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Brown algae (Phaeophyceae) are habitat-forming species in coastal ecosystems and include kelp forests and seaweed beds that support a wide diversity of marine life. Host-associated microbial communities are an integral part of phaeophyte biology, and whereas the bacterial microbial partners have received considerable attention, the microbial eukaryotes associated with brown algae have hardly been studied. Here, we used broadly targeted "pan-eukaryotic" primers (metabarcoding) to investigate brown algal-associated eukaryotes (the eukaryome). Using this approach, we aimed to investigate the eukaryome of seven large brown algae that are important and common species in coastal ecosystems. We also aimed to assess whether these macroalgae harbor novel eukaryotic diversity and to ascribe putative functional roles to the host-associated eukaryome based on taxonomic affiliation and phylogenetic placement. We detected a significant diversity of microeukaryotic and algal lineages associated with the brown algal species investigated. The operational taxonomic units (OTUs) were taxonomically assigned to 10 of the eukaryotic major supergroups, including taxonomic groups known to be associated with seaweeds as epibionts, endobionts, parasites, and commensals. Additionally, we revealed previously unrecorded sequence types, including novel phaeophyte OTUs, particularly in the Fucus spp. samples, that may represent fucoid genomic variants, sequencing artifacts, or undescribed epi-/endophytes. Our results provide baseline data and technical insights that will be useful for more comprehensive seaweed eukaryome studies investigating the evidently lineage-rich and functionally diverse symbionts of brown algae.
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Here, we present new transcriptome sequencing data from seven species of Dasycladales (Ulvophyceae) and a phylogenomic analysis of the Chlorophyta with a particular focus on Ulvophyceae. We have focused on a broad selection of green algal groups and carefully selected genes suitable for reconstructing deep eukaryote evolutionary histories. Increasing the taxon sampling of Dasycladales restructures the Ulvophyceae by identifying Dasycladales as closely related to Scotinosphaerales and Oltmannsiellopsidales. Contrary to previous studies, we do not find support for a close relationship between Dasycladales and a group with Cladophorales and Trentepohliales. Instead, the latter group is sister to the remainder of the Ulvophyceae. Furthermore, our analyses show high and consistent statistical support for a sister relationship between Bryopsidales and Chlorophyceae in trees generated with both homogeneous and heterogeneous (heterotachy) evolutionary models. Our study provides a new framework for interpreting the evolutionary history of Ulvophyceae and the evolution of cellular morphologies.
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Chlorophyceae , Chlorophyta , Chlorophyta/genética , Eucariontes , Evolución Molecular , FilogeniaRESUMEN
In the original publication of this article [1] there was an error in an author name. In this correction article the correct and incorrect name are indicated.
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BACKGROUND: The class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea. RESULTS: We cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S-28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I - III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II + III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms. CONCLUSIONS: This altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.
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Biodiversidad , Eucariontes/clasificación , Eucariontes/genética , Análisis de Secuencia de ADN/métodos , Cartilla de ADN/metabolismo , Ecosistema , Eucariontes/citología , Agua Dulce , Filogenia , ARN Ribosómico 18S/genéticaRESUMEN
The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport, and cytokinesis, and is largely responsible for the diversity of morphologies among eukaryotes. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton, we provide the first single cell transcriptomes from uncultured, free-living unicellular eukaryotes: the polycystine radiolarian Lithomelissa setosa (Nassellaria) and Sticholonche zanclea (Taxopodida). A phylogenomic approach using 255 genes finds Radiolaria and Foraminifera as separate monophyletic groups (together as Retaria), while Cercozoa is shown to be paraphyletic where Endomyxa is sister to Retaria. Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these on the revised phylogeny of Rhizaria reveal lineage-specific gene duplications and neofunctionalization of α and ß tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for resolving deep phylogenies and studying gene evolution in uncultured protist species.
