Is Predominant Clonal Evolution a Common Evolutionary Adaptation to Parasitism in Pathogenic Parasitic Protozoa, Fungi, Bacteria, and Viruses?

We propose that predominant clonal evolution (PCE) in microbial pathogens be defined as restrained recombination on an evolutionary scale, with genetic exchange scarce enough to not break the prevalent pattern of clonal population structure. The main features of PCE are (1) strong linkage disequilibrium, (2) the widespread occurrence of stable genetic clusters blurred by occasional bouts of genetic exchange ('near-clades'), (3) the existence of a "clonality threshold", beyond which recombination is efficiently countered by PCE, and near-clades irreversibly diverge. We hypothesize that the PCE features are not mainly due to natural selection but also chiefly originate from in-built genetic properties of pathogens. We show that the PCE model obtains even in microbes that have been considered as 'highly recombining', such as Neisseria meningitidis, and that some clonality features are observed even in Plasmodium, which has been long described as panmictic. Lastly, we provide evidence that PCE features are also observed in viruses, taking into account their extremely fast genetic turnover. The PCE model provides a convenient population genetic framework for any kind of micropathogen. It makes it possible to describe convenient units of analysis (clones and near-clades) for all applied studies. Due to PCE features, these units of analysis are stable in space and time, and clearly delimited. The PCE model opens up the possibility of revisiting the problem of species definition in these organisms. We hypothesize that PCE constitutes a major evolutionary strategy for protozoa, fungi, bacteria, and viruses to adapt to parasitism.

Phylogenetic character mapping of RADES Probing, a new marker for exploring the clonal evolution of expressed coding sequences in Trypanosoma cruzi, the agent of Chagas disease.

We have tested a new genetic marker, RADES Probing (RADES-P), on a standard sample of 19 laboratory-cloned stocks of Trypanosoma cruzi, the agent of Chagas disease. This set of stocks, fully characterized using multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD), is representative of this parasite's main genetic subdivisions. RADES-P consists in hybridizing RAPD profiles with probes composed of the products of random amplified differentially expressed sequences (RADES). The profiles thus obtained uncover only expressed coding sequences that are as well present on RAPD gels. Direct visual examination and the banding record show that these RADES-P profiles are different of, and not redundant with, both RAPD and RADES patterns obtained on the same set of stocks with the same primers. Phylogenetic character mapping (PCM) of the RADES-P polymorphism fairly confirms the known population structure and phylogenetic diversity of T. cruzi. This suggests that the impact of clonal evolution on T. cruzi has been predominant enough over the long term to carve the polymorphism of all types of DNA sequences, including polymorphisms of expressed coding sequences, although these sequences are subject to natural selection.

Using mitochondrial and nuclear markers to evaluate the degree of genetic cohesion among Echinococcus populations.

Based on the distinctiveness of their mitochondrial haplotypes and other biological features, several recent publications have proposed that some Echinococcus granulosus strains should be regarded as separate species. However, the genetic cohesion of these species has not been extensively evaluated using nuclear markers. We assess the degree of polymorphism of the partial mitochondrial cox1 (366bp), the nuclear mdh (214bp) and EgAgB4 (281-283bp) genes of E. granulosus sensu lato isolates collected from areas where different strains occur sympatrically. Five distinct mitochondrial haplotypes were determined by direct sequencing (G1, G2, G5, G6 and G7). The mdh genotypes were first screened by SSCP: three alleles were identified (Md1-Md3), which were further confirmed by nucleotide sequencing. For EgAgB4, which was analysed by direct sequencing the PCR products, two groups of sequences were found: EgAgB4-1 and EgAgB4-2. No haplotype-specific mdh or EgAgB4 sequences occur. Nevertheless, alleles Md1 and Md2 and type 1 sequences of EgAgB4 showed a higher frequency within the group of haplotypes G1-G2, while allele Md3 and EgAgB4-2 are most frequent in the G5-G7 cluster. By AMOVA it is shown that 79% of the total genetic variability is found among haplotype groups. These findings are compatible with two not mutually exclusive evolutionary hypotheses: (a) that haplotypes share an ancestral polymorphism, or (b) that the reproductive isolation between parasites with distinct haplotypes is not complete, leading to gene introgression. The biologic and epidemiologic consequences of our findings are discussed.

