Gene expression dynamics of natural assemblages of heterotrophic flagellates during bacterivory.

BACKGROUND: Marine heterotrophic flagellates (HF) are dominant bacterivores in the ocean, where they represent the trophic link between bacteria and higher trophic levels and participate in the recycling of inorganic nutrients for regenerated primary production. Studying their activity and function in the ecosystem is challenging since most of the HFs in the ocean are still uncultured. In the present work, we investigated gene expression of natural HF communities during bacterivory in four unamended seawater incubations. RESULTS: The most abundant species growing in our incubations belonged to the taxonomic groups MAST-4, MAST-7, Chrysophyceae, and Telonemia. Gene expression dynamics were similar between incubations and could be divided into three states based on microbial counts, each state displaying distinct expression patterns. The analysis of samples where HF growth was highest revealed some highly expressed genes that could be related to bacterivory. Using available genomic and transcriptomic references, we identified 25 species growing in our incubations and used those to compare the expression levels of these specific genes. Video Abstract CONCLUSIONS: Our results indicate that several peptidases, together with some glycoside hydrolases and glycosyltransferases, are more expressed in phagotrophic than in phototrophic species, and thus could be used to infer the process of bacterivory in natural assemblages.

Taxonomic composition, community structure and molecular novelty of microeukaryotes in a temperate oligomesotrophic lake as revealed by metabarcoding.

Microbial eukaryotes are diverse and ecologically important organisms, yet sampling constraints have hindered the understanding of their distribution and diversity in freshwater ecosystems. Metabarcoding has provided a powerful complement to traditional limnological studies, revealing an unprecedented diversity of protists in freshwater environments. Here, we aim to expand our knowledge of the ecology and diversity of protists in lacustrine ecosystems by targeting the V4 hypervariable region of the 18S rRNA gene in water column, sediment and biofilm samples collected from Sanabria Lake (Spain) and surrounding freshwater ecosystems. Sanabria is a temperate lake, which are relatively understudied by metabarcoding in comparison to alpine and polar lakes. The phylogenetic diversity of microbial eukaryotes detected in Sanabria spans all currently recognized eukaryotic supergroups, with Stramenopiles being the most abundant and diverse supergroup in all sampling sites. Parasitic microeukaryotes account for 21% of the total protist ASVs identified in our study and were dominated by Chytridiomycota, both in terms of richness and abundance, in all sampling sites. Sediments, biofilms and water column samples harbour distinct microbial communities. Phylogenetic placement of poorly assigned and abundant ASVs indicates molecular novelty inside Rhodophyta, Bigyra, early-branching Nucletmycea and Apusomonadida. In addition, we report the first freshwater incidence of the previously exclusively marine genera Abeoforma and Sphaeroforma. Our results contribute to a deeper understanding of microeukaryotic communities in freshwater ecosystems, and provide the first molecular reference for future biomonitoring surveys in Sanabria Lake.

Divergent genomic trajectories predate the origin of animals and fungi.

Animals and fungi have radically distinct morphologies, yet both evolved within the same eukaryotic supergroup: Opisthokonta1,2. Here we reconstructed the trajectory of genetic changes that accompanied the origin of Metazoa and Fungi since the divergence of Opisthokonta with a dataset that includes four novel genomes from crucial positions in the Opisthokonta phylogeny. We show that animals arose only after the accumulation of genes functionally important for their multicellularity, a tendency that began in the pre-metazoan ancestors and later accelerated in the metazoan root. By contrast, the pre-fungal ancestors experienced net losses of most functional categories, including those gained in the path to Metazoa. On a broad-scale functional level, fungal genomes contain a higher proportion of metabolic genes and diverged less from the last common ancestor of Opisthokonta than did the gene repertoires of Metazoa. Metazoa and Fungi also show differences regarding gene gain mechanisms. Gene fusions are more prevalent in Metazoa, whereas a larger fraction of gene gains were detected as horizontal gene transfers in Fungi and protists, in agreement with the long-standing idea that transfers would be less relevant in Metazoa due to germline isolation3-5. Together, our results indicate that animals and fungi evolved under two contrasting trajectories of genetic change that predated the origin of both groups. The gradual establishment of two clearly differentiated genomic contexts thus set the stage for the emergence of Metazoa and Fungi.

Mixotrophy in the bloom-forming genus Phaeocystis and other haptophytes.