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Rhizaria/clasificación , Rhizaria/genética , Teorema de Bayes , Evolución Biológica , Eucariontes/genética , Células Eucariotas , Evolución Molecular , Filogenia , Rhizaria/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Tubulina (Proteína)/genéticaRESUMEN
The Basic Local Alignment Search Tool (BLAST) algorithm remains one of the most widely used bioinformatic programs. For many projects, new sequencing technologies and increased database sizes will increase the BLAST output significantly. Frequently, this output is so large that it is no longer able to be processed manually. As BLAST users are increasingly recruited from mainstream biology without any bioinformatic background, user-friendly programs capable of BLAST output visualization, analysis and post-processing are in demand. In this review, freely available BLAST output processing programs are categorized as BLAST output interpreters, BLAST environments, BLAST output parsers or specialized tools. They are evaluated according to their user-friendliness, analysis features and high-throughput data processing capabilities.
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Alineación de Secuencia , Análisis de Secuencia/métodos , Algoritmos , Biología ComputacionalRESUMEN
Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.
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Alveolados/genética , Sedimentos Geológicos/microbiología , Plancton/clasificación , Plancton/genética , Agua de Mar/microbiología , Estramenopilos/genética , Secuencia de Bases , Biodiversidad , Ecosistema , Europa (Continente) , Hongos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
Long non-coding RNAs (lncRNAs) play important regulatory roles during animal development, and it has been hypothesized that an RNA-based gene regulation was important for the evolution of developmental complexity in animals. However, most studies of lncRNA gene regulation have been performed using model animal species, and very little is known about this type of gene regulation in non-bilaterians. We have therefore analysed RNA-Seq data derived from a comprehensive set of embryogenesis stages in the calcareous sponge Sycon ciliatum and identified hundreds of developmentally expressed intergenic lncRNAs (lincRNAs) in this species. In situ hybridization of selected lincRNAs revealed dynamic spatial and temporal expression during embryonic development. More than 600 lincRNAs constitute integral parts of differentially expressed gene modules, which also contain known developmental regulatory genes, e.g. transcription factors and signalling molecules. This study provides insights into the non-coding gene repertoire of one of the earliest evolved animal lineages, and suggests that RNA-based gene regulation was probably present in the last common ancestor of animals.
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Poríferos/genética , ARN Largo no Codificante/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Poríferos/embriología , Análisis de Componente Principal , TranscriptomaRESUMEN
BACKGROUND: Advances in sequencing efficiency have vastly increased the sizes of biological sequence databases, including many thousands of genome-sequenced species. The BLAST algorithm remains the main search engine for retrieving sequence information, and must consequently handle data on an unprecedented scale. This has been possible due to high-performance computers and parallel processing. However, the raw BLAST output from contemporary searches involving thousands of queries becomes ill-suited for direct human processing. Few programs attempt to directly visualize and interpret BLAST output; those that do often provide a mere basic structuring of BLAST data. RESULTS: Here we present a bioinformatics application named BLASTGrabber suitable for high-throughput sequencing analysis. BLASTGrabber, being implemented as a Java application, is OS-independent and includes a user friendly graphical user interface. Text or XML-formatted BLAST output files can be directly imported, displayed and categorized based on BLAST statistics. Query names and FASTA headers can be analysed by text-mining. In addition to visualizing sequence alignments, BLAST data can be ordered as an interactive taxonomy tree. All modes of analysis support selection, export and storage of data. A Java interface-based plugin structure facilitates the addition of customized third party functionality. CONCLUSION: The BLASTGrabber application introduces new ways of visualizing and analysing massive BLAST output data by integrating taxonomy identification, text mining capabilities and generic multi-dimensional rendering of BLAST hits. The program aims at a non-expert audience in terms of computer skills; the combination of new functionalities makes the program flexible and useful for a broad range of operations.
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Biología Computacional/métodos , Bases de Datos Factuales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Algoritmos , Secuencia de Aminoácidos , Metodologías Computacionales , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²âº-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.