Expression and diversity of Echinococcus multilocularis AgB genes in secondarily infected mice: evaluating the influence of T-cell immune selection on antigenic variation.

The T-cell-mediated immune response exhibits a crucial function in the control of the intrahepatic proliferation of Echinococcus multilocularis larvae in mice and humans, both being natural intermediate hosts of the parasite. Antigen B (AgB), a metabolized Echinococcus spp. lipoprotein, contributes to the modulation of the T-cell immune response, and distinct sites of the corresponding AgB1, AgB3 and AgB4 genes were shown to be under positive selection pressure. Since several AgB gene variants are present in a single Echinococcus metacestode, we used secondary E. multilocularis infections in BALB/c and in athymic nude mice (devoid of T-cell responses) to analyze the effect of the cellular immune response on the expression and diversity of EmAgB1-EmAgB4 genes. We demonstrated hereby that EmAgB transcripts were less abundant in nude mice during the early phase of infection (at one month post-infection), and that EmAgB2 is simultaneously down-regulated when compared to the other three genes. A negative relationship exists between the level of transcription and diversity of EmAgB genes. Moreover, no excess of non-synonymous substitutions was found among the distinct EmAgB alleles from a single host. Together, these results pointed to the effect of purifying selection, which seemed to eliminate the detrimental AgB variants generated during the development of the metacestode within the peritoneal cavity of its intermediate host.

Redundancy and recombination in the Echinococcus AgB multigene family: is there any similarity with protozoan contingency genes?

Numerous genetic variants of the Echinococcus antigen B (AgB) are encountered within a single metacestode. This could be a reflection of gene redundancy or the result of a somatic hypermutation process. We evaluate the complexity of the AgB multigene family by characterizing the upstream promoter regions of the 4 already known genes (EgAgB1-EgAgB4) and evaluating their redundancy in the genome of 3 Echinococcus species (E. granulosus, E. ortleppi and E. multilocularis) using PCR-based approaches. We have ascertained that the number of AgB gene copies is quite variable, both within and between species. The most repetitive gene seems to be AgB3, of which there are more than 110 copies in E. ortleppi. For E. granulosus, we have cloned and characterized 10 distinct upstream promoter regions of AgB3 from a single metacestode. Our sequences suggest that AgB1 and AgB3 are involved in gene conversion. These results are discussed in light of the role of gene redundancy and recombination in parasite evasion mechanisms of host immunity, which at present are known for protozoan organisms, but virtually unknown for multicellular parasites.

Searching for antigen B genes and their adaptive sites in distinct strains and species of the helminth Echinococcus.

Twenty-seven PCR-derived antigen B (AgB) nucleotide sequences from four Echinococcus species (Echinococcus granulosus, Echinococcus multilocularis, Echinococcus oligarthrus and Echinococcus vogeli) were aligned with 78 already published sequences, to generate a maximum likelihood phylogeny of the AgB multigene family. The phylogenetic analysis confirms that the family is constituted by four groups of genes present in each one of the four species (AgB1, AgB2, AgB3 and AgB4), and suggests that it originated by ancient duplication events preceding speciation within the genus. AgB5 sequences, which had been formerly suggested to correspond to a putatively new AgB subunit, cluster with AgB3. Likelihood tests suggest that AgB gene evolution may have been driven by heterogeneous selection pressures acting on particular AgB1, AgB3 and AgB4 codons. No selection is detected in AgB2. We discuss implications of our findings in terms of AgB biology and its use as a diagnostic tool.

"Clonal" population structure of the malaria agent Plasmodium falciparum in high-infection regions.

