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Accurate and timely detection of recombinant lineages is crucial for interpreting genetic variation, reconstructing epidemic spread, identifying selection and variants of interest, and accurately performing phylogenetic analyses1-4. During the SARS-CoV-2 pandemic, genomic data generation has exceeded the capacities of existing analysis platforms, thereby crippling real-time analysis of viral evolution5. Here, we use a new phylogenomic method to search a nearly comprehensive SARS-CoV-2 phylogeny for recombinant lineages. In a 1.6 million sample tree from May 2021, we identify 589 recombination events, which indicate that around 2.7% of sequenced SARS-CoV-2 genomes have detectable recombinant ancestry. Recombination breakpoints are inferred to occur disproportionately in the 3' portion of the genome that contains the spike protein. Our results highlight the need for timely analyses of recombination for pinpointing the emergence of recombinant lineages with the potential to increase transmissibility or virulence of the virus. We anticipate that this approach will empower comprehensive real-time tracking of viral recombination during the SARS-CoV-2 pandemic and beyond.

Heat-shock (HS) assays to understand the connection between standing inversion variation and evolutionary response to climate change in Drosophila subobscura found that "warm-climate" inversion O3+4 exhibits non-HS levels of Hsp70 protein like those of "cold-climate" OST after HS induction. This was unexpected, as overexpression of Hsp70 can incur multiple fitness costs. To understand the genetic basis of this finding, we have determined the genomic sequence organization of the Hsp70 family in four different inversions, including OST , O3+4 , O3+4+8 and O3+4+16 , using as outgroups the remainder of the subobscura species subgroup, namely Drosophila madeirensis and Drosophila guanche. We found (i) in all the assayed lines, the Hsp70 family resides in cytological locus 94A and consists of only two genes, each with four HS elements (HSEs) and three GAGA sites on its promoter. Yet, in OST , the family is comparatively more compact; (ii) the two Hsp70 copies evolve in concert through gene conversion, except in D. guanche; (iii) within D. subobscura, the rate of concerted evolution is strongly structured by inversion, being higher in OST than in O3+4 ; and (iv) in D. guanche, the two copies accumulated multiple differences, including a newly evolved "gap-type" HSE2. The absence of concerted evolution in this species may be related to a long-gone-unnoticed observation that it lacks Hsp70 HS response, perhaps because it has evolved within a narrow thermal range in an oceanic island. Our results point to a previously unrealized link between inversions and concerted evolution, with potentially major implications for understanding genome evolution.

Heat-shock (HS) assays to understand the connection between standing inversion variation and evolutionary response to climate change in Drosophila subobscura found that "warm-climate" inversion O3+4 exhibits non-HS levels of Hsp70 protein like those of "cold-climate" OST after HS induction. This was unexpected, as overexpression of Hsp70 can incur multiple fitness costs. To understand the genetic basis of this finding, we have determined the genomic sequence organization of the Hsp70 family in four different inversions, including OST , O3+4 , O3+4+8 and O3+4+16 , using as outgroups the remainder of the subobscura species subgroup, namely Drosophila madeirensis and Drosophila guanche. We found (i) in all the assayed lines, the Hsp70 family resides in cytological locus 94A and consists of only two genes, each with four HS elements (HSEs) and three GAGA sites on its promoter. Yet, in OST , the family is comparatively more compact; (ii) the two Hsp70 copies evolve in concert through gene conversion, except in D. guanche; (iii) within D. subobscura, the rate of concerted evolution is strongly structured by inversion, being higher in OST than in O3+4 ; and (iv) in D. guanche, the two copies accumulated multiple differences, including a newly evolved "gap-type" HSE2. The absence of concerted evolution in this species may be related to a long-gone-unnoticed observation that it lacks Hsp70 HS response, perhaps because it has evolved within a narrow thermal range in an oceanic island. Our results point to a previously unrealized link between inversions and concerted evolution, with potentially major implications for understanding genome evolution.

