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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 â¼1.7â 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.
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Genoma del Cloroplasto , Genoma Mitocondrial , Animales , Análisis de Secuencia de ADN , Recombinación Genética , Cloroplastos , FilogeniaRESUMEN
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.
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Genoma , Histonas , Animales , Genómica , Histonas/genética , Humanos , Reproducción Asexuada/genética , Caracoles/genéticaRESUMEN
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.
RESUMEN
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.
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Ascomicetos/genética , Chlorophyta/genética , Líquenes/genética , Simbiosis/genética , Transferencia de Gen Horizontal , Genoma FúngicoRESUMEN
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 FST -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.
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Coevolución Biológica , Genética de Población , Interacciones Huésped-Parásitos/genética , Caracoles/genética , Caracoles/parasitología , Trematodos/patogenicidad , Animales , Nueva ZelandaRESUMEN
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.
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Genes de Insecto , Himenópteros/genética , Meiosis/genética , Animales , Evolución Molecular , Duplicación de Gen , FilogeniaRESUMEN
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.
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Células Eucariotas/clasificación , Células Eucariotas/metabolismo , Parásitos/clasificación , Parásitos/citología , Fotosíntesis , Filogenia , Plastidios/metabolismo , Animales , Núcleo Celular/genética , Clorofila/metabolismo , Clorofila A , Codón/genética , Células Eucariotas/citología , Células Eucariotas/ultraestructura , Parásitos/genética , Parásitos/ultraestructura , Plasmodium falciparum/clasificación , Plastidios/genética , ARN Ribosómico/genéticaRESUMEN
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.
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Filogenia , Caracoles , Animales , Caracoles/genética , Nueva Zelanda , Introgresión Genética , Evolución Molecular , Genoma Mitocondrial , GenomaRESUMEN
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.
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Perfilación de la Expresión Génica , Óvulo/fisiología , Reproducción Asexuada/genética , Rotíferos/genética , Rotíferos/fisiología , Animales , Proteínas de Ciclo Celular/genética , Citoesqueleto/genética , Elementos Transponibles de ADN/genética , Gametogénesis/genética , Histonas/genética , Meiosis/genética , Recombinación Genética/genética , Rotíferos/citología , Rotíferos/metabolismoRESUMEN
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.
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Meiosis/genética , Partenogénesis/genética , Filogenia , Rotíferos/genética , Animales , Proteínas de Ciclo Celular/genética , Cromosomas/genética , Replicación del ADN , Etiquetas de Secuencia Expresada , Regulación de la Expresión GénicaAsunto(s)
United States National Aeronautics and Space Administration/tendencias , Astronautas/tendencias , Marte , Planetas Menores , Investigación/economía , Investigación/tendencias , Robótica/economía , Robótica/tendencias , Vuelo Espacial/economía , Vuelo Espacial/tendencias , Nave Espacial/economía , Estados Unidos , United States National Aeronautics and Space Administration/economíaRESUMEN
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.
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Giardia/genética , Recombinación Genética , Animales , Evolución Biológica , Giardia/fisiología , Meiosis/genética , Reproducción/genéticaRESUMEN
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.
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Mutación , Caracoles/genética , Animales , ADN Mitocondrial/genética , Filogenia , Reproducción Asexuada , Caracoles/fisiologíaAsunto(s)
Daphnia/genética , Evolución Molecular , Genoma/genética , Reproducción Asexuada/genética , AnimalesRESUMEN
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.
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Evolución Biológica , Meiosis/genética , Reproducción/genética , Alelos , Animales , Femenino , Hongos/genética , Técnicas Genéticas , Heterocigoto , Humanos , Invertebrados/genética , Masculino , Modelos Genéticos , Filogenia , Plantas/genética , Reproducción Asexuada/genética , Selección GenéticaRESUMEN
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.
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Evolución Biológica , Flagelina/genética , Variación Genética , Salmonella/clasificación , Animales , Eucariontes/microbiología , Eucariontes/fisiología , Regulación Bacteriana de la Expresión Génica , Humanos , Modelos Genéticos , Conducta Predatoria , Salmonella/genética , Salmonella/metabolismo , Salmonella enterica/clasificación , Salmonella enterica/genética , Salmonella enterica/metabolismo , SerotipificaciónRESUMEN
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.
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Daphnia/genética , Evolución Molecular , Genoma/genética , Meiosis/genética , Partenogénesis/genética , Animales , Proteínas de Ciclo Celular/genética , Reparación de la Incompatibilidad de ADN/genética , Drosophila/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Filogenia , Recombinación Genética/genéticaRESUMEN
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.
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Insectos/genética , Insectos/fisiología , Reproducción Asexuada/genética , Animales , Femenino , Hibridación Genética , Masculino , Partenogénesis , SimbiosisRESUMEN
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 â¼400 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.
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Genoma de los Insectos , Avispas/genética , Animales , Femenino , Genes de Insecto , Especiación Genética , Himenópteros/genética , Masculino , Modelos Biológicos , Reproducción Asexuada/genética , Procesos de Determinación del SexoRESUMEN
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.