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DNA Analysis of Algal Endosymbionts of Ciliates Reveals the State of Algal Integration and the Surprising Specificity of the Symbiosis

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Abstract

Many freshwater protists harbor unicellular green algae within their cells, but little is known of their degree of integration and specificity. Using algae-targeted PCR of whole ciliate cells collected at irregular intervals over 15 months from Lake Biwa, Japan, we explored the SSU-ITS rDNA of the endosymbiotic algae and its changes over time, obtaining sequences of algal rDNA fragments from four ciliate species. A high proportion of clonal algae was evident within the ciliate cells. The differences observed in those sequences from the SSU through to the ITS region were less than 1%. The name ‘Chlorb’ is proposed for these algae, with the implication that they represent a single ‘species.’ The sequences of the algal DNA fragments were identical for any given host species throughout the collection period, thus we conclude that these four ciliates stably retain their algae over long term. In contrast, algal DNA fragments obtained from Didinium sp. were variable within each sample, which indicates that this ciliate only temporarily holds its algal cells. The ITS1 sequences of Chlorb populations are close (at intraspecific level) to those of algae isolated from ciliates in Austria, which raises the possibility that Chlorb algae are universally shared as symbionts among various ciliates.

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... These protists usually contain hundreds of algae within a host cell, and we have referred to them collectively as 'multi-algae retaining protists' (MARP). Recently we reported that several ciliate species from Japan shared a single species of symbiotic algae, which we called 'Chlorb' (Hoshina & Kusuoka 2016). Although closely related, each Chlorb alga from a given host ciliate species had a different genotype. ...
... Furthermore, this alga has been also reported as the shared symbiont of several ciliate species from a remote Austrian lake (Summerer et al. 2007(Summerer et al. , 2008. It follows that the Chlorb algae are widely shared as symbionts among various ciliates (Hoshina & Kusuoka 2016). For the present study, we re-collected three MARP ciliates examined by Hoshina and Kusuoka (2016): Pelagodileptus trachelioides, Cyclotrichium viride, and Stokesia vernalis. ...
... It follows that the Chlorb algae are widely shared as symbionts among various ciliates (Hoshina & Kusuoka 2016). For the present study, we re-collected three MARP ciliates examined by Hoshina and Kusuoka (2016): Pelagodileptus trachelioides, Cyclotrichium viride, and Stokesia vernalis. We isolated Chlorb algae from these ciliate cells and described the algae as Brandtia ciliaticola gen. ...
Article
Many freshwater protists harbor unicellular green algae within their cells and these host-symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host-algal genotype combinations remained unchanged throughout a 15-month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate-algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella-like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella-clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU-ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.
... Paramecium bursaria-a ciliate protist that harbors a population of often clonal intracellular green algae, Chlorella spp. (11)(12)(13)-represents a tractable model system to study emergent mechanisms in endosymbiosis (14). The interaction is facultative (11,(14)(15)(16)(17) and based on two-way metabolic exchange (18)(19)(20)(21)(22)(23)(24)(25)(26). ...
... In natural interactions between P. bursaria and its algal endosymbiont, such a cost would only need to occur in the drastic occurrence of mass endosymbiont digestion in order to drive stability of the interaction. Importantly, the endosymbiotic algal population within P. bursaria is largely composed of closely related or clonal lineages (11)(12)(13), and as such, the fate of the algal population should be considered as a collective unit. This cost therefore need only act to suppress large-scale, rapid destruction by the host in order to drive the maintenance of a surviving subsection of the endosymbiont population. ...
Article
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Significance Stable endosymbiosis between eukaryotic microbes has driven the evolution of further cellular complexity. Yet the mechanisms that can act to stabilize an emergent eukaryote–eukaryote endosymbiosis are unclear. Using the model facultative endosymbiotic system, Paramecium bursaria , we demonstrate that endosymbiont–host RNA–RNA interactions can drive a cost to host growth upon endosymbiont digestion. These RNA–RNA interactions are facilitated by the host RNA-interference system. For endosymbiont messenger RNA sharing a high level of sequence identity with host transcripts, this process can result in host gene knockdown. We propose that these endosymbiont–host RNA–RNA interactions—“RNA-interference collisions”—represent an emergent mechanism to sanction the host for breakdown of the endosymbiosis, promoting the stability of the facultative endosymbiotic interaction.