Paratrypanosoma confusum is a monoxenous kinetoplastid flagellate that constitutes the most basal branch of the highly diverse parasitic trypanosomatids, which include human pathogens Trypanosoma and Leishmania This makes Paratrypanosoma uniquely informative for the evolution of obligatory parasitism from free-living lifestyle and the evolution of human parasitism in some trypanosomatid lineages. It has typical promastigote morphology but also forms surface-attached haptomonads and amastigotes. Haptomonads form by attachment to a surface via a large bulge at the base of the flagellum, which is then remodeled into a thin attachment pad associated with flagellum shortening. Promastigotes and haptomonads multiply by binary division, and the progeny of a haptomonad can either remain attached or grow a flagellum and resume swimming. Whole genome sequencing and transcriptome profiling, in combination with analysis of the cell ultrastructure, reveal how the cell surface and metabolism are adapted to parasitism and how characteristic cytoskeletal features are conserved. Our data demonstrate that surface attachment by the flagellum and the flagellar pocket, a Leishmania-like flagellum attachment zone, and a Trypanosoma cruzi-like cytostome are ancestral features, while evolution of extant trypanosomatids, including the human parasites, is associated with genome streamlining and diversification of membrane proteins.

The complete mitochondrial genome was sequenced in two individuals of the Kamchatka grayling Thymallus mertensii. The genome sequences are 16 662 bp in size, and the gene arrangement, composition, and size are very similar to the salmonid fish genomes published previously. The low level of sequence divergence (0.92%) detected between the genome of T. mertensii and the GenBank complete mitochondrial genomes of the Arctic grayling T. arcticus (FJ872559) may likely be due to recent divergence of the species and/or historical hybridization and interspecific replacement of mtDNA.

BackgroundThe sperm gene bindin encodes a gamete recognition protein, which plays an important role in conspecific fertilization and reproductive isolation of sea urchins. Molecular evolution of the gene has been extensively investigated with the attention focused on the protein coding regions. Intron evolution has been investigated to a much lesser extent. We have studied nucleotide variability in the complete bindin locus, including two exons and one intron, in the sea urchin Strongylocentrotus intermedius represented by two morphological forms. We have also analyzed all available bindin sequences for two other sea urchin species, S. pallidus and S. droebachiensis.ResultsThe results show that the bindin sequences from the two forms of S. intermedius are intermingled with no evidence of genetic divergence; however, the forms exhibit slightly different patterns in bindin variability. The level of the bindin nucleotide diversity is close for S. intermedius and S. droebachiensis, but noticeably higher for S. pallidus. The distribution of variability is non-uniform along the gene; however there are striking similarities among the species, indicating similar evolutionary trends in this gene engaged in reproductive function. The patterns of nucleotide variability and divergence are radically different in the bindin coding and intron regions. Positive selection is detected in the bindin coding region. The neutrality tests as well as the maximum likelihood approaches suggest the action of diversifying selection in the bindin intron.ConclusionsSignificant deviation from neutrality has been detected in the bindin coding region and suggested in the intron, indicating the possible functional importance of the bindin intron variability. To clarify the question concerning possible involvement of diversifying selection in the bindin intron evolution more data combining population genetic and functional approaches are necessary.

The complete mitochondrial genome was sequenced in two individuals of white char Salvelinus albus. The genome sequences are 16 653 bp in size, and the gene arrangement, composition, and size are very similar to the salmonid fish genomes published previously. The low level of sequence divergence detected between the genome of S. albus and the GenBank complete mitochondrial genomes of the Northern Dolly Varden char S. malma (KJ746618) and the Arctic char S. alpinus (AF154851) may likely be due to recent divergence of the species and/or historical hybridization and interspecific replacement of mtDNA.

