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Abstract Background The ability of emerging pathogens to infect new species is likely related to the diversity of pathogen variants present in existing reservoirs and their degree of genomic plasticity, which determines their ability to adapt to new environments. Certain simian immunodeficiency viruses (SIVcpz, SIVsm) have demonstrated tremendous success in infecting new species, including humans, resulting in the HIV-1 and HIV-2 epidemics. Although SIV diversification has been studied on a population level, the essential substrates for cross-species transmission, namely SIV sequence diversity and the types and extent of viral diversification present in individual reservoir animals have not been elucidated. To characterize this intra-host SIV diversity, we performed sequence analyses of clonal viral envelope (env) V1V2 and gag p27 variants present in individual SIVsm-infected sooty mangabeys over time. Results SIVsm demonstrated extensive intra-animal V1V2 length variation and amino acid diversity (le38%), and continual variation in V1V2 N-linked glycosylation consensus sequence frequency and location. Positive selection was the predominant evolutionary force. Temporal sequence shifts suggested continual selection, likely due to evolving antibody responses. In contrast, gag p27 was predominantly under purifying selection. SIVsm V1V2 sequence diversification is at least as great as that in HIV-1 infected humans, indicating that extensive viral diversification in and of itself does not inevitably lead to AIDS. Conclusion Positive diversifying selection in this natural reservoir host is the engine that has driven the evolution of the uniquely adaptable SIV/HIV envelope protein. These studies emphasize the importance of retroviral diversification within individual host reservoir animals as a critical substrate in facilitating cross-species transmission.

We have sequenced, assembled, and analyzed the nuclear and mitochondrial genomes and transcriptomes of Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa, two prosobranch snail species native to New Zealand that together span the continuum from estuary to freshwater. These two species are the closest known relatives of the freshwater species Potamopyrgus antipodarum-a model for studying the evolution of sex, host-parasite coevolution, and biological invasiveness-and thus provide key evolutionary context for understanding its unusual biology. The P. estuarinus and P. kaitunuparaoa genomes are very similar in size and overall gene content. Comparative analyses of genome content indicate that these two species harbor a near-identical set of genes involved in meiosis and sperm functions, including seven genes with meiosis-specific functions. These results are consistent with obligate sexual reproduction in these two species and provide a framework for future analyses of P. antipodarum-a species comprising both obligately sexual and obligately asexual lineages, each separately derived from a sexual ancestor. Genome-wide multigene phylogenetic analyses indicate that P. kaitunuparaoa is likely the closest relative to P. antipodarum. We nevertheless show that there has been considerable introgression between P. estuarinus and P. kaitunuparaoa. That introgression does not extend to the mitochondrial genome, which appears to serve as a barrier to hybridization between P. estuarinus and P. kaitunuparaoa. Nuclear-encoded genes whose products function in joint mitochondrial-nuclear enzyme complexes exhibit similar patterns of nonintrogression, indicating that incompatibilities between the mitochondrial and the nuclear genome may have prevented more extensive gene flow between these two species., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits-mode of genomic inheritance-influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S-5.8S-28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA-histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction-with potential functional consequences related to the loss of sexual reproduction., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

How changes in selective regimes affect trait evolution is an important open biological question. We take advantage of naturally occurring and repeated transitions from sexual to asexual reproduction in a New Zealand freshwater snail species, Potamopyrgus antipodarum, to address how evolution in an asexual context-including the potential for relaxed selection on male-specific traits-influences sperm morphology. The occasional production of male offspring by the otherwise all-female asexual P. antipodarum lineages affords a unique and powerful opportunity to assess the fate of sperm traits in a context where males are exceedingly rare. These comparisons revealed that the sperm produced by 'asexual' males are markedly distinct from sexual counterparts. We also found that the asexual male sperm harboured markedly higher phenotypic variation and was much more likely to be morphologically abnormal. Together, these data suggest that transitions to asexual reproduction might be irreversible, at least in part because male function is likely to be compromised. These results are also consistent with a scenario where relaxed selection and/or mutation accumulation in the absence of sex translates into rapid trait degeneration., (© 2020 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2020 European Society For Evolutionary Biology.)