... Although S. pyriformis and P. bursaria share C. variabilis as their endosymbionts, considering the genetic differences depending on their host species, the sharing event has not happened recently. Symbiont sharing among various host species has also been known for some ciliates 41,59 (Carolibrandtia ciliaticola in Fig. S3), and a script to spread a particular algal symbiont has been suggested 41 . Given the physiological characters of C. variabilis (mentioned above), this algal species might be an ideal algal symbiont, and it will be no surprise if the other protists also retained C. variabilis as their algal partners. ...
... Although S. pyriformis and P. bursaria share C. variabilis as their endosymbionts, considering the genetic differences depending on their host species, the sharing event has not happened recently. Symbiont sharing among various host species has also been known for some ciliates 41,59 (Carolibrandtia ciliaticola in Fig. S3), and a script to spread a particular algal symbiont has been suggested 41 . Given the physiological characters of C. variabilis (mentioned above), this algal species might be an ideal algal symbiont, and it will be no surprise if the other protists also retained C. variabilis as their algal partners. ...
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The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor , but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis . The cytoplasm of S. piriformis contains a large number of 1–3 μm refractive granules that turn brown by Lugol’s staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.
... Ciliates (Alveolata) are the most common among such protozoans, along with certain Amoebozoa and Heliozoa 9 . These protists typically contain hundreds of algae within a single host cell, and we referred to them collectively as multi-algae retaining protists (MARP) 10 . In the course of our investigation of MARP on a subtropical island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained between one and two dozens transmission electron microscopy. ...
... If there is a protozoan with algae in the cell, it is difficult to determine if it is truly symbiotic or not without long-term culture. However, regardless of whether it was true symbiosis (i.e., long-time retention) or not (temporally retention), ciliate-algae combinations in natural water environment are usually observed as hundreds of small coccoid algae per one ciliate cell 9,10,21 . In this study, we focused on a Loxodes that retains between a dozen and two dozens of very large coccoid algae (Fig. 1). ...
Article
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Freshwater protists often harbor unicellular green algae within their cells. In ciliates, possibly because of large host cell sizes and the small size of algal coccoids, a single host cell typically contains more than a hundred algal cells. While surveying such algae-bearing protists on Minami Daito Jima Island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained one or two dozens of very large coccoid algae. We isolated one of these algae and analyzed its characteristics in detail. A small subunit (SSU) rDNA phylogeny indicated Pseudodidymocystis species (Scenedesmaceae, Chlorophyceae) to be the taxon closest to the alga, although it was clearly separated from this by 39 or more different sites (inclusive of gaps). SSU rRNA structure analyses indicated that these displacements included eight compensatory base changes (CBCs) and seven hemi-CBCs. We therefore concluded that this alga belongs to a separate genus, and described it as Pediludiella daitoensis gen. et sp. nov. The shape of the isolated and cultured P. daitoensis was nearly spherical and reached up to 30 µm in diameter. Chloroplasts were arranged peripherally and often split and elongated. Cells were often vacuolated and possessed a net-like cytoplasm that resembled a football (soccer ball) in appearance, which was reflected in the genus name.
... To characterize the stage of symbiosis between C. variabilis and P. bursaria, further comparative analysis with other symbiotic endosymbiotic relations is needed. Algae closely related to C. variabilis include several species that, as in C. variabilis, engage in endosymbiosis with speci c protists, which have arisen as phylogenetically independent organisms (Hoshina & Kusuoka 2016). Additional functional analyzes are needed, using techniques such as genome editing of genes related to symbiosis, as identi ed in this study. ...