Haemosporidia parasites have mostly and abundantly been described using mitochondrial genes, and in particular cytochrome b (cytb). Failure to amplify the mitochondrial cytb gene of Nycteria parasites isolated from Nycteridae bats has been recently reported. Bats are hosts to a diverse and profuse array of Haemosporidia parasites that remain largely unstudied. There is a need to obtain more molecular data from chiropteran parasites. Such data would help to better understand the evolutionary history of Haemosporidia, which notably include the Plasmodium parasites, malaria's agents. We use next-generation sequencing to obtain the complete mitochondrial genome of Nycteria parasites from African Nycteris grandis (Nycteridae) and Rhinolophus alcyone (Rhinolophidae) and Asian Megaderma spasma (Megadermatidae). We report four complete mitochondrial genomes, including two rearranged mitochondrial genomes within Haemosporidia. Our results open outlooks into potentially undiscovered Haemosporidian diversity.

The complete mitochondrial genomes were sequenced in anadromous and resident forms of the lamprey Lethenteron camtschaticum. The sizes of the genomes in the two isolates are 16,245 and 16,295 bp. The gene arrangement, base composition, and size of the two sequenced genomes are similar to the lamprey genomes previously published. The total sequence divergence between the two genomes is very low (0.14%), supporting conspecificity of the anadromous and resident forms of L. camtschaticum. Comparison of the genomes sequenced in the present work with other genomes of lampreys available in GenBank, reveals two distinct evolutionary lineages with a genera level of divergence among the lampreys of eastern Eurasia.

The complete mitochondrial genomes were sequenced in two individuals of Siberian taimen Hucho taimen. The sizes of the genomes were 16,833 and 16,914 in the two isolates, representing two haplotype groups previously detected. The gene arrangement, base composition, and size of the two sequenced genomes are very similar to the H. taimen genome previously published (HQ897271), but did not contain introgressed fragments from two subspecies of lenok, Brachymystax lenok and B. l. tsinlingensis.

Comparing the population structure of Trypanosoma cruzi with that of other pathogens, including parasitic protozoa, fungi, bacteria and viruses, shows that the agent of Chagas disease shares typical traits with many other species, related to a predominant clonal evolution (PCE) pattern: statistically significant linkage disequilibrium, overrepresented multilocus genotypes, near-clades (genetic subdivisions somewhat blurred by occasional genetic exchange/hybridization) and "Russian doll" patterns (PCE is observed, not only at the level of the whole species, but also, within the near-clades). Moreover, T. cruzi population structure exhibits linkage with the diversity of several strongly selected genes, with gene expression profiles, and with some major phenotypic traits. We discuss the evolutionary significance of these results, and their implications in terms of applied research (molecular epidemiology/strain typing, analysis of genes of interest, vaccine and drug design, immunological diagnosis) and of experimental evolution. Lastly, we revisit the long-term debate of describing new species within the T. cruzi taxon.

There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this "residual" genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered.

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.

Plasmodium vivax is considered to be absent from Central and West Africa because of the protective effect of Duffy negativity. However, there are reports of persons returning from these areas infected with this parasite and observations suggesting the existence of transmission. Among the possible explanations for this apparent paradox, the existence of a zoonotic reservoir has been proposed. May great apes be this reservoir? We analyze the mitochondrial and nuclear genetic diversity of P. vivax parasites isolated from great apes in Africa and compare it to parasites isolated from travelers returning from these regions of Africa, as well as to human isolates distributed all over the world. We show that the P. vivax sequences from parasites of great apes form a clade genetically distinct from the parasites circulating in humans. We show that this clade's parasites can be infectious to humans by describing the case of a traveler returning from the Central African Republic infected with one of them. The relationship between this P. vivax clade in great apes and the human isolates is discussed.