Parasitoid wasps are among the most speciose animals, yet have relatively few available genomic resources. We report a draft genome assembly of the wasp Diachasma alloeum (Hymenoptera: Braconidae), a host-specific parasitoid of the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae), and a developing model for understanding how ecological speciation can "cascade" across trophic levels. Identification of gene content confirmed the overall quality of the draft genome, and we manually annotated ∼400 genes as part of this study, including those involved in oxidative phosphorylation, chemosensation, and reproduction. Through comparisons to model hymenopterans such as the European honeybee Apis mellifera and parasitoid wasp Nasonia vitripennis, as well as a more closely related braconid parasitoid Microplitis demolitor, we identified a proliferation of transposable elements in the genome, an expansion of chemosensory genes in parasitoid wasps, and the maintenance of several key genes with known roles in sexual reproduction and sex determination. The D. alloeum genome will provide a valuable resource for comparative genomics studies in Hymenoptera as well as specific investigations into the genomic changes associated with ecological speciation and transitions to asexuality., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution., Results: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting., Conclusions: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Boasting a staggering diversity of reproductive strategies, insects provide attractive models for the comparative study of the causes and consequences of transitions to asexuality. We provide an overview of some contemporary studies of reproductive systems in insects and compile an initial database of asexual insect genome resources. Insect systems have already yielded some important insights into various mechanisms by which sex is lost, including genetic, endosymbiont-mediated, and hybridization. Studies of mutation and substitution after loss of sex provide the strongest empirical support for hypothesized effects of asexuality, whereas there is mixed evidence for ecological hypotheses such as increased parasite load and altered niche breadth in asexuals. Most hypotheses have been explored in a select few taxa (e.g. stick insects, aphids), such that much of the great taxonomic breadth of insects remain understudied. Given the variation in the proximate causes of asexuality in insects, we argue for expanding the taxonomic breadth of study systems. Despite some challenges for investigating sex in insects, the increasing cost-effectiveness of genomic sequencing makes data generation for closely-related asexual and sexual lineages increasingly feasible., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Harmful mutations are ubiquitous and inevitable, and the rate at which these mutations are removed from populations is a critical determinant of evolutionary fate. Closely related sexual and asexual taxa provide a particularly powerful setting to study deleterious mutation elimination because sexual reproduction should facilitate mutational clearance by reducing selective interference between sites and by allowing the production of offspring with different mutational complements than their parents. Here, we compared the rate of removal of conservative (i.e., similar biochemical properties) and radical (i.e., distinct biochemical properties) nonsynonymous mutations from mitochondrial genomes of sexual versus asexual Potamopyrgus antipodarum, a New Zealand freshwater snail characterized by coexisting and ecologically similar sexual and asexual lineages. Our analyses revealed that radical nonsynonymous mutations are cleared at higher rates than conservative changes and that sexual lineages eliminate radical changes more rapidly than asexual counterparts. These results are consistent with reduced efficacy of purifying selection in asexual lineages allowing harmful mutations to remain polymorphic longer than in sexual lineages. Together, these data illuminate some of the population-level processes contributing to mitochondrial mutation accumulation and suggest that mutation accumulation could influence the outcome of competition between sexual and asexual lineages., (© 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.)

The cellular mechanisms of meiosis are critical for proper gamete formation in sexual organisms. Functional studies in model organisms have identified genes essential for meiosis, yet the extent to which this core meiotic machinery is conserved across non-model systems is not fully understood. Moreover, it is unclear whether deviation from canonical modes of sexual reproduction is accompanied by modifications in the genetic components involved in meiosis. We used a robust approach to identify and catalogue meiosis genes in Hymenoptera, an insect order typically characterized by haplodiploid reproduction. Using newly available genome data, we searched for 43 genes involved in meiosis in 18 diverse hymenopterans. Seven of eight genes with roles specific to meiosis were found across a majority of surveyed species, suggesting the preservation of core meiotic machinery in haplodiploid hymenopterans. Phylogenomic analyses of the inventory of meiosis genes and the identification of shared gene duplications and losses provided support for the grouping of species within Proctotrupomorpha, Ichneumonomorpha, and Aculeata clades, along with a paraphyletic Symphyta. The conservation of meiosis genes across Hymenoptera provides a framework for studying transitions between reproductive modes in this insect group., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Reciprocal co-evolving interactions between hosts and parasites are a primary source of strong selection that can promote rapid and often population- or genotype-specific evolutionary change. These host-parasite interactions are also a major source of disease. Despite their importance, very little is known about the genomic basis of co-evolving host-parasite interactions in natural populations, especially in animals. Here, we use gene expression and sequence evolution approaches to take critical steps towards characterizing the genomic basis of interactions between the freshwater snail Potamopyrgus antipodarum and its co-evolving sterilizing trematode parasite, Microphallus sp., a textbook example of natural coevolution. We found that Microphallus-infected P. antipodarum exhibit systematic downregulation of genes relative to uninfected P. antipodarum. The specific genes involved in parasite response differ markedly across lakes, consistent with a scenario where population-level co-evolution is leading to population-specific host-parasite interactions and evolutionary trajectories. We also used an F ST -based approach to identify a set of loci that represent promising candidates for targets of parasite-mediated selection across lakes as well as within each lake population. These results constitute the first genomic evidence for population-specific responses to co-evolving infection in the P. antipodarum-Microphallus interaction and provide new insights into the genomic basis of co-evolutionary interactions in nature., (© 2017 John Wiley & Sons Ltd.)