The population genetic structure of Plasmodium falciparum, the agent of malignant malaria, has been shown to be predominantly "clonal" (i.e., highly inbred) in regions of low infectivity; in high-infectivity regions, it is often thought to be panmictic, or nearly so, although there is little supporting evidence for this. The matter can be settled by investigating the parasite's genetic makeup in the midgut oocysts of the mosquito vector, where the products of meiosis can directly be observed. The developmental stages of P. falciparum are haploid, except in the oocysts of infected mosquito vectors, where two gametes fuse, diploidy occurs, and meiosis ensues. We have investigated genetic polymorphisms at seven microsatellite loci located on five chromosomes by assaying 613 oocysts in 145 mosquitoes sampled from 11 localities of Kenya, where malignant malaria is perennial and intense. There is considerable allelic variation, 16.3 +/- 2.1 alleles per locus, and considerable inbreeding, approximately 50% on the average. The inbreeding is caused by selfing (approximately 25%) and nonrandom genotype distribution of oocysts among mosquito guts (35%). The observed frequency of heterozygotes is 0.43 +/- 0.03; the expected frequency, assuming random mating, is 0.80 +/- 0.05. Linkage disequilibrium is statistically significant for all 21 pairwise comparisons between loci, even though 19 comparisons are between loci in different chromosomes, which is consistent with strong deviation from panmixia and the consequent reproduction of genomes as clones, without recombination between gene loci. This is of considerable evolutionary significance and of epidemiological consequence, concerning the spread of multilocus drug and vaccine resistance.

The Echinococcus granulosus antigen B shows a high degree of genetic variability.

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.

[Pseudogenes: structure conservation, expression, and functions]. / Psevdogeny: konservatsiia struktury, ékspressiia i funktsiia.

Pseudogenes have been defined as nonfunctional sequences of genomic DNA (junk DNA) originally derived from functional genes. It is therefore assumed that pseudogenes are not subject to natural selection and consequently pseudogene mutations are selectively neutral and have equal probability to become fixed in the population. We describe some unexpected features of pseudogenes in diverse organisms that are inconsistent with this widely accepted point of view. Pseudogenes are often evolutionary conserved and transcriptionally active. Moreover, pseudogenes that have been suitably investigated often exhibit functional roles, such as gene regulation, generation of genetic diversity, and other features that are expected in genes or DNA sequences that have functional roles. A review of the evidence leads to the conclusion that pseudogenes are important components of genomes, representing a repertoire of sequences available for functional evolution and subject to non-neutral evolutionary changes. Pseudogenes might be considered as potogenes, i.e. DNA sequences with a potentiality for becoming new genes or acquire new functions. Furthermore we conjecture that some pseudogenes along with their parental sequences may constitute sets of indivisible functionally interacting entities (intergenic complexes or "intergenes"), in which all the component elements are required in order to fulfill a collective functional role.

Meager genetic variability of the human malaria agent Plasmodium vivax.

Malaria is a major human parasitic disease caused by four species of Plasmodium protozoa. Plasmodium vivax, the most widespread, affects millions of people across Africa, Asia, the Middle East, and Central and South America. We have studied the genetic variability of 13 microsatellite loci in 108 samples from 8 localities in Asia, Africa, South America, and New Guinea. Only one locus is polymorphic; nine are completely monomorphic, and the remaining three are monomorphic in all but one or two populations, which have a rare second allele. In contrast, Plasmodium falciparum displays extensive microsatellite polymorphism within and among populations. We further have analyzed, in 96 samples from the same 8 localities, 8 tandem repeats (TRs) located on a 100-kb contiguous chromosome segment described as highly polymorphic. Each locus exhibits 2-10 alleles in the whole sample but little intrapopulation polymorphism (1-5 alleles with a prevailing allele in most cases). Eight microsatellite loci monomorphic in P. vivax are polymorphic in three of five Plasmodium species related to P. vivax (two to seven individuals sampled). Plasmodium simium, a parasite of New World monkeys, is genetically indistinguishable from P. vivax. At 13 microsatellite loci and at 7 of the 8 TRs, both species share the same (or most common) allele. Scarce microsatellite polymorphism may reflect selective sweeps or population bottlenecks in recent evolutionary history of P. vivax; the differential variability of the TRs may reflect selective processes acting on particular regions of the genome. We infer that the world expansion of P. vivax as a human parasite occurred recently, perhaps <10,000 years ago.