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Calpaína/química , Proteínas de Plantas/química , Plantas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Calcio/metabolismo , Calpaína/genética , Calpaína/fisiología , Carofíceas/genética , Carofíceas/metabolismo , Secuencia Conservada , Evolución Molecular , Prueba de Complementación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
The current consensus for the eukaryote tree of life consists of several large assemblages (supergroups) that are hypothesized to describe the existing diversity. Phylogenomic analyses have shed light on the evolutionary relationships within and between supergroups as well as placed newly sequenced enigmatic species close to known lineages. Yet, a few eukaryote species remain of unknown origin and could represent key evolutionary forms for inferring ancient genomic and cellular characteristics of eukaryotes. Here, we investigate the evolutionary origin of the poorly studied protist Collodictyon (subphylum Diphyllatia) by sequencing a cDNA library as well as the 18S and 28S ribosomal DNA (rDNA) genes. Phylogenomic trees inferred from 124 genes placed Collodictyon close to the bifurcation of the "unikont" and "bikont" groups, either alone or as sister to the potentially contentious excavate Malawimonas. Phylogenies based on rDNA genes confirmed that Collodictyon is closely related to another genus, Diphylleia, and revealed a very low diversity in environmental DNA samples. The early and distinct origin of Collodictyon suggests that it constitutes a new lineage in the global eukaryote phylogeny. Collodictyon shares cellular characteristics with Excavata and Amoebozoa, such as ventral feeding groove supported by microtubular structures and the ability to form thin and broad pseudopods. These may therefore be ancient morphological features among eukaryotes. Overall, this shows that Collodictyon is a key lineage to understand early eukaryote evolution.
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Eucariontes/clasificación , Tipificación de Secuencias Multilocus , Filogenia , Teorema de Bayes , Eucariontes/enzimología , Eucariontes/genética , Evolución Molecular , Biblioteca de Genes , Especiación Genética , Funciones de Verosimilitud , Modelos Genéticos , ARN Ribosómico 18S/genética , ARN Ribosómico 28S/genética , Alineación de Secuencia , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/genéticaRESUMEN
Many of the eukaryotic phylogenomic analyses published to date were based on alignments of hundreds to thousands of genes. Frequently, in such analyses, the most realistic evolutionary models currently available are often used to minimize the impact of systematic error. However, controversy remains over whether or not idiosyncratic gene family dynamics (i.e., gene duplications and losses) and incorrect orthology assignments are always appropriately taken into account. In this paper, we present an innovative strategy for overcoming orthology assignment problems. Rather than identifying and eliminating genes with paralogy problems, we have constructed a data set comprised exclusively of conserved single-copy protein domains that, unlike most of the commonly used phylogenomic data sets, should be less confounded by orthology miss-assignments. To evaluate the power of this approach, we performed maximum likelihood and Bayesian analyses to infer the evolutionary relationships within the opisthokonts (which includes Metazoa, Fungi, and related unicellular lineages). We used this approach to test 1) whether Filasterea and Ichthyosporea form a clade, 2) the interrelationships of early-branching metazoans, and 3) the relationships among early-branching fungi. We also assessed the impact of some methods that are known to minimize systematic error, including reducing the distance between the outgroup and ingroup taxa or using the CAT evolutionary model. Overall, our analyses support the Filozoa hypothesis in which Ichthyosporea are the first holozoan lineage to emerge followed by Filasterea, Choanoflagellata, and Metazoa. Blastocladiomycota appears as a lineage separate from Chytridiomycota, although this result is not strongly supported. These results represent independent tests of previous phylogenetic hypotheses, highlighting the importance of sophisticated approaches for orthology assignment in phylogenomic analyses.
