18S rDNA gene metabarcoding of microeukaryotes and epi-endophytes in the holobiome of seven species of large brown algae.

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.

Genome sequencing and de novo assembly of the giant unicellular alga Acetabularia acetabulum using droplet MDA.

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.

Phylogenomic analysis restructures the ulvophyceae.

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.

Unicellular Origin of the Animal MicroRNA Machinery.

The emergence of multicellular animals was associated with an increase in phenotypic complexity and with the acquisition of spatial cell differentiation and embryonic development. Paradoxically, this phenotypic transition was not paralleled by major changes in the underlying developmental toolkit and regulatory networks. In fact, most of these systems are ancient, established already in the unicellular ancestors of animals [1-5]. In contrast, the Microprocessor protein machinery, which is essential for microRNA (miRNA) biogenesis in animals, as well as the miRNA genes themselves produced by this Microprocessor, have not been identified outside of the animal kingdom [6]. Hence, the Microprocessor, with the key proteins Pasha and Drosha, is regarded as an animal innovation [7-9]. Here, we challenge this evolutionary scenario by investigating unicellular sister lineages of animals through genomic and transcriptomic analyses. We identify in Ichthyosporea both Drosha and Pasha (DGCR8 in vertebrates), indicating that the Microprocessor complex evolved long before the last common ancestor of animals, consistent with a pre-metazoan origin of most of the animal developmental gene elements. Through small RNA sequencing, we also discovered expressed bona fide miRNA genes in several species of the ichthyosporeans harboring the Microprocessor. A deep, pre-metazoan origin of the Microprocessor and miRNAs comply with a view that the origin of multicellular animals was not directly linked to the innovation of these key regulatory components.

Correction to: Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

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.

Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

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.

Draft Genome Sequences of Two Unclassified Bacteria, Hydrogenophaga sp. Strains IBVHS1 and IBVHS2, Isolated from Environmental Samples.

We report here the draft genome sequences of Hydrogenophaga sp. strains IBVHS1 and IBVHS2, two bacteria assembled from the metagenomes of surface samples from freshwater lakes. The genomes are >95% complete and may represent new species within the Hydrogenophaga genus, indicating a larger diversity than currently identified.

Draft Genome Sequences of Two Unclassified Bacteria, Sphingomonas sp. Strains IBVSS1 and IBVSS2, Isolated from Environmental Samples.

We report here the draft genome sequences of Sphingomonas sp. IBVSS1 and IBVSS2, two bacteria assembled from the metagenomes of surface samples from freshwater lakes. The genomes are >99% complete and may represent new species within the Sphingomonas genus, indicating a larger diversity than currently identified.

Draft Genome Sequences of Two Unclassified Chitinophagaceae Bacteria, IBVUCB1 and IBVUCB2, Isolated from Environmental Samples.

We report here the draft genome sequences of two Chitinophagaceae bacteria, IBVUCB1 and IBVUCB2, assembled from metagenomes of surface samples from freshwater lakes. The genomes are >99% complete and may represent new genera within the Chitinophagaceae family, indicating a larger diversity than currently identified.

X-Cells Are Globally Distributed, Genetically Divergent Fish Parasites Related to Perkinsids and Dinoflagellates.

"X-cells" have long been associated with tumor-like formations (xenomas) in marine fish, including many of commercial interest. The name was first used to refer to the large polygonal cells that were found in epidermal xenomas from flatfish from the Pacific Northwest [1]. Similar looking cells from pseudobranchial xenomas had previously been reported from cod in the Atlantic [2] and Pacific Oceans [3]. X-cell pathologies have been reported from five teleost orders: Pleuronectiformes (flatfish), Perciformes (perch-like fish), Gadiformes (cods), Siluriformes (catfish), and Salmoniformes (salmonids). Various explanations have been elicited for their etiology, including being adenomas or adenocarcinomas [4, 5], virally transformed fish cells [6-8], or products of coastal pollution [9, 10]. It was hypothesized that X-cells were protozoan parasites [1, 11-13], and although recent molecular analyses have confirmed this, they have failed to place them in any phylum [14-18], demonstrating weak phylogenetic associations with the haplosporidians [16] or the alveolates [15]. Here, we sequenced rRNA genes from European and Japanese fish that are known to develop X-cell xenomas. We also generated a metagenomic sequence library from X-cell xenomas of blue whiting and Atlantic cod and assembled 63 X-cell protein-coding genes for a eukaryote-wide phylogenomic analysis. We show that X-cells group in two highly divergent clades, robustly sister to the bivalve parasite Perkinsus. We formally describe these as Gadixcellia and Xcellia and provide a phylogenetic context to catalyze future research. We also screened Atlantic cod populations for xenomas and residual pathologies and show that X-cell infections are more prevalent and widespread than previously known.

Single Cell Transcriptomics, Mega-Phylogeny, and the Genetic Basis of Morphological Innovations in Rhizaria.

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.

Benthic protists: the under-charted majority.

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed.

Regulatory RNA at the root of animals: dynamic expression of developmental lincRNAs in the calcisponge Sycon ciliatum.

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.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

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.

BIR Pipeline for Preparation of Phylogenomic Data.

SUMMARY: We present a pipeline named BIR (Blast, Identify and Realign) developed for phylogenomic analyses. BIR is intended for the identification of gene sequences applicable for phylogenomic inference. The pipeline allows users to apply their own manually curated sequence alignments (seed) in search for homologous genes in sequence databases and available genomes. BIR automatically adds the identified sequences from these databases to the seed alignments and reconstruct a phylogenetic tree from each. The BIR pipeline is an efficient tool for the identification of orthologous gene copies because it expands user-defined sequence alignments and conducts massive parallel phylogenetic reconstruction. The application is also particularly useful for large-scale sequencing projects that require management of a large number of single-gene alignments for gene comparison, functional annotation, and evolutionary analyses. AVAILABILITY: The BIR user manual is available at http://www.bioportal.no/ and can be accessed through Lifeportal at https://lifeportal.uio.no. Access is free but requires a user account registration using the link "Register for BIR access" from the Lifeportal homepage.

BLASTGrabber: a bioinformatic tool for visualization, analysis and sequence selection of massive BLAST data.

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.

Patterns of rare and abundant marine microbial eukaryotes.

BACKGROUND: Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS: Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS: We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.