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Background: Photosynthetic eukaryotes have evolved through the acquisition of plastids by secondary endosymbiosis, a process that requires several steps. Immediately before plastid acquisition, the genome of the symbiont is known to be dramatically reduced, but few studies have focused on the genomic changes in the symbiont at the early stages of secondary endosymbiosis. Methods: To investigate the genetic basis of the transition from facultative to obligate endosymbiosis, we compared the genomes of Chlorella variabilis, a representative symbiotic alga, with that of Paramecium bursaria, to compare closely related free-living species and transcriptomes between organisms in symbiotic and non-symbiotic conditions. Results: We found that the non-reduced genome of C. variabilis and its genes play a crucial role in endosymbiosis, being involved in cell wall biogenesis and degradation, and metabolic exchanges with the host. Our results suggest that the genetic mechanism underlying the enhancement of photosynthesis under symbiosis is the increasing light absorption efficiency and carbon fixation capacity of the endosymbiont, resulting in an increase in the supply of maltose to P. bursaria.
... To characterize the stage of symbiosis between C. variabilis and P. bursaria, further comparative analysis with other symbiotic endosymbiotic relations is needed. Algae closely related to C. variabilis include several species that, as in C. variabilis, engage in endosymbiosis with speci c protists, which have arisen as phylogenetically independent organisms (Hoshina & Kusuoka 2016). Additional functional analyzes are needed, using techniques such as genome editing of genes related to symbiosis, as identi ed in this study. ...
Preprint
Full-text available
Background: Photosynthetic eukaryotes have evolved through the acquisition of plastids by secondary endosymbiosis, a process that requires several steps. Immediately before plastid acquisition, the genome of the symbiont is known to be dramatically reduced, but few studies have focused on the genomic changes in the symbiont at the early stages of secondary endosymbiosis. Methods: To investigate the genetic basis of the transition from facultative to obligate endosymbiosis, we compared the genomes of Chlorella variabilis, a representative symbiotic alga, with that of Paramecium bursaria, to compare closely related free-living species and transcriptomes between organisms in symbiotic and non-symbiotic conditions. Results: We found that the non-reduced genome of C. variabilis and its genes play a crucial role in endosymbiosis, being involved in cell wall biogenesis and degradation, and metabolic exchanges with the host. Our results suggest that the genetic mechanism underlying the enhancement of photosynthesis under symbiosis is the increasing light absorption efficiency and carbon fixation capacity of the endosymbiont, resulting in an increase in the supply of maltose to P. bursaria.
... In order to investigate the genetic basis of an emergent endosymbiotic system, we must develop experimentally tractable endosymbiotic model species [5][6][7]. Paramecium bursaria is a ciliate protist which harbours several hundred cells of the green algae, Chlorella spp., in a nascent and facultative photoendosymbiosis [8][9][10][11][12]. The algae provide sugar and oxygen derived from photosynthesis, in exchange for amino acids, CO 2 , divalent cations and protection from viruses and other predators [5,6,[13][14][15][16][17][18][19]. ...
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Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote–eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1 , which we show to be crucial to host growth. Finally, using simultaneous knock-down ‘paradox’ controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1 , Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria – Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.
... In addition, instead of being inside M. polymorpha directly, some of the detected taxa may be living inside each other. For example, trebouxiophycean algae can be intracellular symbionts of ciliates like Colpoda and Paramecium(Glime 2013a;Hoshina and Kusuoka 2016). ...
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Within their tissues, plants host diverse communities of fungi, termed fungal endophytes. These fungi can affect plant growth, competitiveness, and resistance to stressors, thereby influencing plant community structure. Research characterizing fungal endophyte communities has so far mostly focused on seed plants, but information on the endophytes of other plant lineages is needed to understand how plant microbiomes impact whole ecosystems and how major changes through land plant evolution have affected plant-microbe relationships. In this study, we assess the fungal endophyte community of the model liverwort Marchantia polymorpha L. by both culturing and Illumina amplicon sequencing methods. We detect a very diverse fungal community that is distinct between M. polymorpha patches and only shares a few core fungi between populations across the United States. We also show low overlap in taxa detected by the different methods. This study helps build a foundation for using M. polymorpha and other Marchantia species as models for the ecology and dynamics of bryophyte microbiomes.