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Eucariontes/genética , Evolución Molecular , Hongos/genética , Filogenia , Animales , Teorema de Bayes , Genómica , Funciones de Verosimilitud , Modelos Genéticos , Estructura Terciaria de Proteína/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Recently, phylogenomic analyses have been used to assign the vast majority of eukaryotes into only a handful of supergroups. However, a few enigmatic lineages still do not fit into this simple picture. Such lineages may have originated early in the history of eukaryotes and are therefore of key importance in deduction of cellular evolution. In this study, we focus on two deeply diverging lineages, Diphyllatea and Thecamonadea. They are classified in the same phylum, Sulcozoa, but previous multigene phylogenetic analyses have included only one of these two lineages. It is therefore unclear whether they constitute one group or two distinct lineages. The study of rare genomic changes reveals that both have the fused dihydrofolate reductase (DHFR) and thymidylate synthase (TS) genes (i.e. DHFR-TS), which are separated in all other unikonts that have been investigated, indicating a possible close relationship. Their phylogenetic positions have implications for the classification of Sulcozoa and the early eukaryote evolution. Here we present a phylogenomic analysis of these species that include Illumina and 454 transcriptome data from two Collodictyon strains. A total of 42 mitochondrial proteins, which correspond to orthologs published from Thecamonas trahens (Thecamonadea), were used to reconstruct their phylogenies. In the resulting trees, Collodictyon appears as sister to Amoebozoa, whereas Thecamonas branches as the closest relative of Opisthokonta (i.e. the animal, fungi and unicellular Choanozoa). In contrast, the position of another early diverging eukaryote, Malawimonas, is unresolved. The separation of Collodictyon and Thecamonas in our studies suggests that the recently proposed Sulcozoa group is most likely paraphyletic. Furthermore, the data support the hypothesis that the two supergroups Opisthokonta and Amoebozoa, which comprise a great diversity of eukaryotes, have originated from a sulcozoan ancestor.
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Evolución Biológica , Eucariontes/clasificación , Proteínas Mitocondriales/genética , Filogenia , Teorema de Bayes , Eucariontes/genética , Funciones de Verosimilitud , Modelos Genéticos , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. RESULTS: Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. CONCLUSIONS: The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.
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Calpaína/genética , Células Eucariotas/metabolismo , Variación Genética , Filogenia , Teorema de Bayes , Sitios de Unión/genética , Calpaína/clasificación , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Entamoeba histolytica/enzimología , Entamoeba histolytica/genética , Células Eucariotas/citología , Células Eucariotas/enzimología , Evolución Molecular , Modelos Genéticos , Especificidad de la Especie , Trichomonas vaginalis/enzimología , Trichomonas vaginalis/genéticaRESUMEN
BACKGROUND: The implementation of high throughput sequencing for exploring biodiversity poses high demands on bioinformatics applications for automated data processing. Here we introduce CLOTU, an online and open access pipeline for processing 454 amplicon reads. CLOTU has been constructed to be highly user-friendly and flexible, since different types of analyses are needed for different datasets. RESULTS: In CLOTU, the user can filter out low quality sequences, trim tags, primers, adaptors, perform clustering of sequence reads, and run BLAST against NCBInr or a customized database in a high performance computing environment. The resulting data may be browsed in a user-friendly manner and easily forwarded to downstream analyses. Although CLOTU is specifically designed for analyzing 454 amplicon reads, other types of DNA sequence data can also be processed. A fungal ITS sequence dataset generated by 454 sequencing of environmental samples is used to demonstrate the utility of CLOTU. CONCLUSIONS: CLOTU is a flexible and easy to use bioinformatics pipeline that includes different options for filtering, trimming, clustering and taxonomic annotation of high throughput sequence reads. Some of these options are not included in comparable pipelines. CLOTU is implemented in a Linux computer cluster and is freely accessible to academic users through the Bioportal web-based bioinformatics service (http://www.bioportal.uio.no).