Background: Sexual reproduction is a widely studied biological process because it is critically important to the genetics, evolution, and ecology of eukaryotes. Despite decades of study on this topic, no comprehensive explanation has been accepted that explains the evolutionary forces underlying its prevalence and persistence in nature. Monogonont rotifers offer a useful system for experimental studies relating to the evolution of sexual reproduction due to their rapid reproductive rate and close relationship to the putatively ancient asexual bdelloid rotifers. However, little is known about the molecular underpinnings of sex in any rotifer species., Results: We generated mRNA-seq libraries for obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of the monogonont rotifer, Brachionus calyciflorus, to identify genes specific to both modes of reproduction. Our differential expression analysis identified receptors with putative roles in signaling pathways responsible for the transition from asexual to sexual reproduction. Differential expression of a specific copy of the duplicated cell cycle regulatory gene CDC20 and specific copies of histone H2A suggest that such duplications may underlie the phenotypic plasticity required for reproductive mode switch in monogononts. We further identified differential expression of genes involved in the formation of resting eggs, a process linked exclusively to sex in this species. Finally, we identified transcripts from the bdelloid rotifer Adineta ricciae that have significant sequence similarity to genes with higher expression in CP strains of B. calyciflorus., Conclusions: Our analysis of global gene expression differences between facultatively sexual and exclusively asexual populations of B. calyciflorus provides insights into the molecular nature of sexual reproduction in rotifers. Furthermore, our results offer insight into the evolution of obligate asexuality in bdelloid rotifers and provide indicators important for the use of monogononts as a model system for investigating the evolution of sexual reproduction.

A long-standing question in evolutionary biology is how sexual reproduction has persisted in eukaryotic lineages. As cyclical parthenogens, monogonont rotifers are a powerful model for examining this question, yet the molecular nature of sexual reproduction in this lineage is currently understudied. To examine genes involved in meiosis, we generated partial genome assemblies for 2 distantly related monogonont species, Brachionus calyciflorus and B. manjavacas. Here we present an inventory of 89 meiotic genes, of which 80 homologs were identified and annotated from these assemblies. Using phylogenetic analysis, we show that several meiotic genes have undergone relatively recent duplication events that appear to be specific to the monogonont lineage. Further, we compare the expression of "meiosis-specific" genes involved in recombination and all annotated copies of the cell cycle regulatory gene CDC20 between obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of B. calyciflorus. We show that "meiosis-specific" genes are expressed in both CP and OP strains, whereas the expression of one of the CDC20 genes is specific to cyclical parthenogenesis. The data presented here provide insights into mechanisms of cyclical parthenogenesis and establish expectations for studies of obligate asexual relatives of monogononts, the bdelloid rotifer lineage.

Understanding the evolution and maintenance of sexual reproduction is one of the central challenges of evolutionary biology, yet we know very little about how sex influences molecular evolution. The New Zealand freshwater snail Potamopyrgus antipodarum is ideally suited to address this knowledge gap because obligately sexual individuals often coexist with multiple independently derived obligately asexual lineages. This unusual situation allows direct comparisons both between sexual and asexual P. antipodarum and across populations that differ in the relative frequency of sexual individuals. As such, P. antipodarum has received a great deal of attention as a model system for the maintenance of sex in nature and is also used as a model for environmental toxicology and biological invasions. Molecular genetic resources for P. antipodarum will thus be useful to investigators in a variety of biological fields. We used 454 sequencing of cDNA libraries to generate transcriptomes from two sexual and two asexual P. antipodarum lineages. A de novo assembly of 116.7 Mb of sequence reads produced 41 396 contigs, and sequence similarity-based Gene Ontology annotations were obtained for 3740 contigs. We detected 408 315 SNP loci and 7315 microsatellite loci, which together represent the first genome-scale resource available for P. antipodarum. Raw 454 read sequences, contig sequences, annotation data and polymorphism data are publicly available in a searchable online database and for download at http://www.biology.uiowa.edu/neiman/transcriptome.php., (© 2012 Blackwell Publishing Ltd.)

