and nature of selective forces acting on the coding sequences can be determined by studying the correlation between the different parameters like effective number of codon, codon adaptation index and nucleotide substitution ratios and careful study of all these parameters is a pretty good indicator for determining the functionality of the genes and this can be extended towards the genes of unknown function(s). Keywords: Amino acid biosynthetic pathways, Arthrobacter, CAI, Nc, Ka/Ks, synonymous and non-synonymous nucleotide substitution rates, positive selection, purifying selection.

Allelic Frequency in Human SNPs Predicts the Rate of Non-Synonymous Nucleotide Substitutions between Human and Chimpanzee Genes

The combination of comparative genomics and population genetics may provide important clues regarding human evolution. We have hypothesized that the divergence between various human and chimpanzee orthologs will be reflected in the variability of single nucleotide polymorphisms (SNPs) that are localized in the vicinity of the corresponding loci in different human populations. Consistently with this notion, more diverged genes between humans and chimpanzees are more likely to be associated with human speciation and are anticipated to contain SNPs with reduced variability between different human populations. In order to test this hypothesis, we have compared the rate of non synonymous nucleotide substitutions (Ka) between 255 chimpanzee and human orthologs with the average deviation in the allelic frequencies of corresponding closely linked SNPs in two distinct human populations: The Yoruba people in Ibadan, Nigeria (YRI) and US residents with ancestry from Northern and Western Europe, collected in 1980 by the Centre d’Etude du Polymorphisme Humain (CEU). We found a significant (p

Evolution of a Cell Culture-Derived Genotype 1a Hepatitis C Virus (H77S.2) during Persistent Infection with Chronic Hepatitis in a Chimpanzee

Persistent infection is a key feature of hepatitis C virus (HCV). However, chimpanzee infections with cell culture-derived viruses (JFH1 or related chimeric viruses that replicate efficiently in cell culture) have been limited to acute-transient infections with no pathogenicity. Here, we report persistent infection with chronic hepatitis in a chimpanzee challenged with cell culture-derived genotype 1a virus (H77S.2) containing 6 cell culture-adaptive mutations. Following acute-transient infection with a chimeric H77/JFH1 virus (HJ3-5), intravenous (i.v.) challenge with 10(6) FFU H77S.2 virus resulted in immediate seroconversion and, following an unusual 4- to 6-week delay, persistent viremia accompanied by alanine aminotransferase (ALT) elevation, intrahepatic innate immune responses, and diffuse hepatopathy. This first persistent infection with cell culture-produced HCV provided a unique opportunity to assess evolution of cell culture-adapted virus in vivo. Synonymous and nonsynonymous nucleotide substitution rates were greatest during the first 8 weeks of infection. Of 6 cell culture-adaptive mutations in H77S.2, Q1067R (NS3) had reverted to Q1067 and S2204I (NS5A) was replaced by T2204 within 8 weeks of infection. By 62 weeks, 4 of 6 mutations had reverted to the wild-type sequence, and all reverted to the wild-type sequence by 194 weeks. The data suggest H77S.2 virus has greater potential for persistence and pathogenicity than JFH1 and demonstrate both the capacity of a nonfit virus to persist for weeks in the liver in the absence of detectable viremia as well as strong selective pressure against cell culture-adaptive mutations in vivo. This study shows that mutations promoting the production of infectious genotype 1a HCV in cell culture have the opposite effect and attenuate replication in the liver of the only fully permissive animal species other than humans. It provides the only example to date of persistent infection in a chimpanzee challenged with cell culture-produced virus and provides novel insight into the forces shaping molecular evolution of that virus during 5 years of persistent infection. It demonstrates that a poorly fit virus can replicate for weeks within the liver in the absence of detectable viremia, an observation that expands current concepts of HCV pathogenesis and that is relevant to relapses observed with direct-acting antiviral therapies.