... However, as genome data were not available for symbionts other than C. variabilis and closely related species, it was not possible to compare genomes to clarify the genomic changes associated with becoming a symbiont. To shed light on the genomic change of the early stages of plastid acquisition, it is necessary to sequence the genomes of symbionts and free-living species other than C. variabilis; there are many species of symbiotic green algae, and the host organisms have diversified to Ciliates (Alveolata) as well as Amoebozoa, Heliozoa, and other protists [8]. Thus, we must examine genomic changes in these symbionts and the transcriptomic interactions of hosts and symbionts. ...
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Background: The plastid acquisition by secondary endosymbiosis is a driving force for the algal evolution, and the comparative genomics was required to examine the genomic change of symbiont. Therefore, we established a pipeline of a de novo assembly of middle-sized genomes at a low cost and with high quality using long and short reads. Results: We sequenced symbiotic algae Chlorella variabilis using Oxfofrd Nanopore MinION as the long-read sequencer and Illumina HiSeq 4000 as the short-read sequencer and then assembled the genomes under various conditions. Subsequently, we evaluated these assemblies by the gene model quality and RNA-seq mapping rate. We found that long-read only assembly could not be suitable for the comparative genomics studies, but with short reads, we could obtain the acceptable assembly. On the basis of this result, we established the pipeline of de novo assembly for middle-sized algal genome using MinION. Conclusions: The genomic change during the early stages of plastid acquisition can now be revealed by sequencing and comparing many algal genomes. Moreover, this pipeline offers a solution for the assembly of various middle-sized eukaryotic genomes with high-quality and ease.
... Many freshwater protists harbor unicellular green algae within their cells and these host-symbiont relationships are slowly becoming better understood. Recently, Hoshina and Kusuoka (2016) reported that several ciliate species shared a single species of symbiotic algae belonging to Chlorellaceae, Trebouxiophyceae. On the grounds of phylogenetic independency and unique compensatory base change in the small subunit (SSU) rRNA gene, a new genus name for this alga, Brandtia Hoshina (with a single species, B. ciliaticola ...
Article
Recently, the genus name Brandtia Hoshina was given for a chlorellacean symbiotic alga residing in some ciliate species. However, it was found that Brandtia Hoshina 2017 is an illegitimate later homonym of Brandtia Kunth 1831. Therefore, a replacement name, Carolibrandtia nom. nov., for Brandtia Hoshina is here proposed.
... Comparative analysis of different Chlorella-like symbionts [79] revealed five phylogenetically distinct symbionts, namely Chlorella, Choricystis, Coccomyxa, Scenedesmus and Micractinium, which could be found in several ciliates. Recently representatives of a new green algae group called "Chlorb" (probably forming a new genus) were retrieved from four green ciliates isolated from Lake Biwa in Japan [82]. Now also the genus Meyerella should be added to the list of symbiosis-forming algae after its retrieval in the cytoplasm of both German [12] and Russian P. chlorelligerum. ...
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Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of “green” ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name “Candidatus Holospora parva” for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.
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Previous studies on the colonial coccoid green algal genus Dictyosphaerium have shown a polyphyletic origin of this morphotype within the Chlorellaceae. Recent molecular analyses assigned the type species D. ehrenbergianum to the Parachlorella clade of the Chlorellaceae. In the present study we focused on strains of D. tetrachotomum, one of the most frequent morphospecies of the genus in inland waters. Analyses of combined SSU and ITS rRNA gene sequences revealed that strains with D. tetrachotomum morphology do not share a close phylogenetic relationship with the type species D. ehrenbergianum. These strains are part of a subclade of the Chlorella clade of the Chlorellaceae, nested between members of Didymogenes and a second new lineage of different Dictyosphaerium-like strains. Due to the unique position of these strains in the tree, we here establish the new genera Hindakia and Heynigia. Morphological analysis and phylogenetic and secondary structure analyses of the SSU, ITS1 and ITS2 of Hindakia strains have revealed considerable differences within this subclade, leading to the separation of two distinct species: H. tetrachotoma and H. fallax. Closely related to this new genus are two colony-forming strains with spherical cells, constituting a distinct lineage consisting of Heynigia dictyosphaerioides, sp. nov., and Heynigia riparia, sp. nov. Further, our results have confirmed the polyphyletic origin of the Dictyosphaerium morphotype within the Chlorellaceae.
