The Complete Plastid Genomes of the Two ‘Dinotoms’ Durinskia baltica and Kryptoperidinium foliaceum

In one small group of dinoflagellates, photosynthesis is carried out by a tertiary endosymbiont derived from a diatom, giving rise to a complex cell that we collectively refer to as a 'dinotom'. The endosymbiont is separated from its host by a single membrane and retains plastids, mitochondria, a large nucleus, and many other eukaryotic organelles and structures, a level of complexity suggesting an early stage of integration. Although the evolution of these endosymbionts has attracted considerable interest, the plastid genome has not been examined in detail, and indeed no tertiary plastid genome has yet been sequenced.Here we describe the complete plastid genomes of two closely related dinotoms, Durinskia baltica and Kryptoperidinium foliaceum. The D. baltica (116470 bp) and K. foliaceum (140426 bp) plastid genomes map as circular molecules featuring two large inverted repeats that separate distinct single copy regions. The organization and gene content of the D. baltica plastid closely resemble those of the pennate diatom Phaeodactylum tricornutum. The K. foliaceum plastid genome is much larger, has undergone more reorganization, and encodes a putative tyrosine recombinase (tyrC) also found in the plastid genome of the heterokont Heterosigma akashiwo, and two putative serine recombinases (serC1 and serC2) homologous to recombinases encoded by plasmids pCf1 and pCf2 in another pennate diatom, Cylindrotheca fusiformis. The K. foliaceum plastid genome also contains an additional copy of serC1, two degenerate copies of another plasmid-encoded ORF, and two non-coding regions whose sequences closely resemble portions of the pCf1 and pCf2 plasmids.These results suggest that while the plastid genomes of two dinotoms share very similar gene content and genome organization with that of the free-living pennate diatom P. tricornutum, the K. folicaeum plastid genome has absorbed two exogenous plasmids. Whether this took place before or after the tertiary endosymbiosis is not clear

A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis

Plastid endosymbiosis has been a major force in the evolution of eukaryotic cellular complexity, but how endosymbionts are integrated is still poorly understood at a mechanistic level. Dinoflagellates, an ecologically important protist lineage, represent a unique model to study this process because dinoflagellate plastids have repeatedly been reduced, lost, and replaced by new plastids, leading to a spectrum of ages and integration levels. Here we describe deep-transcriptomic analyses of the Antarctic Ross Sea dinoflagellate (RSD), which harbors long-term but temporary kleptoplasts stolen from haptophyte prey, and is closely related to dinoflagellates with fully integrated plastids derived from different haptophytes. In some members of this lineage, called the Kareniaceae, their tertiary haptophyte plastids have crossed a tipping point to stable integration, but RSD has not, and may therefore reveal the order of events leading up to endosymbiotic integration. We show that RSD has retained its ancestral secondary plastid and has partitioned functions between this plastid and the kleptoplast. It has also obtained genes for kleptoplast-targeted proteins via horizontal gene transfer (HGT) that are not derived from the kleptoplast lineage. Importantly, many of these HGTs are also found in the related species with fully integrated plastids, which provides direct evidence that genetic integration preceded organelle fixation. Finally, we find that expression of kleptoplast-targeted genes is unaffected by environmental parameters, unlike prey-encoded homologs, suggesting that kleptoplast-targeted HGTs have adapted to posttranscriptional regulation mechanisms of the host

The Mitochondrial Genome of the Entomoparasitic Green Alga Helicosporidium

BACKGROUND: Helicosporidia are achlorophyllous, non-photosynthetic protists that are obligate parasites of invertebrates. Highly specialized, these pathogens feature an unusual cyst stage that dehisces inside the infected organism and releases a filamentous cell displaying surface projections, which will penetrate the host gut wall and eventually reproduce in the hemolymph. Long classified as incertae sedis or as relatives of other parasites such as Apicomplexa or Microsporidia, the Helicosporidia were surprisingly identified through molecular phylogeny as belonging to the Chlorophyta, a phylum of green algae. Most phylogenetic analyses involving Helicosporidia have placed them within the subgroup Trebouxiophyceae and further suggested a close affiliation between the Helicosporidia and the genus Prototheca. Prototheca species are also achlorophyllous and pathogenic, but they infect vertebrate hosts, inducing protothecosis in humans. The complete plastid genome of an Helicosporidium species was recently described and is a model of compaction and reduction. Here we describe the complete mitochondrial genome sequence of the same strain, Helicosporidium sp. ATCC 50920 isolated from the black fly Simulium jonesi. METHODOLOGY/PRINCIPAL FINDINGS: The circular mapping 49343 bp mitochondrial genome of Helicosporidium closely resembles that of the vertebrate parasite Prototheca wickerhamii. The two genomes share an almost identical gene complement and display a level of synteny that is higher than any other sequenced chlorophyte mitochondrial DNAs. Interestingly, the Helicosporidium mtDNA feature a trans-spliced group I intron, and a second group I intron that contains two open reading frames that appear to be degenerate maturase/endonuclease genes, both rare characteristics for this type of intron. CONCLUSIONS/SIGNIFICANCE: The architecture, genome content, and phylogeny of the Helicosporidium mitochondrial genome are all congruent with its close relationship to Prototheca within the Trebouxiophyceae. The Helicosporidium mitochondrial genome does, however, contain a number of novel features, particularly relating to its introns