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Clasificación/métodos , Biología Computacional/métodos , Hongos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , ADN de Hongos/genética , ADN Ribosómico/genéticaRESUMEN
The aquatic bacterial group SAR11 is one of the most abundant organisms on Earth, with an estimated global population size of 2.4 x 10(28) cells in the oceans. Members of SAR11 have also been detected in brackish and fresh waters, but the evolutionary relationships between the species present in the different environments have been ambiguous. In particular, it was not clear how frequently this lineage has crossed the saline-freshwater boundary during its evolutionary diversification. Due to the huge population size of SAR11 and the potential of microbes for long-distance dispersal, we hypothesized that environmental transitions could have occurred repeatedly during the evolutionary diversification of this group. Here, we have constructed extensive 16S rDNA-based molecular phylogenies and undertaken metagenomic data analyses to assess the frequency of saline-freshwater transitions in SAR11 and to investigate the evolutionary implications of this process. Our analyses indicated that very few saline-freshwater transitions occurred during the evolutionary diversification of SAR11, generating genetically distinct saline and freshwater lineages that do not appear to exchange genes extensively via horizontal gene transfer. In contrast to lineages from saline environments, extant freshwater taxa from diverse, and sometimes distant, geographic locations were very closely related. This points to a rapid diversification and dispersal in fresh waters or to slower evolutionary rates in fresh water SAR11 when compared with marine counterparts. In addition, the colonization of both saline and fresh waters appears to have occurred early in the evolution of SAR11. We conclude that the different biogeochemical conditions that prevail in saline and fresh waters have likely prevented the environmental transitions in SAR11, promoting the evolution of clearly distinct lineages in each environment.
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Bacterias/genética , Agua Dulce/microbiología , Agua de Mar/microbiología , Bacterias/clasificación , Bacterias/crecimiento & desarrollo , Evolución Molecular , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
The Haptophyta is a common algal group in many marine environments, but only a few species have been observed in freshwaters, with DNA sequences available from just a single species, Crysochromulina parva Lackey, 1939. Here we investigate the haptophyte diversity in a high mountain lake, Lake Finsevatn, Norway, targeting the variable V4 region of the 18S rDNA gene with PCR and 454 pyrosequencing. In addition, the freshwater diversity of Pavlovophyceae was investigated by lineage-specific PCR-primers and clone library sequencing from another Norwegian lake, Lake Svaersvann. We present new freshwater phylotypes belonging to the classes Prymnesiophyceae and Pavlovophyceae, as well as a distinct group here named HAP-1. This is the first molecular evidence of a freshwater species belonging to the class Pavlovophyceae. The HAP-1 and another recently detected marine group (i.e. HAP-2) are separated from both Pavlovophyceae and Prymnesiophyceae and may constitute new higher order taxonomic lineages. As all obtained freshwater sequences of haptophytes are distantly related to the freshwater species C. parva, the phylogeny demonstrates that haptophytes colonized freshwater on multiple independent occasions. One of these colonizations, which gave rise to HAP-1, took place very early in the history of haptophytes, before the radiation of the Prymnesiophyceae.
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Haptophyta/clasificación , Haptophyta/aislamiento & purificación , Lagos , Organismos Acuáticos/clasificación , Organismos Acuáticos/genética , Organismos Acuáticos/aislamiento & purificación , Secuencia de Bases , ADN Ribosómico , Haptophyta/genética , Noruega , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 18S/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The macroscopic single-celled green alga Acetabularia acetabulum has been a model system in cell biology for more than a century. However, no genomic information is available from this species. Since the alga has a long life cycle, is difficult to grow in dense cultures, and has an estimated diploid genome size of almost 2 Gb, obtaining sufficient genomic material for genome sequencing is challenging. Here, we have attempted to overcome these challenges by amplifying genomic DNA using multiple displacement amplification (MDA) combined with microfluidics technology to distribute the amplification reactions across thousands of microscopic droplets. By amplifying and sequencing DNA from five single cells we were able to recover an estimated ~ 7-11% of the total genome, providing the first draft of the A. acetabulum genome. We highlight challenges associated with genome recovery and assembly of MDA data due to biases arising during genome amplification, and hope that our study can serve as a reference for future attempts on sequencing the genome from non-model eukaryotes.