Background: Parabasalia are single-celled eukaryotes (protists) that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA), while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms., Principal Findings: Comparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the Trichomonas vaginalis genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1) in T. vaginalis and other eukaryotes. Here we directly sequenced Rpb1 degenerate PCR products from 10 parabasalid genera, including several T. vaginalis isolates and avian isolates, and compared these data by phylogenetic analyses. Rpb1 genes from parabasalids, diplomonads, Parabodo, Diplonema and Percolomonas were all intronless, unlike intron-rich homologs in Naegleria, Jakoba and Malawimonas., Conclusions/significance: The phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive relationships within Parabasalia.

Approximately half of all eukaryotic genes show signs of prokaryotic origin. Genes derived from eubacteria are more abundant than those from archaebacteria, but the latter are functionally more important. This supports archaebacteria as founding ancestors of the eukaryotic nucleus.

Sexual reproduction is both extremely costly and widespread relative to asexual reproduction, meaning that it must also confer profound advantages in order to persist. One theorized benefit of sex is that it facilitates the clearance of harmful mutations, which would accumulate more rapidly in the absence of recombination. The extent to which ineffective purifying selection and mutation accumulation are direct consequences of asexuality and whether the accelerated buildup of harmful mutations in asexuals can occur rapidly enough to maintain sex within natural populations, however, remain as open questions. We addressed key components of these questions by estimating the rate of mutation accumulation in the mitochondrial genomes of multiple sexual and asexual representatives of Potamopyrgus antipodarum, a New Zealand snail characterized by mixed sexual/asexual populations. We found that increased mutation accumulation is associated with asexuality and occurs rapidly enough to be detected in recently derived asexual lineages of P. antipodarum. Our results demonstrate that increased mutation accumulation in asexuals can differentially affect coexisting and ecologically similar sexual and asexual lineages. The accelerated rate of mutation accumulation observed in asexual P. antipodarum provides some of the most direct evidence to date for a link between asexuality and mutation accumulation and implies that mutational buildup could be rapid enough to contribute to the short-term evolutionary mechanisms that favor sexual reproduction.

The parasitoid jewel wasp Nasonia vitripennis reproduces by haplodiploidy (arrhenotokous parthenogenesis). In diploid females, meiosis occurs during oogenesis, but in haploid males spermatogenesis is ameiotic and involves a single equational division. Here we describe the phylogenomic distribution of meiotic genes in N. vitripennis and in 10 additional arthropods. Homologues for 39 meiosis-related genes (including seven meiosis-specific genes) were identified in N. vitripennis. The meiotic genes missing from N. vitripennis are also sporadically absent in other arthropods, suggesting that certain meiotic genes are dispensable for meiosis. Among an additional set of 15 genes thought to be specific for male meiosis in Drosophila, two genes (bol and crl) were identified in N. vitripennis and Apis mellifera (both for which canonical meiosis is absent in males) and in other arthropods. The distribution of meiotic genes across arthropods and the impact of gene duplications and reproductive modes on meiotic gene evolution are discussed.

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

Background: Thousands of parthenogenetic animal species have been described and cytogenetic manifestations of this reproductive mode are well known. However, little is understood about the molecular determinants of parthenogenesis. The Daphnia pulex genome must contain the molecular machinery for different reproductive modes: sexual (both male and female meiosis) and parthenogenetic (which is either cyclical or obligate). This feature makes D. pulex an ideal model to investigate the genetic basis of parthenogenesis and its consequences for gene and genome evolution. Here we describe the inventory of meiotic genes and their expression patterns during meiotic and parthenogenetic reproduction to help address whether parthenogenesis uses existing meiotic and mitotic machinery, or whether novel processes may be involved., Results: We report an inventory of 130 homologs representing over 40 genes encoding proteins with diverse roles in meiotic processes in the genome of D. pulex. Many genes involved in cell cycle regulation and sister chromatid cohesion are characterized by expansions in copy number. In contrast, most genes involved in DNA replication and homologous recombination are present as single copies. Notably, RECQ2 (which suppresses homologous recombination) is present in multiple copies while DMC1 is the only gene in our inventory that is absent in the Daphnia genome. Expression patterns for 44 gene copies were similar during meiosis versus parthenogenesis, although several genes displayed marked differences in expression level in germline and somatic tissues., Conclusion: We propose that expansions in meiotic gene families in D. pulex may be associated with parthenogenesis. Taking into account our findings, we provide a mechanistic model of parthenogenesis, highlighting steps that must differ from meiosis including sister chromatid cohesion and kinetochore attachment.