The Phylogenetic Position of the Oxymonad Saccinobaculus Based on SSU rRNA

The oxymonads are a group of structurally complex anaerobic flagellates about which we know very little. They are found in association with complex microbial communities in the guts of animals. There are five recognized families of oxymonads; molecular data have been acquired for four of these. Here, we describe the first molecular data from the last remaining group, represented by Saccinobaculus, an organism that is found exclusively in the hindgut of the wood-eating cockroach Cryptocercus. We sequenced small subunit ribosomal RNA (SSU rRNA) from total gut DNA to describe Saccinobaculus SSU rRNA diversity. We also sequenced SSU rRNA from manually isolated cells of the two most abundant and readily identifiable species: the type species Saccinobaculus ambloaxostylus and the taxonomically contentious Saccinobaculus doroaxostylus. We inferred phylogenetic trees including all five known oxymonad subgroups in order to elucidate the internal phylogeny of this poorly studied group, to resolve some outstanding issues of the taxonomy and identification of certain Saccinobaculus species, and to investigate the evolution of character states within it. Our analysis recovered strong support for the existence of the five subgroups of oxymonads, and consistently united the subgroups containing Monocercomonoides and Streblomastix, but was unable to resolve any further higher-order branching patterns

A Lack of Parasitic Reduction in the Obligate Parasitic Green Alga \u3cem\u3eHelicosporidium\u3c/em\u3e

The evolution of an obligate parasitic lifestyle is often associated with genomic reduction, in particular with the loss of functions associated with increasing host-dependence. This is evident in many parasites, but perhaps the most extreme transitions are from free-living autotrophic algae to obligate parasites. The best-known examples of this are the apicomplexans such as Plasmodium, which evolved from algae with red secondary plastids. However, an analogous transition also took place independently in the Helicosporidia, where an obligate parasite of animals with an intracellular infection mechanism evolved from algae with green primary plastids. We characterised the nuclear genome of Helicosporidium to compare its transition to parasitism with that of apicomplexans. The Helicosporidium genome is small and compact, even by comparison with the relatively small genomes of the closely related green algae Chlorella and Coccomyxa, but at the functional level we find almost no evidence for reduction. Nearly all ancestral metabolic functions are retained, with the single major exception of photosynthesis, and even here reduction is not complete. The great majority of genes for light-harvesting complexes, photosystems, and pigment biosynthesis have been lost, but those for other photosynthesis-related functions, such as Calvin cycle, are retained. Rather than loss of whole function categories, the predominant reductive force in the Helicosporidium genome is a contraction of gene family complexity, but even here most losses affect families associated with genome maintenance and expression, not functions associated with host-dependence. Other gene families appear to have expanded in response to parasitism, in particular chitinases, including those predicted to digest the chitinous barriers of the insect host or remodel the cell wall of Helicosporidium. Overall, the Helicosporidium genome presents a fascinating picture of the early stages of a transition from free-living autotroph to parasitic heterotroph where host-independence has been unexpectedly preserved

Evolution of the sex-Related Locus and Genomic Features Shared in Microsporidia and Fungi

Microsporidia are obligate intracellular, eukaryotic pathogens that infect a wide range of animals from nematodes to humans, and in some cases, protists. The preponderance of evidence as to the origin of the microsporidia reveals a close relationship with the fungi, either within the kingdom or as a sister group to it. Recent phylogenetic studies and gene order analysis suggest that microsporidia share a particularly close evolutionary relationship with the zygomycetes.Here we expanded this analysis and also examined a putative sex-locus for variability between microsporidian populations. Whole genome inspection reveals a unique syntenic gene pair (RPS9-RPL21) present in the vast majority of fungi and the microsporidians but not in other eukaryotic lineages. Two other unique gene fusions (glutamyl-prolyl tRNA synthetase and ubiquitin-ribosomal subunit S30) that are present in metazoans, choanoflagellates, and filasterean opisthokonts are unfused in the fungi and microsporidians. One locus previously found to be conserved in many microsporidian genomes is similar to the sex locus of zygomycetes in gene order and architecture. Both sex-related and sex loci harbor TPT, HMG, and RNA helicase genes forming a syntenic gene cluster. We sequenced and analyzed the sex-related locus in 11 different Encephalitozoon cuniculi isolates and the sibling species E. intestinalis (3 isolates) and E. hellem (1 isolate). There was no evidence for an idiomorphic sex-related locus in this Encephalitozoon species sample. According to sequence-based phylogenetic analyses, the TPT and RNA helicase genes flanking the HMG genes are paralogous rather than orthologous between zygomycetes and microsporidians.The unique genomic hallmarks between microsporidia and fungi are independent of sequence based phylogenetic comparisons and further contribute to define the borders of the fungal kingdom and support the classification of microsporidia as unusual derived fungi. And the sex/sex-related loci appear to have been subject to frequent gene conversion and translocations in microsporidia and zygomycetes