Siberian taimen Hucho taimen is the largest representative of the family Salmonidae inhabiting rivers of northern Eurasia. The species is under intensive aquaculture activity. To monitor natural taimen populations we have sequenced a portion (8,141 bp) of the mitochondrial (mt) genome in 28 specimens of H. taimen from six localities in the Amur River basin. Nucleotide variability is low (π = 0.0010), but structured in two divergent haplotype groups. A comparison of the data with the GenBank H. taimen mt genome (HQ897271) reveals significant differences between them in spite of the fact that the fish specimens come from neighboring geographical areas. The distribution of divergence is non-uniform with two highly pronounced divergent regions centered on two genes, ND3 and ND6. To clarify the pattern of divergence we sequenced the corresponding portion of the mt genome of lenok Brachymystax tumensis and analyzed the GenBank complete mt genomes of related species. We have found that the first and second divergent regions are identical between the GenBank H. taimen and two lenok subspecies, B. lenok and B. lenok tsinlingensis, respectively. Consequently, both divergent regions represent introgressed mtDNA resulting from intergeneric hybridization between the two lenok subspecies and H. taimen. Introgression is, however, not detected in our specimens. This plus the precise identity of the introgressed fragments between the donor and the recipient GenBank sequence suggests that the introgression is local and very recent, probably due to artificial manipulations involving taimen-lenok intergeneric hybridization. Human-mediated hybridization may become a major threat to aquatic biodiversity. Consequently we suggest that due attention needs to be given to this threat by means of responsible breeding program management, so as to prevent a potential spread of hybrid fishes that could jeopardize the resilience of locally adapted gene pools of the native H. taimen populations.

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

The evolutionary history of two human malaria parasites, Plasmodium vivax and Plasmodium malariae, remains unresolved. The near genetic identity between human P. vivax and P. malariae, and primate P. simium and P. brasilianum, respectively, suggests that recent host transfers occurred, but questions remain, such as whether the transfer was from humans to New World monkeys or vice versa, and when the transfers occurred. Here, we investigate the phylogenies, haplotype networks, positive selection and genetic diversity among these parasite species by means of four genes. Human P. vivax and primate P. simium recently derived one from the other; at least two host transfers have occurred. Human P. malariae and primate P. brasilianum also have recently derived one from the other by lateral host transfer. The direction of the host transfer cannot be decided in either one of the two pairs of species, owing to the scarcity of available strains from the primate parasites.

Until very recently, only one species (P. reichenowi) was known to be a phylogenetic sister lineage of P. falciparum, the main malignant agent of human malaria. In 2009 and 2010, new studies have revealed the existence of several new phylogenetic species related to this deadly parasite and infecting chimpanzees and gorillas in Africa. These discoveries invite us to explore a whole set of new questions, which we briefly do in this article.

We performed a phylogenetic character mapping on 26 stocks of Trypanosoma cruzi, the parasite responsible for Chagas disease, and 2 stocks of the sister taxon T. cruzi marinkellei to test for possible associations between T. cruzi-subspecific phylogenetic diversity and levels of protein expression, as examined by proteomic analysis and mass spectrometry. We observed a high level of correlation (P < 10(-4)) between genetic distance, as established by multilocus enzyme electrophoresis, and proteomic dissimilarities estimated by proteomic Euclidian distances. Several proteins were found to be specifically associated to T. cruzi phylogenetic subdivisions (discrete typing units). This study explores the previously uncharacterized links between infraspecific phylogenetic diversity and gene expression in a human pathogen. It opens the way to searching for new vaccine and drug targets and for identification of specific biomarkers at the subspecific level of pathogens.