The predominance of sexual reproduction indicates that it must confer profound benefits, considering its significant costs relative to asexuality. However, definitively determining whether a lineage engages in sex is often complicated by the potential for cryptic sex, especially among unfamiliar organisms. Here we consider the strengths and weaknesses of various molecular- and organismal-based approaches for recognizing signs of sex and describe their applications and relevance to evolutionary biology. We review recent studies that use these methods; some analyses even dispute several 'ancient' asexual taxa, and suggest they are recently derived or might be covertly sexual. More broadly, a better understanding of which organisms have sex and how they do it will deepen our understanding of the distribution, maintenance and evolution of sexual reproduction.

The salmonellae are a diverse group of bacteria within the family Enterobacteriaceae that includes two species, Salmonella enterica and Salmonella bongori. In order to characterize the phylogenetic relationships of the species and subspecies of Salmonella, we analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongori were included in this study. We present a robust phylogeny of the Salmonella species and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of intersubspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.

At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

Sexual reproduction is the dominant reproductive mode in eukaryotes but, in many taxa, it has never been observed. Molecular methods that detect evidence of sex are largely based on the genetic consequences of sexual reproduction. Here we describe a powerful new approach to directly search genomes for genes that function in meiosis. We describe a "meiosis detection toolkit", a set of meiotic genes that represent the best markers for the presence of meiosis. These genes are widely present in eukaryotes, function only in meiosis and can be isolated by degenerate PCR. The presence of most, or all, of these genes in a genome would suggest they have been maintained for meiosis and, implicitly, sexual reproduction. In contrast, their absence would be consistent with the loss of meiosis and asexuality. This approach will help to understand both meiotic gene evolution and the capacity for meiosis and sex in putative obligate asexuals.

Salmonellae are enterobacteria that have the unique ability to change their flagellar composition by switching expression among two loci that encode the major flagellin protein. This property is not available to all Salmonella, but is species, subspecies and serotype specific. Curiously, the subsequent loss of the second locus in some lineages of Salmonella has apparently been tolerated and, indeed, has led to considerable success for some lineages. We discuss here an evolutionary model for maintenance of this unique function and the possible evolutionary advantages of loss or preservation of this mechanism. We hypothesize that the second flagellin locus is a genetic 'spare tyre' used in particular environmental circumstances.

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

Previous analyses of the Giardia genome exposed numerous genes required for meiosis, suggesting that sexual reproduction is occurring in this early-diverging eukaryote. A new study now uncovers direct genetic evidence for recombination in Giardia populations.

Background: Our understanding of the eukaryotic tree of life and the tremendous diversity of microbial eukaryotes is in flux as additional genes and diverse taxa are sampled for molecular analyses. Despite instability in many analyses, there is an increasing trend to classify eukaryotic diversity into six major supergroups: the 'Amoebozoa', 'Chromalveolata', 'Excavata', 'Opisthokonta', 'Plantae', and 'Rhizaria'. Previous molecular analyses have often suffered from either a broad taxon sampling using only single-gene data or have used multigene data with a limited sample of taxa. This study has two major aims: (1) to place taxa represented by 72 sequences, 61 of which have not been characterized previously, onto a well-sampled multigene genealogy, and (2) to evaluate the support for the six putative supergroups using two taxon-rich data sets and a variety of phylogenetic approaches., Results: The inferred trees reveal strong support for many clades that also have defining ultrastructural or molecular characters. In contrast, we find limited to no support for most of the putative supergroups as only the 'Opisthokonta' receive strong support in our analyses. The supergroup 'Amoebozoa' has only moderate support, whereas the 'Chromalveolata', 'Excavata', 'Plantae', and 'Rhizaria' receive very limited or no support., Conclusion: Our analytical approach substantiates the power of increased taxon sampling in placing diverse eukaryotic lineages within well-supported clades. At the same time, this study indicates that the six supergroup hypothesis of higher-level eukaryotic classification is likely premature. The use of a taxon-rich data set with 105 lineages, which still includes only a small fraction of the diversity of microbial eukaryotes, fails to resolve deeper phylogenetic relationships and reveals no support for four of the six proposed supergroups. Our analyses provide a point of departure for future taxon- and gene-rich analyses of the eukaryotic tree of life, which will be critical for resolving their phylogenetic interrelationships.

