A practical assembly guideline for genomes with various levels of heterozygosity.

Although current long-read sequencing technologies have a long-read length that facilitates assembly for genome reconstruction, they have high sequence errors. While various assemblers with different perspectives have been developed, no systematic evaluation of assemblers with long reads for diploid genomes with varying heterozygosity has been performed. Here, we evaluated a series of processes, including the estimation of genome characteristics such as genome size and heterozygosity, de novo assembly, polishing, and removal of allelic contigs, using six genomes with various heterozygosity levels. We evaluated five long-read-only assemblers (Canu, Flye, miniasm, NextDenovo and Redbean) and five hybrid assemblers that combine short and long reads (HASLR, MaSuRCA, Platanus-allee, SPAdes and WENGAN) and proposed a concrete guideline for the construction of haplotype representation according to the degree of heterozygosity, followed by polishing and purging haplotigs, using stable and high-performance assemblers: Redbean, Flye and MaSuRCA.

Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis.

Gene loss, pseudogenization, and independent genome reduction in non-photosynthetic species of Cryptomonas (Cryptophyceae) revealed by comparative nucleomorph genomics.

BACKGROUND: Cryptophytes are ecologically important algae of interest to evolutionary cell biologists because of the convoluted history of their plastids and nucleomorphs, which are derived from red algal secondary endosymbionts. To better understand the evolution of the cryptophyte nucleomorph, we sequenced nucleomorph genomes from two photosynthetic and two non-photosynthetic species in the genus Cryptomonas. We performed a comparative analysis of these four genomes and the previously published genome of the non-photosynthetic species Cryptomonas paramecium CCAP977/2a. RESULTS: All five nucleomorph genomes are similar in terms of their general architecture, gene content, and gene order and, in the non-photosynthetic strains, loss of photosynthesis-related genes. Interestingly, in terms of size and coding capacity, the nucleomorph genome of the non-photosynthetic species Cryptomonas sp. CCAC1634B is much more similar to that of the photosynthetic C. curvata species than to the non-photosynthetic species C. paramecium. CONCLUSIONS: Our results reveal fine-scale nucleomorph genome variation between distantly related congeneric taxa containing photosynthetic and non-photosynthetic species, including recent pseudogene formation, and provide a first glimpse into the possible impacts of the loss of photosynthesis on nucleomorph genome coding capacity and structure in independently evolved colorless strains.

Single-cell genomics unveiled a cryptic cyanobacterial lineage with a worldwide distribution hidden by a dinoflagellate host.

Cyanobacteria are one of the most important contributors to oceanic primary production and survive in a wide range of marine habitats. Much effort has been made to understand their ecological features, diversity, and evolution, based mainly on data from free-living cyanobacterial species. In addition, symbiosis has emerged as an important lifestyle of oceanic microbes and increasing knowledge of cyanobacteria in symbiotic relationships with unicellular eukaryotes suggests their significance in understanding the global oceanic ecosystem. However, detailed characteristics of these cyanobacteria remain poorly described. To gain better insight into marine cyanobacteria in symbiosis, we sequenced the genome of cyanobacteria collected from a cell of a pelagic dinoflagellate that is known to host cyanobacterial symbionts within a specialized chamber. Phylogenetic analyses using the genome sequence revealed that the cyanobacterium represents an underdescribed lineage within an extensively studied, ecologically important group of marine cyanobacteria. Metagenomic analyses demonstrated that this cyanobacterial lineage is globally distributed and strictly coexists with its host dinoflagellates, suggesting that the intimate symbiotic association allowed the cyanobacteria to escape from previous metagenomic studies. Furthermore, a comparative analysis of the protein repertoire with related species indicated that the lineage has independently undergone reductive genome evolution to a similar extent as Prochlorococcus, which has the most reduced genomes among free-living cyanobacteria. Discovery of this cyanobacterial lineage, hidden by its symbiotic lifestyle, provides crucial insights into the diversity, ecology, and evolution of marine cyanobacteria and suggests the existence of other undiscovered cryptic cyanobacterial lineages.

Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Microbial Eukaryotes that Lack Sterols.

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen-dependent multienzyme pathways. However, there are known exceptions-ciliated protozoans, such as Tetrahymena, along with diverse low-oxygen-adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol-like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol-like lipids in eukaryotes.

Complete genome of a nonphotosynthetic cyanobacterium in a diatom reveals recent adaptations to an intracellular lifestyle.