Phaeocystis is a globally widespread marine phytoplankton genus, best known for its colony-forming species that can form large blooms and odorous foam during bloom decline. In the North Sea, Phaeocystis globosa typically becomes abundant towards the end of the spring bloom, when nutrients are depleted and the share of mixotrophic protists increases. Although mixotrophy is widespread across the eukaryotic tree of life and is also found amongst haptophytes, a mixotrophic nutrition has not yet been demonstrated in Phaeocystis. Here, we sampled two consecutive Phaeocystis globosa spring blooms in the coastal North Sea. In both years, bacterial cells were observed inside 0.6 - 2% of P. globosa cells using double CARD-FISH hybridizations in combination with laser scanning confocal microscopy. Incubation experiments manipulating light and nutrient availability showed a trend towards higher occurrence of intracellular bacteria under P-deplete conditions. Based on counts of bacteria inside P. globosa cells in combination with theoretical values of prey digestion times, maximum ingestion rates of up to 0.08 bacteria cell-1 h-1 were estimated. In addition, a gene-based predictive model was applied to the transcriptome assemblies of seven Phaeocystis strains and 24 other haptophytes to assess their trophic mode. This model predicted a phago-mixotrophic feeding strategy in several (but not all) strains of P. globosa, P. antarctica and other haptophytes that were previously assumed to be autotrophic. The observation of bacterial cells inside P. globosa and the gene-based model predictions strongly suggest that the phago-mixotrophic feeding strategy is widespread among members of the Phaeocystis genus and other haptophytes, and might contribute to their remarkable success to form nuisance blooms under nutrient-limiting conditions.

Metagenome assembled genomes are for eukaryotes too.

Environmental community sequencing is suitable for producing metagenome assembled genomes (MAGs) of prokaryotes, but there is the perception that it cannot work for eukaryotes. In this issue of Cell Genomics, Delmont et al1 process a massive sequencing dataset from marine plankton to produce 683 eukaryotic MAGs; the study also includes useful functional information.

Comparative genomics reveals new functional insights in uncultured MAST species.

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles.

Gene expression during bacterivorous growth of a widespread marine heterotrophic flagellate.

Phagocytosis is a fundamental process in marine ecosystems by which prey organisms are consumed and their biomass incorporated in food webs or remineralized. However, studies searching for the genes underlying this key ecological process in free-living phagocytizing protists are still scarce, in part due to the lack of appropriate ecological models. Our reanalysis of recent molecular datasets revealed that the cultured heterotrophic flagellate Cafeteria burkhardae is widespread in the global oceans, which prompted us to design a transcriptomics study with this species, grown with the cultured flavobacterium Dokdonia sp. We compared the gene expression between exponential and stationary phases, which were complemented with three starvation by dilution phases that appeared as intermediate states. We found distinct expression profiles in each condition and identified 2056 differentially expressed genes between exponential and stationary samples. Upregulated genes at the exponential phase were related to DNA duplication, transcription and translational machinery, protein remodeling, respiration and phagocytosis, whereas upregulated genes in the stationary phase were involved in signal transduction, cell adhesion, and lipid metabolism. We identified a few highly expressed phagocytosis genes, like peptidases and proton pumps, which could be used to target this ecologically relevant process in marine ecosystems.

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

Reconstruction of protein domain evolution using single-cell amplified genomes of uncultured choanoflagellates sheds light on the origin of animals.

Understanding the origins of animal multicellularity is a fundamental biological question. Recent genome data have unravelled the role that co-option of pre-existing genes played in the origin of animals. However, there were also some important genetic novelties at the onset of Metazoa. To have a clear understanding of the specific genetic innovations and how they appeared, we need the broadest taxon sampling possible, especially among early-branching animals and their unicellular relatives. Here, we take advantage of single-cell genomics to expand our understanding of the genomic diversity of choanoflagellates, the sister-group to animals. With these genomes, we have performed an updated and taxon-rich reconstruction of protein evolution from the Last Eukaryotic Common Ancestor (LECA) to animals. Our novel data re-defines the origin of some genes previously thought to be metazoan-specific, like the POU transcription factor, which we show appeared earlier in evolution. Moreover, our data indicate that the acquisition of new genes at the stem of Metazoa was mainly driven by duplications and protein domain rearrangement processes at the stem of Metazoa. Furthermore, our analysis allowed us to reveal protein domains that are essential to the maintenance of animal multicellularity. Our analyses also demonstrate the utility of single-cell genomics from uncultured taxa to address evolutionary questions. This article is part of a discussion meeting issue 'Single cell ecology'.

Metabarcoding analysis on European coastal samples reveals new molecular metazoan diversity.

