Three New Freshwater Cochliopodium Species (Himatismenida, Amoebozoa) from the Southeastern United States.

Cochliopodium is a lens-shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often-used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum-likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, "crystal UK-YT2" (Cochliopodium crystalli n. sp.) and "crystal-like UK-YT3" (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical-shaped crystals. The "Marrs Spring UK-YT4" isolate, C. marrii n. sp., was 100% identical to "Cochliopodium sp. SG-2014 KJ569724." These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.

A practical implementation of large transcriptomic data analysis to resolve cryptic species diversity problems in microbial eukaryotes.

BACKGROUND: Transcriptome sequencing has become a method of choice for evolutionary studies in microbial eukaryotes due to low cost and minimal sample requirements. Transcriptome data has been extensively used in phylogenomic studies to infer ancient evolutionary histories. However, its utility in studying cryptic species diversity is not well explored. An empirical investigation was conducted to test the applicability of transcriptome data in resolving two major types of discordances at lower taxonomic levels. These include cases where species have the same morphology but different genetics (cryptic species) and species of different morphologies but have the same genetics. We built a species comparison bioinformatic pipeline that takes into account the nature of transcriptome data in amoeboid microbes exemplifying such discordances. RESULT: Our analyses of known or suspected cryptic species yielded consistent results regardless of the methods of culturing, RNA collection or sequencing. Over 95% of the single copy genes analyzed in samples of the same species sequenced using different methods and cryptic species had intra- and interspecific divergences below 2%. Only a minority of groups (2.91-4.87%) had high distances exceeding 2% in these taxa, which was likely caused by low data quality. This pattern was also observed in suspected genetically similar species with different morphologies. Transcriptome data consistently delineated all taxa above species level, including cryptically diverse species. Using our approach we were able to resolve cryptic species problems, uncover misidentification and discover new species. We also identified several potential barcode markers with varying evolutionary rates that can be used in lineages with different evolutionary histories. CONCLUSION: Our findings demonstrate that transcriptome data is appropriate for understanding cryptic species diversity in microbial eukaryotes.

Longamoebia is not monophyletic: Phylogenomic and cytoskeleton analyses provide novel and well-resolved relationships of amoebozoan subclades.

Longamoebia is one of the most morphologically diverse member of Amoebozoa. It includes the human pathogen Acanthamoeba, which causes minor skin and serious eye infections as well as fatal central nervous system complications. The taxonomy and phylogeny of Longamoebia is poorly understood partly due to the growing number of molecular studies that report unsuspected affiliations of lineages with extremely different morphotypes in the group. A recent molecular study questioned the monophyly of Longamoebia. In this study, we conducted a more comprehensive phylogenomic analysis including all of putative members of Longamoebia to assess its monophyly. We conducted extensive analyses to see effects of outgroup choice, missing data, and gene and taxon sampling on resulting phylogenies. We also collected morphological characters derived from the cytoskeleton using immunocytochemistry to assess homologies of pseudopodia at a finer scale. Our phylogenomic analysis yielded a well-resolved tree of Amoebozoa and highly supported novel relationships. Discosea is recovered as a monophyletic group with all of its known taxonomic orders. However, its within-group relationships dramatically differed from those originally proposed. Our study strongly demonstrates that Longamoebia sensu Smirnov et al. (2011) is not monophyletic and an invalid taxon. Thecamoebida forms a strongly supported sister group relationship with clade Flabellinea (Dactylopodida and Vannellida), while Dermamoebida (Mayorella+Dermamoeba) form an independent branch basal to other members of Discosea. The remaining groups including members of Centramoebida form a consistently well-supported clade that was shown to form a sister group relationship with Himatismenida. This robust clade shares the unique cytoskeletal features of coiled cytoplasmic microtubule network and F-actin characters. Our analyses demonstrated that placement of unstable taxa in large-scale analysis with varying levels of missing data might be compromised by some confounding factors such as outgroup choice and gene and taxon sampling.

Genetic Evidence for Sexuality in Cochliopodium (Amoebozoa).