The evolution of mitochondria and plastids from bacterial endosymbionts were key events in the origin and diversification of eukaryotic cells. Although the ancient nature of these organelles makes it difficult to understand the earliest events that led to their establishment, the study of eukaryotic cells with recently evolved obligate endosymbiotic bacteria has the potential to provide important insight into the transformation of endosymbionts into organelles. Diatoms belonging to the family Rhopalodiaceae and their endosymbionts of cyanobacterial origin (i.e., "spheroid bodies") are emerging as a useful model system in this regard. The spheroid bodies, which appear to enable rhopalodiacean diatoms to use gaseous nitrogen, became established after the divergence of extant diatom families. Here we report what is, to our knowledge, the first complete genome sequence of a spheroid body, that of the rhopalodiacean diatom Epithemia turgida. The E. turgida spheroid body (EtSB) genome was found to possess a gene set for nitrogen fixation, as anticipated, but is reduced in size and gene repertoire compared with the genomes of their closest known free-living relatives. The presence of numerous pseudogenes in the EtSB genome suggests that genome reduction is ongoing. Most strikingly, our genomic data convincingly show that the EtSB has lost photosynthetic ability and is metabolically dependent on its host cell, unprecedented characteristics among cyanobacteria, and cyanobacterial symbionts. The diatom-spheroid body endosymbiosis is thus a unique system for investigating the processes underlying the integration of a bacterial endosymbiont into eukaryotic cells.

Heme pathway evolution in kinetoplastid protists.

BACKGROUND: Kinetoplastea is a diverse protist lineage composed of several of the most successful parasites on Earth, organisms whose metabolisms have coevolved with those of the organisms they infect. Parasitic kinetoplastids have emerged from free-living, non-pathogenic ancestors on multiple occasions during the evolutionary history of the group. Interestingly, in both parasitic and free-living kinetoplastids, the heme pathway-a core metabolic pathway in a wide range of organisms-is incomplete or entirely absent. Indeed, Kinetoplastea investigated thus far seem to bypass the need for heme biosynthesis by acquiring heme or intermediate metabolites directly from their environment. RESULTS: Here we report the existence of a near-complete heme biosynthetic pathway in Perkinsela spp., kinetoplastids that live as obligate endosymbionts inside amoebozoans belonging to the genus Paramoeba/Neoparamoeba. We also use phylogenetic analysis to infer the evolution of the heme pathway in Kinetoplastea. CONCLUSION: We show that Perkinsela spp. is a deep-branching kinetoplastid lineage, and that lateral gene transfer has played a role in the evolution of heme biosynthesis in Perkinsela spp. and other Kinetoplastea. We also discuss the significance of the presence of seven of eight heme pathway genes in the Perkinsela genome as it relates to its endosymbiotic relationship with Paramoeba.

Proposal of a Twin Arginine Translocator System-Mediated Constraint against Loss of ATP Synthase Genes from Nonphotosynthetic Plastid Genomes. [Corrected].

Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes-such as those coding for ATP synthase-remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Complex evolution of two types of cardiolipin synthase in the eukaryotic lineage stramenopiles.

The phospholipid cardiolipin is indispensable for eukaryotes to activate mitochondria, and it was previously reported that two phylogenetically distinct types of enzyme synthesizing cardiolipin, one with two phospholipase D domains (CLS_pld) and the other with a CDP-alcohol phosphatidyltransferase domain (CLS_cap), are patchily and complementarily distributed at higher taxonomic (e.g., supergroup) levels of eukaryotes. Stramenopiles, one of the major eukaryotic clades, were considered to exclusively possess CLS_cap. However, through our present surveys with genome or transcriptome data from a broad range of stramenopile taxa, species with both CLS_cap and CLS_pld and species with only CLS_pld or CLS_cap were discovered among this group. Because these homologues of CLS_cap and CLS_pld retrieved from stramenopiles were likely inherited from the last eukaryotic common ancestor, it is reasonable to assume that a common ancestor of all stramenopiles harbored both CLS_cap and CLS_pld. Furthermore, based on the robust organismal phylogeny of stramenopiles unveiled with large-scale phylogenetic analyses, the earliest diverging lineage of stramenopiles (including bicosoecids, placidids, etc.) was found to comprise species with both CLS_cap and CLS_pld along with species with only either CLS_cap or CLS_pld. These findings suggest that a common ancestor of the most basal stramenopile lineage retained these two vertically inherited enzymes and that differential losses of either CLS_cap or CLS_pld occurred in this lineage. On the other hand, in the other stramenopile lineage composed of Ochrophyta, Pseudofungi, and Labyrinthulomycetes (to the exclusion of the most basal lineage), only CLS_cap was found, and therefore a common ancestor of these three groups likely lost CLS_pld. Based on our findings, the evolution of CLS_cap/CLS_pld in stramenopiles appears to be more complex than previously thought.

Reduced nuclear genomes maintain high gene transcription levels.

Reductive genome evolution is seen in organisms living in close association with each other, such as in endosymbiosis, symbiosis, and parasitism. The reduced genomes of endosymbionts and parasites often exhibit similar features such as high gene densities and A+T compositional bias. Little is known about how the regulation of gene expression has been affected in organisms with highly compacted genomes. We studied gene transcription patterns in "nucleomorph" genomes, which are relic nuclear genomes of algal endosymbionts found in cryptophytes and chlorarachniophytes. We examined nuclear and nucleomorph gene transcription patterns using RNA-Seq transcriptome and genome mapping analyses in representatives of both lineages. In all four examined genomes, the most highly transcribed nucleomorph gene category was found to be plastid-associated genes. Remarkably, only 0.49-3.37% of the nucleomorph genomes of these organisms did not have any mRNA counterpart in our RNA-Seq data sets, and nucleomorph genes show equal or higher levels of transcription than their counterparts in the nuclear genomes. We hypothesize that elevated levels of nucleomorph gene transcription may serve to counteract the degradation or modification of protein function due to the loss of interacting proteins in the nucleomorph and nucleomorph-associated subcellular compartments.