Although animals are among the best studied organisms, we still lack a full description of their diversity, especially for microscopic taxa. This is partly due to the time-consuming and costly nature of surveying animal diversity through morphological and molecular studies of individual taxa. A powerful alternative is the use of high-throughput environmental sequencing, providing molecular data from all organisms sampled. We here address the unknown diversity of animal phyla in marine environments using an extensive dataset designed to assess eukaryotic ribosomal diversity among European coastal locations. A multi-phylum assessment of marine animal diversity that includes water column and sediments, oxic and anoxic environments, and both DNA and RNA templates, revealed a high percentage of novel 18S rRNA sequences in most phyla, suggesting that marine environments have not yet been fully sampled at a molecular level. This novelty is especially high among Platyhelminthes, Acoelomorpha, and Nematoda, which are well studied from a morphological perspective and abundant in benthic environments. We also identified, based on molecular data, a potentially novel group of widespread tunicates. Moreover, we recovered a high number of reads for Ctenophora and Cnidaria in the smaller fractions suggesting their gametes might play a greater ecological role than previously suspected.

Parvularia atlantis gen. et sp. nov., a Nucleariid Filose Amoeba (Holomycota, Opisthokonta).

The opisthokonts constitute a eukaryotic supergroup divided into two main clades: the holozoans, which include animals and their unicellular relatives, and the holomycotans, which include fungi, opisthosporidians, and nucleariids. Nucleariids are phagotrophic filose amoebae that phenotypically resemble more their distant holozoan cousins than their holomycotan phylogenetic relatives. Despite their evolutionary interest, the diversity and internal phylogenetic relationships within the nucleariids remain poorly studied. Here, we formally describe and characterize by molecular phylogeny and microscopy observations Parvularia atlantis gen. et sp. nov. (formerly Nuclearia sp. ATCC 50694), and compare its features with those of other nucleariid genera. Parvularia is an amoebal genus characterized by radiating knobbed and branching filopodia. It exhibits prominent vacuoles observable under light microscopy, a cyst-like stage, and completely lacks cilia. P. atlantis possesses one or two nuclei with a central nucleolus, and mitochondria with flat or discoid cristae. These morphological features, although typical of nucleariids, represent a combination of characters different to those of any other described Nuclearia species. Likewise, 18S rRNA-based phylogenetic analyses show that P. atlantis represents a distinct lineage within the nucleariids.

Evaluation of single-cell genomics to address evolutionary questions using three SAGs of the choanoflagellate Monosiga brevicollis.

Single-cell genomics (SCG) appeared as a powerful technique to get genomic information from uncultured organisms. However, SCG techniques suffer from biases at the whole genome amplification step that can lead to extremely variable numbers of genome recovery (5-100%). Thus, it is unclear how useful can SCG be to address evolutionary questions on uncultured microbial eukaryotes. To provide some insights into this, we here analysed 3 single-cell amplified genomes (SAGs) of the choanoflagellate Monosiga brevicollis, whose genome is known. Our results show that each SAG has a different, independent bias, yielding different levels of genome recovery for each cell (6-36%). Genes often appear fragmented and are split into more genes during annotation. Thus, analyses of gene gain and losses, gene architectures, synteny and other genomic features can not be addressed with a single SAG. However, the recovery of phylogenetically-informative protein domains can be up to 55%. This means SAG data can be used to perform accurate phylogenomic analyses. Finally, we also confirm that the co-assembly of several SAGs improves the general genomic recovery. Overall, our data show that, besides important current limitations, SAGs can still provide interesting and novel insights from poorly-known, uncultured organisms.

Hidden diversity of Acoelomorpha revealed through metabarcoding.

Animals with bilateral symmetry comprise the majority of the described species within Metazoa. However, the nature of the first bilaterian animal remains unknown. As most recent molecular phylogenies point to Xenacoelomorpha as the sister group to the rest of Bilateria, understanding their biology, ecology and diversity is key to reconstructing the nature of the last common bilaterian ancestor (Urbilateria). To date, sampling efforts have focused mainly on coastal areas, leaving potential gaps in our understanding of the full diversity of xenacoelomorphs. We therefore analysed 18S rDNA metabarcoding data from three marine projects covering benthic and pelagic habitats worldwide. Our results show that acoels have a greater richness in planktonic environments than previously described. Interestingly, we also identified a putative novel clade of acoels in the deep benthos that branches as sister group to the rest of Acoela, thus representing the earliest-branching acoel clade. Our data highlight deep-sea environments as an ideal habitat to sample acoels with key phylogenetic positions, which might be useful for reconstructing the early evolution of Bilateria.