We investigated two mitochondrial genes (cytb and cox1), one plastid gene (tufA), and one nuclear gene (ldh) in blood samples from 12 chimpanzees and two gorillas from Cameroon and one lemur from Madagascar. One gorilla sample is related to Plasmodium falciparum, thus confirming the recently reported presence in gorillas of this parasite. The second gorilla sample is more similar to the recently defined Plasmodium gaboni than to the P. falciparum-Plasmodium reichenowi clade, but distinct from both. Two chimpanzee samples are P. falciparum. A third sample is P. reichenowi and two others are P. gaboni. The other chimpanzee samples are different from those in the ape clade: two are Plasmodium ovale, and one is Plasmodium malariae. That is, we have found three human Plasmodium parasites in chimpanzees. Four chimpanzee samples were mixed: one species was P. reichenowi; the other species was P. gaboni in three samples and P. ovale in the fourth sample. The lemur sample, provisionally named Plasmodium malagasi, is a sister lineage to the large cluster of primate parasites that does not include P. falciparum or ape parasites, suggesting that the falciparum + ape parasite cluster (Laverania clade) may have evolved from a parasite present in hosts not ancestral to the primates. If malignant malaria were eradicated from human populations, chimpanzees, in addition to gorillas, might serve as a reservoir for P. falciparum.

Plasmodium reichenowi, a chimpanzee parasite, was until very recently the only known close relative of Plasmodium falciparum, the most virulent agent of human malaria. Recently, Plasmodium gaboni, another closely related chimpanzee parasite, was discovered, suggesting that the diversity of Plasmodium circulating in great apes in Africa might have been underestimated. It was also recently shown that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite and that the world diversity of P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. The evidence indicates that all extant populations of P. falciparum originated from P. reichenowi, likely by a single transfer from chimpanzees. In this work, we have studied the diversity of Plasmodium species infecting chimpanzees and gorillas in Central Africa (Cameroon and Gabon) from both wild-living and captive animals. The studies in wild apes used noninvasive sampling methods. We confirm the presence of P. reichenowi and P. gaboni in wild chimpanzees. Moreover, our results reveal the existence of an unexpected genetic diversity of Plasmodium lineages circulating in gorillas. We show that gorillas are naturally infected by two related lineages of parasites that have not been described previously, herein referred to as Plasmodium GorA and P. GorB, but also by P. falciparum, a species previously considered as strictly human specific. The continuously increasing contacts between humans and primate populations raise concerns about further reciprocal host transfers of these pathogens.

Plasmodium falciparum, the causative agent of malignant malaria, is among the most severe human infectious diseases. The closest known relative of P. falciparum is a chimpanzee parasite, Plasmodium reichenowi, of which one single isolate was previously known. The co-speciation hypothesis suggests that both parasites evolved separately from a common ancestor over the last 5-7 million years, in parallel with the divergence of their hosts, the hominin and chimpanzee lineages. Genetic analysis of eight new isolates of P. reichenowi, from wild and wild-born captive chimpanzees in Cameroon and Côte d'Ivoire, shows that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite. The genetic lineage comprising the totality of global P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. This finding is inconsistent with the co-speciation hypothesis. Phylogenetic analysis indicates that all extant P. falciparum populations originated from P. reichenowi, likely by a single host transfer, which may have occurred as early as 2-3 million years ago, or as recently as 10,000 years ago. The evolutionary history of this relationship may be explained by two critical genetic mutations. First, inactivation of the CMAH gene in the human lineage rendered human ancestors unable to generate the sialic acid Neu5Gc from its precursor Neu5Ac, and likely made humans resistant to P. reichenowi. More recently, mutations in the dominant invasion receptor EBA 175 in the P. falciparum lineage provided the parasite with preference for the overabundant Neu5Ac precursor, accounting for its extreme human pathogenicity.

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.

The time of origin of the animal phyla is controversial. Abundant fossils from the major animal phyla are found in the Cambrian, starting 544 million years ago. Many paleontologists hold that these phyla originated in the late Neoproterozoic, during the 160 million years preceding the Cambrian fossil explosion. We have analyzed 18 protein-coding gene loci and estimated that protostomes (arthropods, annelids, and mollusks) diverged from deuterostomes (echinoderms and chordates) about 670 million years ago, and chordates from echinoderms about 600 million years ago. Both estimates are consistent with paleontological estimates. A published analysis of seven gene loci that concludes that the corresponding divergence times are 1,200 and 1,000 million years ago is shown to be flawed because it extrapolates from slow-evolving vertebrate rates to faster-evolving invertebrate rates, as well as in other ways.

