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.

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.

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.

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.

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.

Detailed process of shell construction in the photosynthetic testate amoeba Paulinella chromatophora (euglyphid, Rhizaria).

Most euglyphids, a group of testate amoebae, have a shell that is constructed from numerous siliceous scales. The euglyphid Paulinella chromatophora has photosynthetic organelles (termed cyanelles or chromatophores), allowing it to be cultivated more easily than other euglyphids. Like other euglyphids, P. chromatophora has a siliceous shell made of brick-like scales. These scales are varied in size and shape. How a P. chromatophora cell makes this shell is still a mystery. We examined shell construction process in P. chromatophora in detail using time-lapse video microscopy. The new shell was constructed by a specialized pseudopodium that laid out each scale into correct position, one scale at a time. The present study inferred that the sequence of scale production and secretion was well controlled.

Convergent reductive evolution of cyanobacteria in symbiosis with Dinophysiales dinoflagellates.

The diversity of marine cyanobacteria has been extensively studied due to their vital roles in ocean primary production. However, little is understood about the diversity of cyanobacterial species involved in symbiotic relationships. In this study, we successfully sequenced the complete genome of a cyanobacterium in symbiosis with Citharistes regius, a dinoflagellate species thriving in the open ocean. A phylogenomic analysis revealed that the cyanobacterium (CregCyn) belongs to the marine picocyanobacterial lineage, akin to another cyanobacterial symbiont (OmCyn) of a different dinoflagellate closely related to Citharistes. Nevertheless, these two symbionts are representing distinct lineages, suggesting independent origins of their symbiotic lifestyles. Despite the distinct origins, the genome analyses of CregCyn revealed shared characteristics with OmCyn, including an obligate symbiotic relationship with the host dinoflagellates and a degree of genome reduction. In contrast, a detailed analysis of genome subregions unveiled that the CregCyn genome carries genomic islands that are not found in the OmCyn genome. The presence of the genomic islands implies that exogenous genes have been integrated into the CregCyn genome at some point in its evolution. This study contributes to our understanding of the complex history of the symbiosis between dinoflagellates and cyanobacteria, as well as the genomic diversity of marine picocyanobacteria.

Detection of evolutionary conserved and accelerated genomic regions related to adaptation to thermal niches in Anolis lizards.

Understanding the genetic basis for adapting to thermal environments is important due to serious effects of global warming on ectothermic species. Various genes associated with thermal adaptation in lizards have been identified mainly focusing on changes in gene expression or the detection of positively selected genes using coding regions. Only a few comprehensive genome-wide analyses have included noncoding regions. This study aimed to identify evolutionarily conserved and accelerated genomic regions using whole genomes of eight Anolis lizard species that have repeatedly adapted to similar thermal environments in multiple lineages. Evolutionarily conserved genomic regions were extracted as regions with overall sequence conservation (regions with fewer base substitutions) across all lineages compared with the neutral model. Genomic regions that underwent accelerated evolution in the lineage of interest were identified as those with more base substitutions in the target branch than in the entire background branch. Conserved elements across all branches were relatively abundant in "intergenic" genomic regions among noncoding regions. Accelerated regions (ARs) of each lineage contained a significantly greater proportion of noncoding RNA genes than the entire multiple alignment. Common genes containing ARs within 5 kb of their vicinity in lineages with similar thermal habitats were identified. Many genes associated with circadian rhythms and behavior were found in hot-open and cool-shaded habitat lineages. These genes might play a role in contributing to thermal adaptation and assist future studies examining the function of genes involved in thermal adaptation via genome editing.

Evolving a photosynthetic organelle.

The evolution of plastids from cyanobacteria is believed to represent a singularity in the history of life. The enigmatic amoeba Paulinella and its 'recently' acquired photosynthetic inclusions provide a fascinating system through which to gain fresh insight into how endosymbionts become organelles.The plastids, or chloroplasts, of algae and plants evolved from cyanobacteria by endosymbiosis. This landmark event conferred on eukaryotes the benefits of photosynthesis--the conversion of solar energy into chemical energy--and in so doing had a huge impact on the course of evolution and the climate of Earth 1. From the present state of plastids, however, it is difficult to trace the evolutionary steps involved in this momentous development, because all modern-day plastids have fully integrated into their hosts. Paulinella chromatophora is a unicellular eukaryote that bears photosynthetic entities called chromatophores that are derived from cyanobacteria and has thus received much attention as a possible example of an organism in the early stages of organellogenesis. Recent studies have unlocked the genomic secrets of its chromatophore 23 and provided concrete evidence that the Paulinella chromatophore is a bona fide photosynthetic organelle 4. The question is how Paulinella can help us to understand the process by which an endosymbiont is converted into an organelle.

Three-dimensional architecture and assembly mechanism of the egg-shaped shell in testate amoeba Paulinella micropora.

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

A novel structural maintenance of chromosomes (SMC)-related protein family specific to Archaea.