Microbial eukaryotes, including amoeboids, display diverse and complex life cycles that may or may not involve sexual reproduction. A recent comprehensive gene inventory study concluded that the Amoebozoa are ancestrally sexual. However, the detection of sex genes in some lineages known for their potentially sexual life cycle was very low. Particularly, the genus Cochliopodium, known to undergo a process of cell fusion, karyogamy, and subsequent fission previously described as parasexual, had no meiosis genes detected. This is likely due to low data representation, given the extensive nuclear fusion observed in the genus. In this study, we generate large amounts of transcriptome data for 2 species of Cochliopodium, known for their high frequency of cellular and nuclear fusion, in order to study the genetic basis of the complex life cycle observed in the genus. We inventory 60 sex-related genes, including 11 meiosis-specific genes, and 31 genes involved in fusion and karyogamy. We find a much higher detection of sex-related genes, including 5 meiosis-specific genes not previously detected in Cochliopodium, in this large transcriptome data. The expressed genes form a near-complete recombination machinery, indicating that Cochliopodium is an actively recombining sexual lineage. We also find 9 fusion-related genes in Cochliopodium, although no conserved fusion-specific genes were detected in the transcriptomes. Cochliopodium thus likely uses lineage specific genes for the fusion and depolyploidization processes. Our results demonstrate that Cochliopodium possess the genetic toolkit for recombination, while the mechanism involving fusion and genome reduction remains to be elucidated.

Phylogenomics of 'Discosea': A new molecular phylogenetic perspective on Amoebozoa with flat body forms.

The majority of amoeboid lineages with flattened body forms are placed under a taxonomic hypothetical class 'Discosea' sensu Smirnov et al. (2011), which encompasses some of the most diverse morphs within Amoebozoa. However, its taxonomy and phylogeny is poorly understood. This is partly due to lack of support in studies that are based on limited gene sampling. In this study we use a phylogenomic approach including newly-generated RNA-Seq data and comprehensive taxon sampling to resolve the phylogeny of 'Discosea'. Our analysis included representatives from all orders of 'Discosea' and up to 550 genes, the largest gene sampling in Amoebozoa to date. We conducted extensive analyses to assess the robustness of our resulting phylogenies to effects of missing data and outgroup choice using probabilistic methods. All of our analyses, which explore the impact of varying amounts of missing data, consistently recover well-resolved and supported groups of Amoebozoa. Our results neither support the monophyly nor dichotomy of 'Discosea' as defined by Smirnov et al. (2011). Rather, we recover a robust well-resolved clade referred to as Eudiscosea encompassing the majority of discosean orders (seven of the nine studied here), while the Dactylopodida, Thecamoebida and Himatismenida, previously included in 'Discosea,' are non-monophyletic. We also recover novel relationships within the Eudiscosea that are largely congruent with morphology. Our analyses enabled us to place some incertae sedis lineages and previously unstable lineages such as Vermistella, Mayorella, Gocevia, and Stereomyxa. We recommend some phylogeny-based taxonomic amendments highlighting the new findings of this study and discuss the evolution of the group based on our current understanding.

Cochliopodium arabianum n. sp. (Amorphea, Amoebozoa).

A new species of Cochliopodium isolated from freshwater at Arabia Lake in Lithonia, GA, USA is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium arabianum n. sp., previously labeled as "isolate Con1" in prior publications, has been shown to group within the genus Cochliopodium in our molecular phylogenetic analysis. Light microscopy and fine structure evidence indicates the new isolate not only shares characters of the genus but also unique distinctive features. Cochliopodium arabianum n. sp. is typically round when stationary; or oval to sometimes broadly flabellate or triangular in shape during locomotion, with average length of 35 µm and breadth of 51 µm. Fine structure evidence indicates C. arabianum n. sp. has tower-like scales, lacking a terminal spine, sharing high similarity with its closest relative C. actinophorum. However, the scales of C. arabianum n. sp. are unique in height and the breadth of the base plate. Both morphological and molecular data, including SSU-rDNA and COI, indicate that this new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.

A Small Genome amidst the Giants: Evidence of Genome Reduction in a Small Tubulinid Free-Living Amoeba.

This study investigates the genomic characteristics of Echinamoeba silvestris, a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce. E. silvestris presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in E. silvestris shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. Additionally, our investigation into noncoding and repetitive elements sheds light on the evolution of genome size in amoebae, with E. silvestris distinguished by low percentage of repetitive elements. Furthermore, the analysis reveals that E. silvestris has the lowest mean number of introns per gene among the species studied, providing further support for its observed compact genome. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions E. silvestris as a valuable addition to genomic data sets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.

Phylogenetic signal and noise: predicting the power of a data set to resolve phylogeny.