Strongylocentrotus intermedius (A. Agassiz, 1863) is an economically important sea urchin inhabiting the northwest Pacific region of Asia. The northern Primorye (Sea of Japan) populations of S. intermedius consist of two sympatric morphological forms, "usual" (U) and "gray" (G). The two forms are significantly different in morphology and preferred bathymetric distribution, the G form prevailing in deeper-water settlements. We have analyzed the genetic composition of the S. intermedius forms using the nucleotide sequences of the mitochondrial gene encoding the cytochrome c oxidase subunit I and the nuclear gene encoding bindin to evaluate the possibility of cryptic species within S. intermedius. We have examined the presence of symbiont microorganisms by means of 16S rRNA sequences. The nucleotide sequence divergence between the morphological forms is low: 0.74% and 0.70% for cytochrome c oxidase subunit I and nuclear gene encoding bindin, respectively, which is significantly below average intrageneric sequence divergence among Strongylocentrotus species. We thus have found no genetic evidence of cryptic species within S. intermedius. Phylogenetic analysis shows that the bacteria symbionts of S. intermedius belong to the phylum Bacteroidetes, but the U and G forms predominantly harbor highly divergent bacterial lineages belonging to two different taxonomic classes, Flavobacteria and Sphingobacteria. We propose that the U and G forms of S. intermedius represent distinct ecomorphological adaptations to contrasting shallow- and deep-water marine environments and might be considered incipient species. We also propose that the symbiotic bacteria likely play an important role in the evolution of morphological divergence of S. intermedius.

Spliceosomal introns, a hallmark of eukaryotic gene organization, were an unexpected discovery. After three decades, crucial issues such as when and how introns first appeared in evolution remain unsettled. An issue yet to be answered is how intron positions arise de novo. Phylogenetic investigations concur that intron positions continue to emerge, at least in some lineages. Yet genomic scans for the sources of introns occupying new positions have been fruitless. Two alternative solutions to this paradox are: (i) formation of new intron positions halted before the recent past and (ii) it continues to occur, but through processes different from those generally assumed. One process generally dismissed is intron sliding--the relocation of a preexisting intron over short distances--because of supposed associated deleterious effects. The puzzle of intron gain arises owing to a pervasive operational definition of introns, which sees them as precisely demarcated segments of the genome separated from the neighboring nonintronic DNA by unmovable limits. Intron homology is defined as position homology. Recent studies of pre-mRNA processing indicate that this assumption needs to be revised. We incorporate recent advances on the evolutionarily frequent process of alternative splicing, by which exons of primary transcripts are spliced in different patterns, into a new model of intron sliding that accounts for the diversity of intron positions. We posit that intron positional diversity is driven by two overlapping processes: (i) background process of continuous relocation of preexisting introns by sliding and (ii) spurts of extensive gain/loss of new intron sequences.

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of approximately 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged approximately 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa.

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.

We examine the pattern of molecular evolution of the beta-esterase gene cluster, including the Est-6 and psiEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For psiEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of psiEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of psiEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the beta-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function.

We examine the pattern of molecular evolution of the beta-esterase gene cluster, including the Est-6 and psiEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For psiEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of psiEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of psiEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the beta-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function.

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

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.

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.

The relative merits of molecular and paleontological dates of major branching points in the tree of life are currently debated. In some cases, molecular date estimates are up to twice as old as paleontological dates. However, although it is true that paleontological dates are often too young (missing fossils), molecular dates are often too old (statistical bias). Intense study of the dating of major splits in the tree of mammals has shown rapprochement as fossil dates become older and molecular dates become younger.