Spo11 is a meiotic protein of fundamental importance as it is a conserved meiosis-specific transesterase required for meiotic recombination initiation in fungi, animals, and plants. Spo11 is homologous to the archaebacterial topoisomerase VIA (Top6A) gene, and its homologs are broadly distributed among eukaryotes, with some eukaryotes having more than one homolog. However, the evolutionary relationships among these genes are unclear, with some debate as to whether eukaryotic homologs originated by lateral gene transfer. We have identified and characterized protist Spo11 homologs by degenerate polymerase chain reaction (PCR) and sequencing and by analyses of sequences from public databases. Our phylogenetic analyses show that Spo11 homologs evolved by two ancient eukaryotic gene duplication events prior to the last common ancestor of extant eukaryotes, resulting in three eukaryotic paralogs: Spo11-1, Spo11-2, and Spo11-3. Spo11-1 orthologs encode meiosis-specific proteins and are distributed broadly among eukaryotic lineages, though Spo11-1 is absent from some protists. This absence coincides with the presence of Spo11-2 orthologs, which are meiosis-specific in Arabidopsis and are found in plants, red algae, and some protists but absent in animals and fungi. Spo11-3 encodes a Top6A subunit that interacts with topoisomerase VIB (Top6B) subunits, which together play a role in vegetative growth in Arabidopsis. We identified Spo11-3 (Top6A) and Top6B homologs in plants, red algae, and a few protists, establishing a broader distribution of these genes among eukaryotes, indicating their likely vertical descent followed by lineage-specific loss.

Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.

Simian immunodeficiency viruses (SIV) have had considerable success at crossing species barriers; both human immunodeficiency virus (HIV)-1 and HIV-2 have been transmitted on multiple occasions from SIV-infected natural host species. However, the precise evolutionary and ecological mechanisms characterizing a successful cross-species transmission event remain to be elucidated. Here, in addition to expanding and clarifying our previous description of the adaptation of a diverse, naturally occurring SIVsm inoculum to a new rhesus macaque host, we present an analytical framework for understanding the selective forces driving viral adaptation to a new host. A preliminary analysis of large-scale changes in virus population structure revealed that viruses replicating in the macaques were subject to increasing levels of selection through day 70 postinfection (p.i.), whereas contemporaneous viruses in the mangabeys remained similar to the source inoculum. Three different site-by-site methods were employed to identify the amino acid sites responsible for this macaque-specific selection. Of 124 amino acid sites analyzed, 3 codons in V2, a 2-amino acid shift in an N-linked glycosylation site, and variation at 2 sites in the highly charged region were consistently evolving under either directional or diversifying selection at days 40 and 70 p.i. This strong macaque-specific selection on the V2 loop underscores the importance of this region in the adaptation of SIVsm to rhesus macaques. Due to the extreme viral diversity already extant in the naturally occurring viral inoculum, we employed a broad range of phylogenetic and numerical tools in order to distinguish the signatures of past episodes of selection in viral sequences from more recent selection pressures.

Background: Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus)., Results: The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes--mostly encoding metabolic proteins--that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals., Conclusion: Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution.

We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

In Drosophila males, homologous chromosomes segregate by an unusual process involving physical connections not dependent on recombination. We have identified two meiotic proteins specifically required for this process. Stromalin in Meiosis (SNM) is a divergent member of the SCC3/SA/STAG family of cohesin proteins, and Modifier of Mdg4 in Meiosis (MNM) is one of many BTB-domain proteins expressed from the mod(mdg4) locus. SNM and MNM colocalize along with a repetitive rDNA sequence known to function as an X-Y pairing site to nucleolar foci during meiotic prophase and to a compact structure associated with the X-Y bivalent during prometaphase I and metaphase I. Additionally, MNM localizes to autosomal foci throughout meiosis I. These proteins are mutually dependent for their colocalization, and at least MNM requires the function of teflon, another meiotic gene. SNM and MNM do not colocalize with SMC1, suggesting that the homolog conjunction mechanism is independent of cohesin.