A principal objective for phylogenetic experimental design is to predict the power of a data set to resolve nodes in a phylogenetic tree. However, proactively assessing the potential for phylogenetic noise compared with signal in a candidate data set has been a formidable challenge. Understanding the impact of collection of additional sequence data to resolve recalcitrant internodes at diverse historical times will facilitate increasingly accurate and cost-effective phylogenetic research. Here, we derive theory based on the fundamental unit of the phylogenetic tree, the quartet, that applies estimates of the state space and the rates of evolution of characters in a data set to predict phylogenetic signal and phylogenetic noise and therefore to predict the power to resolve internodes. We develop and implement a Monte Carlo approach to estimating power to resolve as well as deriving a nearly equivalent faster deterministic calculation. These approaches are applied to describe the distribution of potential signal, polytomy, or noise for two example data sets, one recent (cytochrome c oxidase I and 28S ribosomal rRNA sequences from Diplazontinae parasitoid wasps) and one deep (eight nuclear genes and a phylogenomic sequence for diverse microbial eukaryotes including Stramenopiles, Alveolata, and Rhizaria). The predicted power of resolution for the loci analyzed is consistent with the historic use of the genes in phylogenetics.

A new freshwater amoeba: Cochliopodium pentatrifurcatum n. sp. (Amoebozoa, Amorphea).

Cochliopodium pentatrifurcatum n. sp. (ATCC(©) 30935(TM) ) is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium pentatrifurcatum n. sp. (length ~ 25 µm) is characterized by surface microscales (0.3 µm tall) containing a circular porous base (~ 0.6 µm diam.) with a thin peripheral rim. Five radially arranged feet, emanating from the base, support a short central column terminating apically as a funnel-shaped collar (~ 0.5 µm diam.) composed of five radial, trifurcate rays extending from the center toward a thin peripheral rim. The central spine is 0.5-0.6 µm long. The comparative morphologies and combined molecular genetic evidence, SSU-rDNA and COI, indicate that the new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.

A Small Genome Amidst the Giants: Evidence of Genome Reduction in a Small Tubulinid Free-Living Amoeba.

This study investigates the genomic characteristics of Echinamoeba silvestris , a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce . E. silvestris presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in E. silvestris shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. The investigation delves into genomic structural evidence, including gene content and repetitive elements, illuminating the distinctive genomic traits of E. silvestris and providing reinforcement for its compact genome size. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions E. silvestris as a valuable addition to genomic datasets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.

Omics of an Enigmatic Marine Amoeba Uncovers Unprecedented Gene Trafficking from Giant Viruses and Provides Insights into Its Complex Life Cycle.

Amoebozoa include lineages of diverse ecology, behavior, and morphology. They are assumed to encompass members with the largest genome sizes of all living things, yet genomic studies in the group are limited. Trichosphaerium, a polymorphic, multinucleate, marine amoeba with a complicated life cycle, has puzzled experts for over a century. In an effort to explore the genomic diversity and investigate extraordinary behavior observed among the Amoebozoa, we used integrated omics approaches to study this enigmatic marine amoeba. Omics data, including single-cell transcriptomics and cytological data, demonstrate that Trichosphaerium sp. possesses the complete meiosis toolkit genes. These genes are expressed in life stages of the amoeba including medium and large cells. The life cycle of Trichosphaerium sp. involves asexual processes via binary fission and multiple fragmentation of giant cells, as well as sexual-like processes involving genes implicated in sexual reproduction and polyploidization. These findings are in stark contrast to a life cycle previously reported for this amoeba. Despite the extreme morphological plasticity observed in Trichosphaerium, our genomic data showed that populations maintain a species-level intragenomic variation. A draft genome of Trichosphaerium indicates elevated lateral gene transfer (LGT) from bacteria and giant viruses. Gene trafficking in Trichosphaerium is the highest within Amoebozoa and among the highest in microbial eukaryotes.

Variation of natural selection in the Amoebozoa reveals heterogeneity across the phylogeny and adaptive evolution in diverse lineages.

The evolution and diversity of the supergroup Amoebozoa is complex and poorly understood. The supergroup encompasses predominantly amoeboid lineages characterized by extreme diversity in phenotype, behavior and genetics. The study of natural selection, a driving force of diversification, within and among species of Amoebozoa will play a crucial role in understanding the evolution of the supergroup. In this study, we searched for traces of natural selection based on a set of highly conserved protein-coding genes in a phylogenetic framework from a broad sampling of amoebozoans. Using these genes, we estimated substitution rates and inferred patterns of selective pressure in lineages and sites with various models. We also examined the effect of selective pressure on codon usage bias and potential correlations with observed biological traits and habitat. Results showed large heterogeneity of selection across lineages of Amoebozoa, indicating potential species-specific optimization of adaptation to their diverse ecological environment. Overall, lineages in Tubulinea had undergone stronger purifying selection with higher average substitution rates compared to Discosea and Evosea. Evidence of adaptive evolution was observed in some representative lineages and in a gene (Rpl7a) within Evosea, suggesting potential innovation and beneficial mutations in these lineages. Our results revealed that members of the fast-evolving lineages, Entamoeba and Cutosea, all underwent strong purifying selection but had distinct patterns of codon usage bias. For the first time, this study revealed an overall pattern of natural selection across the phylogeny of Amoebozoa and provided significant implications on their distinctive evolutionary processes.