Efforts to understand the genetic basis of evolutionary change have concentrated on proteins and their encoding DNA sequences. These studies have brought to light patterns and processes at the nucleotide level, yet the complex functional relationships between genetic variants and phenotypes remain poorly known. The realization that even a complete description of proteins and the effects of their activity will not suffice to understand the conditions under which they are time- and tissue-specifically expressed or repressed during development has refocused attention on cis-regulatory regions. In particular, promoter sequences are thought to hold the key for understanding the evolution of phenotypic differences between species. This is because of their complex organization into independent modules such that, unlike coding sequences in which mutations affect protein function every time the protein is expressed, mutations in cis-regulatory sequences may have minor or no pleiotropic effects. Complex information-encoding makes cis-regulatory regions poorly amenable to comparative methods designed for coding sequences. Some general conclusions are emerging as to how genetic variation is distributed across regulatory networks and the processes modulating the structure of this variation. We bring into this emerging scenario several recent findings pointing to different ways in which spliceosomal introns, pseudogenes and patterns of point mutation can be active players for the evolution of novel transcriptional profiles.

The phylogenetic relationships among the Drosophila melanogaster subgroup species were analyzed using approximately 1550-nucleotide-long sequences of the Cu,Zn SOD gene. Phylogenetic analysis was performed using separately the whole region and the intron sequences of the gene. The resulting phylogenetic trees reveal virtually the same topology, separating the species into distinct clusters. The inferred topology generally agrees with previously proposed classifications based on morphological and molecular data. The amino acid sequences of the Cu,Zn SOD of the D. melanogaster subgroup species reveal a high-conservation pattern. Only 3.9% of the total amino acid sites are variable, and none affects the major structural elements. Comparison of the Drosophila Cu,Zn SOD amino acid sequences with the Cu,Zn SOD of Bos taurus and Xenopus laevis (whose three-dimensional structure has been elucidated) reveals conservation of all the protein's functionally important amino acids and no substitutions that dramatically change the charge or the polarity of the amino acids.

We have examined the patterns of polymorphism at two linked loci, Sod and Est-6, separated by nearly 1000 kb on the left arm of chromosome 3 of Drosophila melanogaster. The evidence suggests that natural selection has been involved in shaping the polymorphisms. At the Sod locus, a fairly strong (s>0.01) selective sweep, started >or=2600 years ago, increased the frequency of a rare haplotype, F(A), to about 50% frequency in populations of Europe, Asia, and the Americas. More recently, an F(A) allele mutated to an S allele, which has increased to frequencies 5-15% in populations of Europe, Asia and North America. All S alleles are identical (or very nearly) in sequence and differ by one nucleotide substitution (which accounts for the F-->S electrophoretic difference) from F(A) alleles. At the Est-6 locus, the evidence indicates both directional and balancing selection impacting separately the promoter and the coding regions of the gene, with linkage disequilibrium occurring within each region. Some linkage disequilibrium also exists between the two genes.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) and trypanothione reductase (TR) are important enzymes for the metabolism of protozoan parasites from the family Trypanosomatidae (e.g. Trypanosoma spp., Leishmania spp.) that are targets of current drug-design studies. Very limited information exists on the levels of genetic polymorphism of these enzymes in natural populations of any trypanosomatid parasite. We present results of a survey of nucleotide variation in the genes coding for those enzymes in a large sample of strains from Trypanosoma cruzi, the agent of Chagas' disease. We discuss the results from an evolutionary perspective. A sample of 31 strains show 39 silent and five amino acid polymorphisms in DHFR-TS, and 35 silent and 11 amino acid polymorphisms in TR. No amino acid replacements occur in regions that are important for the enzymatic activity of these proteins, but some polymorphisms occur in sites previously assumed to be invariant. The sequences from both genes cluster in four major groups, a result that is not fully consistent with the current classification of T. cruzi in two major groups of strains. Most polymorphisms correspond to fixed differences among the four sequence groups. Two tests of neutrality show that there is no evidence of adaptive divergence or of selective events having shaped the distribution of polymorphisms and fixed differences in these genes in T. cruzi. However, one nearly significant reduction of variation in the TR sequences from one sequence group suggests a recent selective event at, or close to, that locus.

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.