Contingent, non-neutral evolution in a multicellular parasite: natural selection and gene conversion in the Echinococcus granulosus antigen B gene family.

Recent studies have demonstrated that the Echinococcus granulosus antigen B (AgB) interferes with the intermediate hosts' immune response and is encoded by a multigene family. The number of members within the family is still uncertain, but there are several evidences of a large genetic variability. The E. granulosus AgB genomic sequences available in nucleotide databases can be grouped into four clades, corresponding to genes EgAgB1, EgAgB2, EgAgB3 and EgAgB4. In the present study, we use PCR amplifications followed by cloning and sequencing to evaluate the genetic variability for AgB isoforms. Two pairs of primers were independently used for PCR amplification. Both PCR reactions from each of three isolated protoscolex (larvae) were cloned in a plasmid vector and the plasmid inserts of 30 colonies from each cloning experiment were sequenced. Using phylogenetic tools, the 113 EgAgB clones are classified as follows: 25 are related to EgAgB1, 24 to EgAgB2, 9 to EgAgB3 and 39 to EgAgB4. The remaining 16 clones form a separate cluster, which we name EgAgB5, more closely related to EgAgB3 than to any of the other genes. Within each gene group, a number of variant sequences occur, which differ from one another by one or few nucleotides. One EgAgB3 clone has a premature stop codon (pseudogene) and an EgAgB2 clone lacks the region corresponding to the intron. The overall variation cannot be explained by differences among the asexual protoscoleces, or by experimental artifacts. Using Echinococcuss AgB genes from other species/strains as outgroups, neutrality is rejected for EgAgB2, and balancing selection is detected for EgAgB5, which also seems to be involved in gene conversion. We suggest that EgAgB1-EgAgB5 represent a family of contingency genes, that is, genes that are variably expressed, so that some but not others are expressed in each individual parasite. Contingency genes are common in parasitic protozoa and other microparasites, but the EgAgB family is the first set identified in a multicellular parasite.

Livestock trade history, geography, and parasite strains: the mitochondrial genetic structure of Echinococcus granulosus in Argentina.

A sample of 114 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from different host species and sites in Argentina has been sequenced for 391 bp from the mitochondrial cytochrome c oxidase subunit I gene to analyze genetic variability and population structure. Nine different haplotypes were identified, 5 of which correspond to already characterized strains. Analysis of molecular variance and nested clade analysis of the distribution of haplotypes among localities within 3 main geographic regions indicate that geographic differentiation accounts for the overall pattern of genetic variability in E. granulosus populations. Significant geographic differentiation is also present when the sheep strain alone is considered. Our results suggest that geographic patterns are not due to actual restricted gene flow between regions but are rather a consequence of past history, probably related to the time and origin of livestock introduction in Argentina.

Complex evolution of orthologous and paralogous decarboxylase genes.

The decarboxylases are involved in neurotransmitter synthesis in animals, and in pathways of secondary metabolism in plants. Different decarboxylase proteins are characterized for their different substrate specificities, but are encoded by homologous genes. We study, within a maximum-likelihood framework, the evolutionary relationships among dopa decarboxylase (Ddc), histidine decarboxylase (Hdc) and alpha-methyldopa hypersensitive (amd) in animals, and tryptophan decarboxylase (Wdc) and tyrosine decarboxylase (Ydc) in plants. The evolutionary rates are heterogeneous. There are differences between paralogous genes in the same lineages: 4.13 x 10(-10) nucleotide substitutions per site per year in mammalian Ddc vs. 1.95 in Hdc; between orthologous genes in different lineages, 7.62 in dipteran Ddc vs. 4.13 in mammalian Ddc; and very large temporal variations in some lineages, from 3.7 up to 54.9 in the Drosophila Ddc lineage. Our results are inconsistent with the molecular clock hypothesis.

Xanthine dehydrogenase (XDH): episodic evolution of a "neutral" protein.