Despite the potential for infectious agents harbored by other species to become emerging human pathogens, little is known about why some agents establish successful cross-species transmission, while others do not. The simian immunodeficiency viruses (SIVs), certain variants of which gave rise to the human HIV-1 and HIV-2 epidemics, have demonstrated tremendous success in infecting new host species, both simian and human. SIVsm from sooty mangabeys appears to have infected humans on several occasions, and was readily transmitted to nonnatural Asian macaque species, providing animal models of AIDS. Here we describe the first in-depth analysis of the tremendous SIVsm quasispecies sequence variation harbored by individual sooty mangabeys, and how this diverse quasispecies adapts to two different host species-new nonnatural rhesus macaque hosts and natural sooty mangabey hosts. Viral adaptation to rhesus macaques was associated with the immediate amplification of a phylogenetically related subset of envelope (env) variants. These variants contained a shorter variable region 1 loop and lacked two specific glycosylation sites, which may be selected for during acute infection. In contrast, transfer of SIVsm to its natural host did not subject the quasispecies to any significant selective pressures or bottleneck. After 100 d postinfection, variants more closely representative of the source inoculum reemerged in the macaques. This study describes an approach for elucidating how pathogens adapt to new host species, and highlights the particular importance of SIVsm env diversity in enabling cross-species transmission. The replicative advantage of a subset of SIVsm variants in macaques may be related to features of target cells or receptors that are specific to the new host environment, and may involve CD4-independent engagement of a viral coreceptor conserved among primates.

According to current thinking, a parasite's transmission mode will be a major determinant of virulence, defined as the harm induced by parasites to their hosts. With horizontal transmission, virulence will increase as a byproduct of a trade-off between fitness gained through increased among-host transmission (infectivity) and fitness lost through increased virulence. With vertical transmission, virulence will decrease because a parasite's reproductive potential will be maximized only by decreasing harm to the host, allowing parasite transmission to more host offspring. To test both predictions, we transmitted barley stripe mosaic virus (BSMV) horizontally and then vertically in its host, barley (Hordeum vulgare). After four generations of horizontal transmission, we observed a nearly twofold increase in horizontal infectivity and nearly tripled virulence. After three generations of subsequent vertical transmission, we observed a modest (16%) increase in vertical transmissibility and a large (40%) reduction in virulence. Increased horizontal transmission is often due to increased pathogen replication which, in turn, causes increased virulence. However, we found no correlation between within-host virus concentration and virulence, indicating that the observed changes in virulence were not due to changes in viral titer. Finally, horizontally transmitted BSMV had reduced vertical transmission and vertically transmitted BSMV had reduced horizontal infectivity. These two observations suggest that, in nature, in different host populations with varying opportunities for horizontal and vertical transmission, different viral strains may be favored.

Background: The ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) proteins are a family of metalloproteases with sequence similarity to the ADAM proteases, that contain the thrombospondin type 1 sequence repeat motifs (TSRs) common to extracellular matrix proteins. ADAMTS proteins have recently gained attention with the discovery of their role in a variety of diseases, including tissue and blood disorders, cancer, osteoarthritis, Alzheimer's and the genetic syndromes Weill-Marchesani syndrome (ADAMTS10), thrombotic thrombocytopenic purpura (ADAMTS13), and Ehlers-Danlos syndrome type VIIC (ADAMTS2) in humans and belted white-spotting mutation in mice (ADAMTS20)., Results: Phylogenetic analysis and comparison of the exon/intron organization of vertebrate (Homo, Mus, Fugu), chordate (Ciona) and invertebrate (Drosophila and Caenorhabditis) ADAMTS homologs has elucidated the evolutionary relationships of this important gene family, which comprises 19 members in humans., Conclusions: The evolutionary history of ADAMTS genes in vertebrate genomes has been marked by rampant gene duplication, including a retrotransposition that gave rise to a distinct ADAMTS subfamily (ADAMTS1, -4, -5, -8, -15) that may have distinct aggrecanase and angiogenesis functions.

Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.