Evidence for the Retention of Two Evolutionary Distinct Plastids in Dinoflagellates with Diatom Endosymbionts

Dinoflagellates harboring diatom endosymbionts (termed “dinotoms”) have undergone a process often referred to as “tertiary endosymbiosis”—the uptake of algae containing secondary plastids and integration of those plastids into the new host. In contrast to other tertiary plastids, and most secondary plastids, the endosymbiont of dinotoms is distinctly less reduced, retaining a number of cellular features, such as their nucleus and mitochondria and others, in addition to their plastid. This has resulted in redundancy between host and endosymbiont, at least between some mitochondrial and cytosolic metabolism, where this has been investigated. The question of plastidial redundancy is particularly interesting as the fate of the host dinoflagellate plastid is unclear. The host cytosol possesses an eyespot that has been postulated to be a remnant of the ancestral peridinin plastid, but this has not been tested, nor has its possible retention of plastid functions. To investigate this possibility, we searched for plastid-associated pathways and functions in transcriptomic data sets from three dinotom species. We show that the dinoflagellate host has indeed retained genes for plastid-associated pathways and that these genes encode targeting peptides similar to those of other dinoflagellate plastid-targeted proteins. Moreover, we also identified one gene encoding an essential component of the dinoflagellate plastid protein import machinery, altogether suggesting the presence of a functioning plastid import system in the host, and by extension a relict plastid. The presence of the same plastid-associated pathways in the endosymbiont also extends the known functional redundancy in dinotoms, further confirming the unusual state of plastid integration in this group of dinoflagellates

EFL GTPase in Cryptomonads and the Distribution of EFL and EF-1a in Chromalveolates

EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1a, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1a. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1a (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1a PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1a in neither. On balance, we suggest the previously reported EF-1a from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group of chromalveolates, cryptomonads. Of the six major subgroups of chromalveolates, EFL is found in half and EF-1a in the other half, and none as yet unambiguously possess both genes. Phylogenetic analysis indicates EFL likely arose early within each subgroup where it is found, but suggests it may have originated multiple times within chromalveolates as a whole

A Bacterial Homolog of a Eukaryotic Inositol Phosphate Signaling Enzyme Mediates Cross-kingdom Dialog in the Mammalian Gut

Dietary InsP6 can modulate eukaryotic cell proliferation and has complex nutritive consequences, but its metabolism in the mammalian gastrointestinal tract is poorly understood. Therefore, we performed phylogenetic analyses of the gastrointestinal microbiome in order to search for candidate InsP6 phosphatases. We determined that prominent gut bacteria express homologs of the mammalian InsP6 phosphatase (MINPP) and characterized the enzyme from Bacteroides thetaiotaomicron (BtMinpp). We show that BtMinpp has exceptionally high catalytic activity, which we rationalize on the basis of mutagenesis studies and by determining its crystal structure at 1.9 Å resolution. We demonstrate that BtMinpp is packaged inside outer membrane vesicles (OMVs) protecting the enzyme from degradation by gastrointestinal proteases. Moreover, we uncover an example of cross-kingdom cell-to-cell signaling, showing that the BtMinpp-OMVs interact with intestinal epithelial cells to promote intracellular Ca2+ signaling. Our characterization of BtMinpp offers several directions for understanding how the microbiome serves human gastrointestinal physiology

Biogeography and Character Evolution of the Ciliate Genus Euplotes (Spirotrichea, Euplotia), with Description of Euplotes curdsi sp. nov.

Ciliates comprise a diverse and ecologically important phylum of unicellular protists. One of the most specious and best-defined genera is Euplotes, which constitutes more than 70 morphospecies, many of which have never been molecularly tested. The increasing number of described Euplotes taxa emphasizes the importance for detailed characterizations of new ones, requiring standardized morphological observations, sequencing of molecular markers and careful comparison with previous literature. Here we describe Euplotes curdsi sp. nov., distinguishable by the combination of the following features: 45±65 μm length, oval or elongated shape with both ends rounded, narrow peristome with 25±34 adoral membranelles, conspicuous paroral membrane, double-eurystomus dorsal argyrome type, 6±7 dorsolateral kineties and 10 frontoventral cirri. Three populations of the novel species have been found in brackish and marine samples in the Mediterranean and the White Sea. We provide the SSU rRNA gene sequences of these populations, and an updated phylogeny of the genus Euplotes. Using the molecular phylogenetic tree, we inferred aspects of the biogeographical history of the genus and the evolution of its most important taxonomic characters in order to provide a frame for future descriptions. Ultimately, these data reveal recurrent trends of freshwater invasion and highlight the dynamic, yet convergent, morphological evolution of Euplotes