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Nuclear-encoded small subunit ribosomal RNA gene (18SrDNA) sequences were determined for Chlamydomonas moewusii Gerloff and five chlorococcalean algae (Chlorococcum hypnosporum Starr; Chlorococcum oleofaciens Trainor et Bold; Chlorococcum sp.; Tetracystis aeria Brown et Bold; Protosiphon botryoides (Kützingl Klebs). All these algae are characterized by a clockwise CCW) flagellar apparatus. Phylogenetic trees were constructed from sequences from these algae together with 20 green algae. All algae with a CW flagellar apparatus form a monophyletic clade (CW group). Three principal clades can be recognized in the CW group, although no morphological character supports monophyly of any of these three clades. The 18SrDNA trees clearly demonstrate the non-monophyly of the Chlamydomonadales and Chlorococcales, suggesting that vegetative morphology does not reflect phylogenetic relationships in the CW group. The paraphyly or polyphyly of the genus Chlamydomonas and Chlorococcum are also revealed. Present analysis suggests that the presence or absence of a zoospore's cell wall and the multinucleate condition have limited taxonomic values at higher taxonomic ranks.
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Symbiotic algae of the ciliate Paramecium bursaria (Ehrenberg) Focker are key species in the fields of virology and molecular evolutionary biology as well as in the biology of symbiotic relationships. These symbiotic algae were once identified as Zoochlorella conductrix Brandt by the Dutch microbiologist, Beijerinck 120 years ago. However, after many twists and turns, the algae are today treated as nameless organisms. Recent molecular analyses have revealed several different algal partners depending on P. bursaria strains, but nearly all P. bursaria contains a symbiont belonging to either the so-called ‘American’ or ‘European’ group. The absence of proper names for these algae is beginning to provoke ill effects in the above-mentioned study areas. In the present study, we confirmed the genetic autonomy of the ‘American’ and ‘European’ groups and described the symbionts as Chlorella variabilis Shihira et Krauss and Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov., respectively (Chlorellaceae, Trebouxiophyceae).
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The nature of Chlorella symbioses in invertebrates and protists has attracted much interest, but the uncertain taxonomy of the algal partner has constrained a deeper ecological understanding of this symbiosis. We sequenced parts of the nuclear 18S rDNA, the internal transcribed spacer (ITS)-1 region, and the chloroplast 16S rDNA of several Chlorella isolated from pelagic ciliate species of different lakes, Paramecium bursaria symbionts, and free-living Chlorella to elucidate phylogenetic relationships of Chlorella-like algae and to assess their host specificity. Sequence analyses resulted in well-resolved phylogenetic trees providing strong statistical support for a homogenous ‘zoochlorellae’ group of different ciliate species from one lake, but clearly different Chlorella in one of those ciliate species occurring in another lake. The two Chlorella strains isolated from the same ciliate species, but from lakes having a 10-fold difference in underwater UV transparency, also presented a distinct physiological trait, such as the ability to synthesize UV-absorbing substances known as mycosporine-like amino acids (MAAs). Algal symbionts of all P. bursaria strains of different origin resolved in one clade apart from the other ciliate symbionts but split into two distinct lineages, suggesting the existence of a biogeographic pattern. Overall, our results suggest a high degree of species specificity but also hint at the importance of physiological adaptation in symbiotic Chlorella.