ADP-ribosylation factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The approximately 100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). Injection of double-stranded RNA (dsRNA) encoding Arf-1 or Arf-3 into N2 hermaphrodites produced embryonic lethality in their offspring and, later, sterility in the injected animals themselves. Injection of Arl-2 dsRNA resulted in a disorganized germline and sterility in early offspring, with later offspring exhibiting an early embryonic arrest. Thus, of the six Arf family members examined in C. elegans, at least three are required for embryogenesis. These data represent the first analysis of the role(s) of multiple members of this family in the development of a multicellular organism.

The commercial space sector to date has failed to develop comprehensive regulations--"rules of the road"--for its international activities. Within the next 5 years, conflicts with respect to international trade in satellite sales and launch services could emerge, highlighting the need for such a regulatory framework. If the commercial space sector is to continue to develop, it is important to begin discussions now, before these conflicts become significant, on the elements of an appropriate international regulatory framework. The existing framework for space activities was developed when government, not commercial, space activities were dominant, or was adapted from regulations in other sectors such as terrestrial telecommunications., (c2003 Elsevier Ltd. All rights reserved.)

We have identified three novel members of the zebrafish forkhead class I gene family, which we have named foxi2, foxi3a, and foxi3b. We have reported previously the identification of zebrafish foxi1, which is required for otic placode and jaw development. Expression analysis shows that foxi2 is expressed within the chordamesoderm during early somitogenesis and the retina and pharyngeal arches during later stages. The foxi3a and foxi3b genes likely represent a recently duplicated pair, and they are similarly expressed in epidermal mucous cells throughout embryogenesis and early larval stages. None of these newly identified FoxI genes are expressed in otic precursor cells and, therefore, are unlikely to share functional overlap with foxi1 in the development of the inner ear. In addition to these zebrafish FoxI paralogs, we have identified 16 new FoxI sequences in species ranging from Ciona intestinalis to Homo sapiens. We present an extensive phylogenetic analysis of the FoxI class that includes these new sequences together with those previously reported. This analysis supports the existence of three subfamilies within the FoxI class, each containing at least one zebrafish member., (Copyright 2003 Wiley-Liss, Inc.)

When the International Human Genome Sequencing Consortium (IHGSC) published its draft of the human genome in February 2001, several genes were identified as possible bacteria-to-vertebrate transfers (BVTs). These genes were identified by their highly significant sequence similarity to bacterial genes in BLAST searches, and by their lack of matches among non-vertebrate eukaryote genes. Many were later rejected as BVTs by several methods, including recovery of probable orthologs from the genomes of incompletely sequenced eukaryotes. Whereas the BVT issue has received considerable attention, there has been no compilation of all potential BVTs considered to date, nor any proposal of a single comprehensive method for rigorously establishing the veracity of a putative BVT. In reviewing the work to date, we list all of the proteins examined and propose systematic tests to investigate whether a vertebrate gene proposed as a BVT is indeed of bacterial origin. We use the proposed strategy to test--and reject--one of the BVTs from the original IHGSC list.

The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant obstacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Polymerase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Ochromonas danica (a heterokont); we have also analyzed the RPB1 gene from the nucleomorph (nm) genome of Guillardia theta (a cryptomonad). Using a variety of phylogenetic methods our analysis shows that RPB1s from Giardia intestinalis and Trichomonas vaginalis are probably subject to intense LBA effects. Thus, the deep branching of these taxa on RPB1 trees is questionable and should not be interpreted as evidence favoring their early divergence. Similar effects are discernable, to a lesser extent, with the Mastigamoeba invertens RPB1 sequence. Upon removal of the outgroup and these problematic sequences, analyses of the remaining RPB1s indicate some resolution among major eukaryotic groups. The most robustly supported higher-level clades are the opisthokonts (animals plus fungi) and the red algae plus the cryptomonad nm-the latter result gives added support to the red algal origin of cryptomonad chloroplasts. Clades comprising Dictyostelium discoideum plus Acanthamoeba castellanii (Amoebozoa) and Ochromonas plus Plasmodium falciparum (chromalveolates) are consistently observed and moderately supported. The clades supported by our RPB1 analyses are congruent with other data, suggesting that bona fide phylogenetic relationships are being resolved. Thus, the RPB1 gene has apparently retained some phylogenetically meaningful signal, making it worthwhile to obtain sequences from more diverse protist taxa. Additional RPB1 data, especially in combination with other genes, should provide further resolution of branching orders among protist groups within the apparently rapid early divergence of eukaryotes.

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.