The ATPases belonging to the structural maintenance of chromosomes (SMC) superfamily are involved in the maintenance of chromosome organization and dynamics, as well as DNA repair. The major proteins in this superfamily recognized to date are either conserved among the three domains of Life (i.e., SMC and Rad50) or specific to Bacteria (i.e., RecF, RecN, and MukB). In Archaea, no protein related to SMC (SMC-related protein) with a broad taxonomic distribution has been reported. Nevertheless, two SMC-related proteins, namely coalescin and Sph, have been identified in crenarchaea Sulfolobus spp. and the euryarchaeon Halobacterium salinarum, respectively, hinting that the diversity of SMC-related proteins has been overlooked in Archaea. In this study, we report a novel SMC-related protein that is distributed among broad archaeal lineages and termed "Archaea-specific SMC-related proteins" or "ASRPs." We further demonstrate that the ASRP family encloses both coalescin and Sph but the two proteins represent only a tip of the diversity of this family.

Comparative Plastid Genomics of Green-Colored Dinoflagellates Unveils Parallel Genome Compaction and RNA Editing.

Dinoflagellates possess plastids that are diverse in both pigmentation and evolutionary background. One of the plastid types found in dinoflagellates is pigmented with chlorophylls a and b (Chl a + b) and originated from the endosymbionts belonging to a small group of green algae, Pedinophyceae. The Chl a + b-containing plastids have been found in three distantly related dinoflagellates Lepidodinium spp., strain MGD, and strain TGD, and were proposed to be derived from separate partnerships between a dinoflagellate (host) and a pedinophycean green alga (endosymbiont). Prior to this study, a plastid genome sequence was only available for L. chlorophorum, which was reported to bear the features that were not found in that of the pedinophycean green alga Pedinomonas minor, a putative close relative of the endosymbiont that gave rise to the current Chl a + b-containing plastid. In this study, we sequenced the plastid genomes of strains MGD and TGD to compare with those of L. chlorophorum as well as pedinophycean green algae. The mapping of the RNA-seq reads on the corresponding plastid genome identified RNA editing on plastid gene transcripts in the three dinoflagellates. Further, the comparative plastid genomics revealed that the plastid genomes of the three dinoflagellates achieved several features, which are not found in or much less obvious than the pedinophycean plastid genomes determined to date, in parallel.

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.

Differential gene retention in plastids of common recent origin.

The cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in a prokaryote-eukaryote (primary) endosymbiosis that occurred approximately 60 mya. Here we sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and compared the sequence with the existing data from the sister taxon Paulinella M0880/a. Alignment of the two plastid genomes shows significant conservation of gene order and only a handful of minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that appear to be outright gene deletions rather than endosymbiotic gene transfers to the host nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the Synechococcus-Prochlorococcus group, and the cyanobacterial provenance of all plastid genes suggests that these organelles were not targets of interphylum gene transfers after endosymbiosis. Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have dN/dS ratios <<1, providing evidence for purifying selection. Our study demonstrates that plastid genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during the earlier phases of organelle evolution.

Spheroid bodies in rhopalodiacean diatoms were derived from a single endosymbiotic cyanobacterium.

Members of the diatom family rhopalodiaceae possess cyanobacteria-derived intracellular structures called spheroid bodies (SBs) that very likely carry out nitrogen fixation. Due to the shortage of molecular data from SBs and rhopalodiacean diatoms, it remains unclear how SBs were established and spread in rhopalodiacean diatoms. We here amplified the small subunit ribosomal DNA sequences from both host and SB in three rhopalodiacean diatom species, Epithemia turgida, E. sorex, and Rhopalodia gibba. Phylogenetic analyses considering these new sequences clearly indicate that the SBs were acquired by a common ancestor of rhopalodiacean diatoms and have been retained during host speciation.

Detection of genes positively selected in Cuban Anolis lizards that naturally inhabit hot and open areas and currently thrive in urban areas.

Species of Anolis lizards of the West Indies that naturally inhabit hot and open areas also tend to thrive in urban areas. In this study, transcriptome was sequenced for nine species of Cuban Anolis lizards that are closely related to each other, but inhabit different thermal microhabitats. Using PAML and HyPhy software, we attempted to identify genes and amino acid sites under positive selection in the common ancestral branch of A. porcatus and A. allisoni, and the branch of A. sagrei, which inhabit hot and open areas, and thrive in urban areas. Although there were no genes where positive selection was commonly detected on both of the tested branches, positive selection was detected in genes involved in the stress response (e.g., DNA damage and oxidative stress) and cardiac function, which could be related to adaptive evolution of tolerance to heat or ultraviolet radiation, on both branches. These findings suggest that adaptive evolution of the response to stress caused by heat or ultraviolet radiation might have occurred in ancestors of Anolis species inhabiting hot and open areas and might be related to the current thriving in urban areas of them.