Nucleomorph and plastid genome sequences of the chlorarachniophyte Lotharella oceanica: convergent reductive evolution and frequent recombination in nucleomorph-bearing algae.

BACKGROUND: Nucleomorphs are residual nuclei derived from eukaryotic endosymbionts in chlorarachniophyte and cryptophyte algae. The endosymbionts that gave rise to nucleomorphs and plastids in these two algal groups were green and red algae, respectively. Despite their independent origin, the chlorarachniophyte and cryptophyte nucleomorph genomes share similar genomic features such as extreme size reduction and a three-chromosome architecture. This suggests that similar reductive evolutionary forces have acted to shape the nucleomorph genomes in the two groups. Thus far, however, only a single chlorarachniophyte nucleomorph and plastid genome has been sequenced, making broad evolutionary inferences within the chlorarachniophytes and between chlorarachniophytes and cryptophytes difficult. We have sequenced the nucleomorph and plastid genomes of the chlorarachniophyte Lotharella oceanica in order to gain insight into nucleomorph and plastid genome diversity and evolution. RESULTS: The L. oceanica nucleomorph genome was found to consist of three linear chromosomes totaling ~610 kilobase pairs (kbp), much larger than the 373 kbp nucleomorph genome of the model chlorarachniophyte Bigelowiella natans. The L. oceanica plastid genome is 71 kbp in size, similar to that of B. natans. Unexpectedly long (~35 kbp) sub-telomeric repeat regions were identified in the L. oceanica nucleomorph genome; internal multi-copy regions were also detected. Gene content analyses revealed that nucleomorph house-keeping genes and spliceosomal intron positions are well conserved between the L. oceanica and B. natans nucleomorph genomes. More broadly, gene retention patterns were found to be similar between nucleomorph genomes in chlorarachniophytes and cryptophytes. Chlorarachniophyte plastid genomes showed near identical protein coding gene complements as well as a high level of synteny. CONCLUSIONS: We have provided insight into the process of nucleomorph genome evolution by elucidating the fine-scale dynamics of sub-telomeric repeat regions. Homologous recombination at the chromosome ends appears to be frequent, serving to expand and contract nucleomorph genome size. The main factor influencing nucleomorph genome size variation between different chlorarachniophyte species appears to be expansion-contraction of these telomere-associated repeats rather than changes in the number of unique protein coding genes. The dynamic nature of chlorarachniophyte nucleomorph genomes lies in stark contrast to their plastid genomes, which appear to be highly stable in terms of gene content and synteny.

Fascinating strategies of marine benthic organisms to cope with emerging pollutant: Titanium dioxide nanoparticles.

Titanium dioxide nanoparticles (NPs) have numerous applications, and their demands have increased as an alternative for banned sunscreen filters. However, the underlying mechanisms of their toxicity, remain largely unknown. Here, we investigate the mechanism of TiO2 NP cytotoxicity and detoxification through time-course experiments (1, 6, and 24 h) based on cellular observations and single-cell transcriptome analyses in a marine benthic foraminifer strain, derived from a common unicellular eukaryotic organism worldwide. After exposure for 1 h, cells enhanced the production of reactive oxygen species (ROS) in acidic endosomes containing TiO2 NPs as well as in mitochondria. In acidic endosomes, ROS were produced through the Fenton reaction on the surface of charged TiO2 NPs. In mitochondria, ROS were associated with porphyrin synthesis that chelated metal ions. Glutathione peroxide and neutral lipids acted as a sink for free radicals, whereas lipid peroxides were excreted to prevent further radical chain reactions. By 24 h, aggregated TiO2 NPs were encapsulated in organic compounds, possibly ceramide, and excreted as mucus, thereby preventing their further uptake. Thus, we reveal that foraminifers can tolerate the toxicity of TiO2 NPs and even prevent their further phagocytosis and uptake by trapping TiO2 NPs inside mucus. This previously unknown strategy could be applied in bioremediation to sequester NPs from the marine environment and can guide management of TiO2 pollution.

Transcriptome data sets of free-living diplomonads, Trepomonas sp. and Hexamita sp.

Most species belonging to the diplomonad genera, Trepomonas and Hexamita, are considered to have secondarily adapted to free-living lifestyles from the parasitic ancestor. Here, we report the annotated transcriptome data of Trepomonas sp. NIES-1444 and Hexamita sp. NIES-1440, the analysis of which will provide insights into the lifestyle transitions.

Genomic characterization of Neoparamoeba pemaquidensis (Amoebozoa) and its kinetoplastid endosymbiont.

We have performed a genomic characterization of a kinetoplastid protist living within the amoebozoan Neoparamoeba pemaquidensis. The genome of this "Ichthyobodo-related organism" was found to be unexpectedly large, with at least 11 chromosomes between 1.0 and 3.5 Mbp and a total genome size of at least 25 Mbp.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.