We investigated the evolution of xanthine dehydrogenase (Xdh) in 34 species from the three multicellular kingdoms, including one plant, two fungi, and three animal phyla, two classes of vertebrates, four orders of mammals, and two orders of insects. We adopted a model-based maximum-likelihood framework of inference. After accounting for among-site rate variation and heterogeneous nucleotide composition of the sequences using the discrete gamma distribution, and using nonhomogeneous nonstationary representations of the substitution process, the rate of amino acid replacement is 30.4 x 10(-10)/site/year when Drosophila species are compared but only approximately 18 x 10(-10)/site/year when comparisons are made between mammal orders, between insect orders, or between different animal phyla and approximately 11 x 10(-10)/site/year when comparisons are made between birds and mammals, between fungi, or between the three multicellular kingdoms. To account for these observations, the rate of amino acid replacement must have been eight or more times higher in some lineages and at some times than in others. Spastic evolution of Xdh appears to be related to the particularities of the genomes in which the locus is embedded.

Erratic overdispersion of three molecular clocks: GPDH, SOD, and XDH.

The neutrality theory predicts that the rate of neutral molecular evolution is constant over time, and thus that there is a molecular clock for timing evolutionary events. It has been observed that the variance of the rate of evolution is generally larger than expected according to the neutrality theory, which has raised the question of how reliable the molecular clock is or, indeed, whether there is a molecular clock at all. We have carried out an extensive investigation of three proteins, glycerol-3-phosphate dehydrogenase (GPDH), superoxide dismutase (SOD), and xanthine dehydrogenase (XDH). We have observed that (i) the three proteins evolve erratically through time and across lineages and (ii) the erratic patterns of acceleration and deceleration differ from locus to locus, so that one locus may evolve faster in one than another lineage, whereas the opposite may be the case for another locus. The observations are inconsistent with the predictions made by various subsidiary hypotheses proposed to account for the overdispersion of the molecular clock.

Shared nucleotide composition biases among species and their impact on phylogenetic reconstructions of the Drosophilidae.

Compositional changes are a major feature of genome evolution. Overlooking nucleotide composition differences among sequences can seriously mislead phylogenetic reconstructions. Large compositional variation exists among the members of the family Drosophilidae. Until now, however, base composition differences have been largely neglected in the formulations of the nucleotide substitution process used to reconstruct the phylogeny of this important group of species. The present study adopts a maximum-likelihood framework of phylogenetic inference in order to analyze five nuclear gene regions and shows that (1) the pattern of compositional variation in the Drosophilidae does not match the phylogeny of the species; (2) accounting for the heterogeneous GC content with Galtier and Gouy's nucleotide substitution model leads to a tree that differs in significant aspects from the tree inferred when the nucleotide composition differences are ignored, even though both phylogenetic hypotheses attain strong nodal support in the bootstrap analyses; and (3) the LogDet distance correction cannot completely overcome the distorting effects of the compositional variation that exists among the species of the Drosophilidae. Our analyses confidently place the Chymomyza genus as an outgroup closer than the genus Scaptodrosophila to the Drosophila genus and conclusively support the monophyly of the Sophophora subgenus.

Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi.

Simple phylogenetic tests were applied to a large data set of nucleotide sequences from two nuclear genes and a region of the mitochondrial genome of Trypanosoma cruzi, the agent of Chagas' disease. Incongruent gene genealogies manifest genetic exchange among distantly related lineages of T. cruzi. Two widely distributed isoenzyme types of T. cruzi are hybrids, their genetic composition being the likely result of genetic exchange between two distantly related lineages. The data show that the reference strain for the T. cruzi genome project (CL Brener) is a hybrid. Well-supported gene genealogies show that mitochondrial and nuclear gene sequences from T. cruzi cluster, respectively, in three or four distinct clades that do not fully correspond to the two previously defined major lineages of T. cruzi. There is clear genetic differentiation among the major groups of sequences, but genetic diversity within each major group is low. We estimate that the major extant lineages of T. cruzi have diverged during the Miocene or early Pliocene (3-16 million years ago).