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Acetabularia/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Secuenciación Completa del Genoma , Acetabularia/citología , Acetabularia/aislamiento & purificación , ADN/genética , Genoma , Anotación de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Plastid replacements through secondary endosymbioses include massive transfer of genes from the endosymbiont to the host nucleus and require a new targeting system to enable transport of the plastid-targeted proteins across 3-4 plastid membranes. The dinoflagellates are the only eukaryotic lineage that has been shown to have undergone several plastid replacement events, and this group is thus highly relevant for studying the processes involved in plastid evolution. In this study, we analyzed the phylogenetic origin and N-terminal extensions of plastid-targeted proteins from Lepidodinium chlorophorum, a member of the only dinoflagellate genus that harbors a green secondary plastid rather than the red algal-derived, peridinin-containing plastid usually found in photosynthetic dinoflagellates. RESULTS: We sequenced 4,746 randomly picked clones from a L. chlorophorum cDNA library. 22 of the assembled genes were identified as genes encoding proteins functioning in plastids. Some of these were of green algal origin. This confirms that genes have been transferred from the plastid to the host nucleus of L. chlorophorum and indicates that the plastid is fully integrated as an organelle in the host. Other nuclear-encoded plastid-targeted protein genes, however, are clearly not of green algal origin, but have been derived from a number of different algal groups, including dinoflagellates, streptophytes, heterokonts, and red algae. The characteristics of N-terminal plastid-targeting peptides of all of these genes are substantially different from those found in peridinin-containing dinoflagellates and green algae. CONCLUSIONS: L. chlorophorum expresses plastid-targeted proteins with a range of different origins, which probably arose through endosymbiotic gene transfer (EGT) and horizontal gene transfer (HGT). The N-terminal extension of the genes is different from the extensions found in green alga and other dinoflagellates (peridinin- and haptophyte plastids). These modifications have likely enabled the mosaic proteome of L. chlorophorum.
Asunto(s)
Dinoflagelados/genética , Filogenia , Plastidios/genética , Proteoma/genética , Secuencia de Aminoácidos , Dinoflagelados/clasificación , Evolución Molecular , Biblioteca de Genes , Transferencia de Gen Horizontal , Datos de Secuencia Molecular , Plastidios/metabolismo , ARN Protozoario/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Simbiosis/genéticaRESUMEN
BACKGROUND: Recent surveys of eukaryote 18S rDNA diversity in marine habitats have uncovered worldwide distribution of the heterotrophic eukaryote phylum Telonemia. Here we investigate the diversity and geographic distribution of Telonemia sequences by in-depth sequencing of several new 18S rDNA clone libraries from both marine and freshwater sites by using a Telonemia-specific PCR strategy. RESULTS: In contrast to earlier studies that have employed eukaryote-wide PCR design, we identified a large and unknown diversity of phylotypes and the first rigorous evidence for several freshwater species, altogether comprising 91 unique sequences. Phylogenies of these and publicly available sequences showed 20 statistically supported sub-clades as well as several solitary phylotypes with no clear phylogenetic affiliation. Most of these sub-clades were composed of phylotypes from different geographic regions. CONCLUSIONS: By using specific PCR primers we reveal a much larger diversity of Telonemia from environmental samples than previously uncovered by eukaryote-wide primers. The new data substantially diminish the geographic structuring of clades identified in earlier studies. Nevertheless, since these clades comprise several distinct phylotypes we cannot exclude endemicity at species level. We identified two freshwater clades and a few solitary phylotypes, implying that Telonemia have colonized freshwater habitats and adapted to the different environmental and ecological conditions at independent occasions.