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Symbiosis of green algae with protozoa and invertebrates has been studied for more than 100 years. Endosymbiotic green algae are widely distributed in ciliates (e.g. Paramecium, Stentor, Climacostomum, Coleps, Euplotes), heliozoa (e.g. Acanthocystis) and invertebrates (e.g. Hydra, Spongilla), and have traditionally been identified as named or unnamed species of Chlorella Beij. or Zoochlorella K. Brandt or referred to as Chlorella-like algae or zoochlorellae. We studied 17 strains of endosymbionts isolated from various hosts and geographical localities using an integrative approach (nuclear encoded small subunit and internal transcribed spacer regions of rRNA gene sequences including their secondary structures, morphology, physiology and virus sensitivity). Phylogenetic analyses have revealed them to be polyphyletic. The strains examined belong to five independent clades within the Trebouxiophyceae (Choricystis-, Elliptochloris-, Auxenochlorella- and Chlorella-clades) and Chlorophyceae (Scenedesmus-clade). The most studied host organism, Paramecium bursaria, harbours endosymbionts representing at least five different species. On the basis of our results, we propose a taxonomic revision of endosymbiotic 'Chlorella'-like green algae. Zoochlorella conductrix K. Brandt is transferred to Micractinium Fresen. and Zoochlorella parasitica K. Brandt to Choricystis (Skuja) Fott. It was shown that Choricystis minor (Skuja) Fott, the generitype, is a later heterotypic synonym of Choricystis parasitica (K. Brandt) comb. nov. A new species, Chlorella heliozoae, is proposed to accommodate the endosymbiont of Acanthocystis turfacea.
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Using a combined set of sequences of SSU and ITS regions of nuclear-encoded ribosomal DNA, the concept of the experimental algal genus Chlorella was evaluated. Conventionally in the genus Chlorella, only coccoid, solitary algae with spherical morphology that do not possess any mucilaginous envelope were included. All Chlorella species reproduce asexually by autospores. However, phylogenetic analyses showed that within the clade of 'true'Chlorella species (Chlorella vulgaris, C. lobophora, and C. sorokiniana), taxa with a mucilaginous envelope and colonial lifeform have also evolved. These algae, formerly designated as Dictyosphaerium, are considered as members of the genus Chlorella. In close relationship to Chlorella, five different genera were supported by the phylogenetic analyses: Micractinium (spherical cells, colonial, with bristles), Didymogenes (ellipsoidal cells, two-celled coenobia, with or without two spines per cell), Actinastrum (ellipsoidal cells within star-shaped coenobia), Meyerella (spherical cells, solitary, without pyrenoids), and Hegewaldia (spherical cells, colonial, with or without bristles, oogamous propagation). Based on the secondary structures of SSU and ITS rDNA sequences, molecular signatures are provided for each genus of the Chlorella clade.
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Marine diatoms rose to prominence about 100 million years ago and today generate most of the organic matter that serves as food for life in the sea. They exist in a dilute world where compounds essential for growth are recycled and shared, and they greatly influence global climate, atmospheric carbon dioxide concentration and marine ecosystem function. How these essential organisms will respond to the rapidly changing conditions in today's oceans is critical for the health of the environment and is being uncovered by studies of their genomes.
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The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS)
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Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.
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Paramecium bursaria is composed of a "host" ciliate and a "symbiont" green alga. Based upon physiology, DNA hybridization and virus infection, two types of symbionts, called "American" type and "European" type, have been reported to date. Here, we determined the 18S rDNA and internal transcribed spacer 2 (ITS2) regions for both "American" and "European" types. Sequence features clearly separated into two lineages; NC64A (USA), Syngen 2-3 (USA), Cs2 (Chinese), MRBG1 (Australian), and Japanese strains belong to the "American", whereas PB-SW1 (German) and CCAP 1660/11 (British) strains belong to the "European". In "American" 18S rDNA, three introns were inserted in the same positions as for previously described Japanese symbionts. In "European" 18S rDNA, a single intron occurred in a different position than in the "American". Between the types, sequence differences were seven or eight nucleotides (0.39 %) in the 18S rDNA exon, and more than 48 nucleotides (19.2 %) in ITS2 regions. We subsequently sequenced the host 18S rDNA. As a result, two groups: Cs2, MRBG1, and Japanese strains, and PB-SW1 and CCAP 1660/11 strains, were separated (with 23 substitutions and 4 insertions or deletions between the groups). The congruent separations between hosts and symbionts may imply that the type of symbiont depends on the host type.
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The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.
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The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems. Availability: The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/ Contact: clustalw{at}ucd.ie