The draft genome of Cochliopodium minus reveals a complete meiosis toolkit and provides insight into the evolution of sexual mechanisms in Amoebozoa.

To date, genomic analyses in amoebozoans have been mostly limited to model organisms or medically important lineages. Consequently, the vast diversity of Amoebozoa genomes remain unexplored. A draft genome of Cochliopodium minus, an amoeba characterized by extensive cellular and nuclear fusions, is presented. C. minus has been a subject of recent investigation for its unusual sexual behavior. Cochliopodium's sexual activity occurs during vegetative stage making it an ideal model for studying sexual development, which is sorely lacking in the group. Here we generate a C. minus draft genome assembly. From this genome, we detect a substantial number of lateral gene transfer (LGT) instances from bacteria (15%), archaea (0.9%) and viruses (0.7%) the majority of which are detected in our transcriptome data. We identify the complete meiosis toolkit genes in the C. minus genome, as well as the absence of several key genes involved in plasmogamy and karyogamy. Comparative genomics of amoebozoans reveals variation in sexual mechanism exist in the group. Similar to complex eukaryotes, C. minus (some amoebae) possesses Tyrosine kinases and duplicate copies of SPO11. We report a first example of alternative splicing in a key meiosis gene and draw important insights on molecular mechanism of sex in C. minus using genomic and transcriptomic data.

New insights on the evolutionary relationships between the major lineages of Amoebozoa.

The supergroup Amoebozoa unites a wide diversity of amoeboid organisms and encompasses enigmatic lineages that have been recalcitrant to modern phylogenetics. Deep divergences, taxonomic placement of some key taxa and character evolution in the group largely remain poorly elucidated or controversial. We surveyed available Amoebozoa genomes and transcriptomes to mine conserved putative single copy genes, which were used to enrich gene sampling and generate the largest supermatrix in the group to date; encompassing 824 genes, including gene sequences not previously analyzed. We recovered a well-resolved and supported tree of Amoebozoa, revealing novel deep level relationships and resolving placement of enigmatic lineages congruent with morphological data. In our analysis the deepest branching group is Tubulinea. A recent proposed major clade Tevosa, uniting Evosea and Tubulinea, is not supported. Based on the new phylogenetic tree, paleoecological and paleontological data as well as data on the biology of presently living amoebozoans, we hypothesize that the evolution of Amoebozoa probably was driven by adaptive responses to a changing environment, where successful survival and predation resulted from a capacity to disrupt and graze on microbial mats-a dominant ecosystem of the mid-Proterozoic period of the Earth history.

Broadly sampled multigene analyses yield a well-resolved eukaryotic tree of life.

An accurate reconstruction of the eukaryotic tree of life is essential to identify the innovations underlying the diversity of microbial and macroscopic (e.g., plants and animals) eukaryotes. Previous work has divided eukaryotic diversity into a small number of high-level "supergroups," many of which receive strong support in phylogenomic analyses. However, the abundance of data in phylogenomic analyses can lead to highly supported but incorrect relationships due to systematic phylogenetic error. Furthermore, the paucity of major eukaryotic lineages (19 or fewer) included in these genomic studies may exaggerate systematic error and reduce power to evaluate hypotheses. Here, we use a taxon-rich strategy to assess eukaryotic relationships. We show that analyses emphasizing broad taxonomic sampling (up to 451 taxa representing 72 major lineages) combined with a moderate number of genes yield a well-resolved eukaryotic tree of life. The consistency across analyses with varying numbers of taxa (88-451) and levels of missing data (17-69%) supports the accuracy of the resulting topologies. The resulting stable topology emerges without the removal of rapidly evolving genes or taxa, a practice common to phylogenomic analyses. Several major groups are stable and strongly supported in these analyses (e.g., SAR, Rhizaria, Excavata), whereas the proposed supergroup "Chromalveolata" is rejected. Furthermore, extensive instability among photosynthetic lineages suggests the presence of systematic biases including endosymbiotic gene transfer from symbiont (nucleus or plastid) to host. Our analyses demonstrate that stable topologies of ancient evolutionary relationships can be achieved with broad taxonomic sampling and a moderate number of genes. Finally, taxon-rich analyses such as presented here provide a method for testing the accuracy of relationships that receive high bootstrap support (BS) in phylogenomic analyses and enable placement of the multitude of lineages that lack genome scale data.