Polymorphisms in major histocompatibility complex class IIα genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)

Summary Infectious hematopoietic necrosis is a serious viral disease of salmonids, including rainbow trout Oncorhynchus mykiss, and causes tremendous economic losses to the rainbow trout farming industry. Major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity in vertebrate organisms and have been linked with the resistance to numerous pathogenic diseases. In this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by cloning and sequencing were used to examine polymorphisms in the DAA genes (specifically DAA exon 2 of MHC class IIα) of rainbow trout and investigate their association with the infectious hematopoietic necrosis virus (IHNV) resistance in rainbow trout. Seventeen alleles were resolved, including 13 novel alleles. Individuals possessed between two and five alleles, indicating that the genome harbours at least three closely-related DAA exon 2 loci. The ratio of non-synonymous to synonymous nucleotide substitutions suggested that DAA exon 2 is under positive selection. A greater variability of amino acids and non-synonymous nucleotide substitution rate was evident in the peptide-binding region (PBR) than in the non-PBR (27.75%). Importantly, the analyses revealed that certain MHC class IIα alleles appear to confer resistance to IHNV in rainbow trout, while others confer susceptibility. The most common alleles in the resistant populations of rainbow trout, Onmy-DAA*1301 and Onmy-DAA*0304, confer resistance to IHNV and were not present in the susceptible population. Hence, these alleles may be ideal molecular markers that can assist the breeding of IHNV resistance in rainbow trout.

A population genetics-based approach reveals candidate gene regions important in the determination of resistance specificity for the Pik family of rice blast resistance genes

Allelic resistance (R) genes at the Pik locus for rice blast resistance consist of two genes, designated as the first gene and the second gene, which each have a nucleotide-binding site and a leucine-rich repeat. We investigated allelic nucleotide sequence diversity at this locus in 36 rice lines and searched for gene regions showing high rates of nonsynonymous nucleotide substitutions. We detected such regions within and near the coiled-coil domain of the first gene but detected only a few nucleotide substitutions in the second gene, suggesting the importance of the N-terminal part of the first protein in the determination of R specificity.

Aphid Protein Effectors Promote Aphid Colonization in a Plant Species-Specific Manner

Microbial pathogens and pests produce effectors to modulate host processes. Aphids are phloem-feeding insects, which introduce effectors via saliva into plant cells. However, it is not known if aphid effectors have adapted to modulate processes in specific plant species. Myzus persicae is a polyphagous insect that colonizes Arabidopsis thaliana and Nicotiana benthamiana, while the pea aphid Acyrthosiphon pisum specializes on colonizing plant species of the family Fabaceae. We found that M. persicae reproduction increased on transgenic Arabidopsis, producing the M. persicae effectors C002, PIntO1 (Mp1), and PIntO2 (Mp2), whereas reproduction of M. persicae did not increase on Arabidopsis producing the A. pisum orthologs of these three proteins. Plant-mediated RNA interference experiments showed that c002- and PIntO2-silenced M. persicae produce less progeny on Arabidopsis and N. benthamiana than nonsilenced aphids. Orthologs of c002, PIntO1, and PIntO2 were identified in multiple aphid species with dissimilar plant host ranges. We revealed high nonsynonymous versus synonymous nucleotide substitution rates within the effector orthologs, indicating that the effectors are fast evolving. Application of maximum likelihood methods identified specific sites with high probabilities of being under positive selection in PIntO1, whereas those of C002 and PIntO2 may be located in alignment gaps. In support of the latter, a M. persicae c002 mutant without the NDNQGEE repeat region, which overlaps with an alignment gap in C002, does not promote M. persicae colonization on Arabidopsis. Taken together, these results provide evidence that aphid effectors are under positive selection to promote aphid colonization on specific plant species.

LEC-2, a highly variable lectin in the lichen Peltigera membranacea.

Lectins are a diverse group of carbohydrate binding proteins often involved in cellular interactions. A lectin gene, lec-2, was identified in the mycobiont of the lichen Peltigera membranacea. Sequencing of lec-2 open reading frames from 21 individual samples showed an unexpectedly high level of polymorphism in the deduced protein (LEC-2), which was sorted into nine haplotypes based on amino acid sequence. Calculations showed that the rates of nonsynonymous versus synonymous nucleotide substitutions deviated significantly from the null hypothesis of neutrality, indicating strong positive selection. Molecular modeling revealed that most amino acid replacements were around the putative carbohydrate-binding pocket, indicating changes in ligand binding. Lectins have been thought to be involved in the recognition of photobiont partners in lichen symbioses, and the hypothesis that positive selection of LEC-2 is driven by variation in the Nostoc photobiont partner was tested by comparing mycobiont LEC-2 haplotypes and photobiont genotypes, as represented by the rbcLX region. It was not possible to pair up the two types of marker sequences without conflicts, suggesting that positive selection of LEC-2 was not due to variation in photobiont partners.

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.