Signs of the plastid: Enzymes involved in plastid-localized metabolic pathways in a eugregarine species.

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study.

Rappemonads are haptophyte phytoplankton.

Rapidly accumulating genetic data from environmental sequencing approaches have revealed an extraordinary level of unsuspected diversity within marine phytoplankton,1-11 which is responsible for around 50% of global net primary production.12,13 However, the phenotypic identity of many of the organisms distinguished by environmental DNA sequences remains unclear. The rappemonads represent a plastid-bearing protistan lineage that to date has only been identified by environmental plastid 16S rRNA sequences.14-17 The phenotypic identity of this group, which does not confidently cluster in any known algal clades in 16S rRNA phylogenetic reconstructions,15 has remained unknown since the first report of environmental sequences over two decades ago. We show that rappemonads are closely related to a haptophyte microalga, Pavlomulina ranunculiformis gen. nov. et sp. nov., and belong to a new haptophyte class, the Rappephyceae. Organellar phylogenomic analyses provide strong evidence for the inclusion of this lineage within the Haptophyta as a sister group to the Prymnesiophyceae. Members of this new class have a cosmopolitan distribution in coastal and oceanic regions. The relative read abundance of Rappephyceae in a large environmental barcoding dataset was comparable to, or greater than, those of major haptophyte species, such as the bloom-forming Gephyrocapsa huxleyi and Prymnesium parvum, and this result indicates that they likely have a significant impact as primary producers. Detailed characterization of Pavlomulina allowed for reconstruction of the ancient evolutionary history of the Haptophyta, a group that is one of the most important components of extant marine phytoplankton communities.

Putative genome features of relic green alga-derived nuclei in dinoflagellates and future perspectives as model organisms.

Nucleomorphs, relic endosymbiont nuclei, have been studied as a model to elucidate the evolutionary process of integrating a eukaryotic endosymbiont into a host cell organelle. Recently, we reported two new dinoflagellates possessing nucleomorphs, and proposed them as new models in this research field based on the following findings: genome integration processes are incomplete, and the origins of the endosymbiont lineages were pinpointed. Here, we focused on the nucleomorph genome features in the two green dinoflagellates and compared them with those of the known nucleomorph genomes of cryptophytes and chlorarachniophytes. All nucleomorph genomes showed similar trends suggesting convergent evolution. However, the number of nucleomorph genes that are unrelated to housekeeping machineries in the two green dinoflagellates are greater than the numbers in cryptophytes and chlorarachniophytes, providing additional evidence that their genome reduction has not progressed much compared with those of cryptophytes and chlorarachniophytes. Finally, potential future work is discussed.

A single origin of the photosynthetic organelle in different Paulinella lineages.

BACKGROUND: Gaining the ability to photosynthesize was a key event in eukaryotic evolution because algae and plants form the base of the food chain on our planet. The eukaryotic machines of photosynthesis are plastids (e.g., chloroplast in plants) that evolved from cyanobacteria through primary endosymbiosis. Our knowledge of plastid evolution, however, remains limited because the primary endosymbiosis occurred more than a billion years ago. In this context, the thecate "green amoeba" Paulinella chromatophora is remarkable because it very recently (i.e., minimum age of approximately 60 million years ago) acquired a photosynthetic organelle (termed a "chromatophore"; i.e., plastid) via an independent primary endosymbiosis involving a Prochlorococcus or Synechococcus-like cyanobacterium. All data regarding P. chromatophora stem from a single isolate from Germany (strain M0880/a). Here we brought into culture a novel photosynthetic Paulinella strain (FK01) and generated molecular sequence data from these cells and from four different cell samples, all isolated from freshwater habitats in Japan. Our study had two aims. The first was to compare and contrast cell ultrastructure of the M0880/a and FK01 strains using scanning electron microscopy. The second was to assess the phylogenetic diversity of photosynthetic Paulinella to test the hypothesis they share a vertically inherited plastid that originated in their common ancestor. RESULTS: Comparative morphological analyses show that Paulinella FK01 cells are smaller than M0880/a and differ with respect to the number of scales per column. There are more distinctive, multiple fine pores on the external surface of FK01 than in M0880/a. Molecular phylogenetic analyses using multiple gene markers demonstrate these strains are genetically distinct and likely comprise separate species. The well-supported monophyly of the Paulinella chromatophora strains analyzed here using plastid-encoded 16S rRNA suggests strongly that they all share a common photosynthetic ancestor. The strain M0880/a is most closely related to Japanese isolates (Kanazawa-1, -2, and Kaga), whereas FK01 groups closely with a Kawaguchi isolate. CONCLUSION: Our results indicate that Paulinella chromatophora comprises at least two distinct evolutionary lineages and likely encompasses a broader taxonomic diversity than previously thought. The finding of a single plastid origin for both lineages shows these taxa to be valuable models for studying post-endosymbiotic cell and genome evolution.