Comprehensive comparative genomics reveals over 50 phyla of free-living and pathogenic bacteria are associated with diverse members of the amoebozoa.

The Amoebozoa, a group containing predominantly amoeboid unicellular protists has been shown to play an important ecological role in controlling environmental bacteria. Amoebozoans not only graze bacteria but also serve as a safe niche for bacterial replication and harbor endosymbiotic bacteria including dangerous human pathogens. Despite their importance, only a few lineages of Amoebozoa have been studied in this regard. In this research, we conducted a comprehensive genomic and transcriptomic study with expansive taxon sampling by including representatives from the three known clades of the Amoebozoa. We used culture independent whole culture and single cell genomics/transcriptomics to investigate the association of bacteria with diverse amoebozoans. Relative to current published evidence, we recovered the largest number of bacterial phyla (64) and human pathogen genera (51) associated with the Amoebozoa. Using single cell genomics/transcriptomics we were able to determine up to 24 potential endosymbiotic bacterial phyla, some potentially endosymbionts. This includes the majority of multi-drug resistant pathogens designated as major public health threats. Our study demonstrates amoebozoans are associated with many more phylogenetically diverse bacterial phyla than previously recognized. It also shows that all amoebozoans are capable of harboring far more dangerous human pathogens than presently documented, making them of primal public health concern.

Identification of new molecular markers for assembling the eukaryotic tree of life.

Six eukaryotic supergroups have been proposed based on both morphological and molecular data. However, some of these supergroups are contentious and the deep relationships among them are poorly resolved. This is due to a limited number of morphological characters and few molecular markers in current use. The lack of resolution in most multigene analyses, including phylogenomic analyses, necessitates a search for additional, appropriate molecular markers to enable targeted sampling of taxa in key phylogenetic positions. We evaluated the phylogenetic signal of 860 proteins obtained from the Clusters of Orthologous Groups of proteins (COGs) database. We report a total of 17 markers that resulted in well-resolved topologies that are congruent with well-established components of the eukaryotic tree. To establish their utility, we designed universal degenerate primers for six markers, some of which showed promising results in unicellular eukaryotes. Finally, we present phylogenetic informativeness profiles for seven selected markers, revealing that the markers contain phylogenetic signal that spans the whole tree including the deeper branches.

A description of a new "Amoebozoan" isolated from the American lobster, Homarus americanus.

Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus. During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba-like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata, this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."

Differential gene expression analysis and cytological evidence reveal a sexual stage of an amoeba with multiparental cellular and nuclear fusion.

Sex is a hallmark of eukaryotes but its evolution in microbial eukaryotes is poorly elucidated. Recent genomic studies revealed microbial eukaryotes possess a genetic toolkit necessary for sexual reproduction. However, the mechanism of sexual development in a majority of microbial eukaryotes including amoebozoans is poorly characterized. The major hurdle in studying sex in microbial eukaryotes is a lack of observational evidence, primarily due to its cryptic nature. In this study, we used a tractable fusing amoeba, Cochliopodium, to investigate sexual development using stage-specific Differential Gene Expression (DGE) and cytological analyses. Both DGE and cytological results showed that most of the meiosis and sex-related genes are upregulated in Cochliopodium undergoing fusion in laboratory culture. Relative gene ontology (GO) category representations in unfused and fused cells revealed a functional skew of the fused transcriptome toward DNA metabolism, nucleus and ligases that are suggestive of a commitment to sexual development. However, the GO categories of unfused cells were dominated by metabolic pathways and other processes indicative of a vegetative phase. Our study provides strong evidence that the fused cells represent a sexual stage in Cochliopodium. Our findings have further implications in understanding the evolution and mechanism of inheritance involving multiparents in other eukaryotes with a similar reproductive strategy.

Molecular Data are Transforming Hypotheses on the Origin and Diversification of Eukaryotes.

The explosion of molecular data has transformed hypotheses on both the origin of eukaryotes and the structure of the eukaryotic tree of life. Early ideas about the evolution of eukaryotes arose through analyses of morphology by light microscopy and later electron microscopy. Though such studies have proven powerful at resolving more recent events, theories on origins and diversification of eukaryotic life have been substantially revised in light of analyses of molecular data including gene and, increasingly, whole genome sequences. By combining these approaches, progress has been made in elucidating both the origin and diversification of eukaryotes. Yet many aspects of the evolution of eukaryotic life remain to be illuminated.