Endosymbiosis is a major driver of evolutionary innovation and underpins the function of diverse ecosystems. The origins and evolution of endosymbiosis are challenging to study experimentally due to the short-lived culturability of many microbial strains derived from endosymbiotic interactions. The facultative endosymbiosis between the ciliate, Paramecium bursaria, and the green alga, Micractinium conductrix (Chlorellaceae, Trebouxiophyceae), is ecologically widespread and has emerged as a powerful lab-tractable model system. This endosymbiosis is founded upon a reciprocal nutrient exchange, but each of the species can be cultured independently enabling quantification of symbiotic fitness effects, new partnerships to be generated in the lab, and co-associations to be subject to experimental evolution. To date, evolve-and-resequence approaches have been limited due to a lack of high-quality genome assemblies enabling gene variants to be identified. Here, we report a near telomere-to-telomere genome assembly for M. conductrix 186b, using a range of sequencing technologies. Comparative analysis shows that this is one of the most complete Chlorellaceae algal genome assemblies available to date. To aid accurate gene calling and annotation, we conducted both RNAseq and Iso-Seq transcriptome sequencing experiments. Collectively, these 'omics datasets will facilitate: (i) comparative genomics studies of endosymbiont evolution, (ii) evolve-and-resequence experiments, (iii) genome-scale metabolic modeling studies, and (iv) identification of targets for genetic modification experiments and biotechnological applications.

Abstract Parasites of the Perkinsea lineage infect both unicellular eukaryotic and metazoan hosts. The Pathogenic Perkinsea Clade (PPC) has been associated with Severe Perkinsea Infections (SPI) and mortality events in wild tadpole populations across the US and in captive European Tree Frogs (Hyla arborea) in the UK. To investigate the spread of this emergent disease and identify additional putative parasites, we launched a UK-based citizen science project called ‘Tadpole Doctor’. Volunteer participants collected tadpoles, sediment samples from ponds suspected to contain tadpoles, or alternatively, sediment from ponds where there was evidence of recent loss of a tadpole population. In addition to the UK samples, tadpoles with SPI symptoms were collected in Alaska and Florida, along with asymptomatic tadpoles in the Czech Republic. To identify traces of PPC and related lineages, we developed a semi-targeted long-read SSU-ITS rDNA sequencing approach, revealing an unprecedented molecular diversity of the wider Perkinsea clade NAG01, where PPC branches in SSU rDNA phylogenies. Seven groups of sequences other than PPC were found associated to tadpole livers, potentially reflecting novel parasitic infections. We also identified three sister groups to the PPC clade, among which one group was found in a tadpole, and another was frequently encountered in environmental DNA (eDNA) metabarcoding surveys. Deeper characterisation of this diversity may identify new threats to amphibians. Overall, these data illustrate how citizen-based research can be useful for assessing organismal distribution patterns, enabling further targeted studies, while also raising public awareness of the vulnerability of amphibians to infectious disease.

Summary Symbioses are widespread (1) and underpin the function of diverse ecosystems (2–6), but their evolutionary stability is challenging to explain (7,8). Fitness trade-offs between contrasting intracellular and extracellular niches could act to stabilise endosymbioses because adaptation to either niche is predicted to reduce fitness in the alternate niche, thus reinforcing symbiosis (8,9). Here, we experimentally evolved four diverse Chlorella green algal endosymbionts of Paramecium bursaria to free-living conditions supplying either an amino acid, as provisioned by hosts (10,11), or nitrate, as available in freshwater (12), as the sole nitrogen source. Experimental algal populations adapted to free-living environments, generally increasing in population density and cellular chlorophyll content over time. In one of the four endosymbiont strains, adaptation to the nitrate free-living environment, but not the amino acid environments, drove the loss of fitness benefits to the host in reconstituted symbioses. This loss was not associated with reduced ability to grow on host-provisioned amino acids, nor lost ability to release the sugars provisioned to the host (10,13). Genome sequencing of evolved algal lines revealed genomic divergence between nitrate-adapted and amino acid-adapted lines, affecting genes involved with metabolic organisation and intracellular resource transport. Untargeted metabolomic profiling further showed extensive changes to membrane remodelling and turnover in N-evolved lines. Together, our data support a role for metabolic trade-offs driving divergence between contrasting intracellular and extracellular niches, with nitrogen as a key environmental axis driving divergence. Fitness trade-offs may, therefore, be a general, simple mechanism acting to reinforce symbiosis, contributing to evolutionary stability.

Endosymbiosis underpins the evolution of complex life and the function of diverse ecosystems, but the mechanisms driving the origin and stability of endosymbiosis remain unclear. Fitness trade-offs between intracellular and extracellular niches could drive the emergence of stable endosymbiosis if adaptation to the intracellular niche reduces fitness in the extracellular niche, reinforcing endosymbiosis through niche specialisation by the endosymbiont. We tested this hypothesis by quantifying phenotypic divergence between endosymbiotic and free-living populations of a facultative algal endosymbiont along trait axes predicted to contrast between its intracellular and extracellular lifestyles. Consistent with endosymbiont specialisation, we observed strong divergence involving multiple traits between endosymbiotic and free-living populations. Specifically, endosymbionts showed convergent losses of multiple nitrogen metabolism pathways, increased export of maltose and glucose, increased sensitivity to acidification, higher and less variable photosynthetic efficiency, and reduced free-living growth rate. Intracellular environments are stable, safe from natural enemies, and come with a reliable supply of specific nutrients, but are acidic and place demands upon endosymbionts to provision their hosts; our data suggest that adapting to this niche is likely to reduce free-living growth and survival of endosymbionts. Our findings support fitness trade-offs between contrasting intracellular and extracellular environments as a mechanism stabilising endosymbiosis by driving evolutionary entrapment of niche-specialist endosymbionts. ### Competing Interest Statement The authors have declared no competing interest. Natural Environment Research Council, https://ror.org/02b5d8509, NE/V000128/1 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/X016439/1

Across the tree of life, DNA damage response (DDR) proteins play a pivotal, yet dichotomous role in organismal development and evolution. Here, we present a comprehensive analysis of 432 DDR proteins encoded by 68 genomes, including that of Nucleospora cyclopteri, an intranuclear microsporidia sequenced in this study. We compared the DDR proteins encoded by these genomes to those of humans to uncover the DNA repair-ome across phylogenetically distant eukaryotes. We also performed further analyses to understand if organismal complexity and lifestyle play a role in the evolution of DDR protein length and conserved domain architecture. We observed that the genomes of extreme parasites such as Paramicrocytos, Giardia, Spironucleus, and certain microsporidian lineages encode the smallest eukaryotic repertoire of DDR proteins and that pathways involved in modulation of nucleotide pools and nucleotide excision repair are the most preserved DDR pathways in the eukaryotic genomes analysed here. We found that DDR and DNA repair proteins are consistently longer than housekeeping and metabolic proteins. This is likely due to the higher number of physical protein-protein interactions which DDR proteins are involved. We find that although DNA repair proteins are generally longer than housekeeping proteins, their functional domains occupy a relatively smaller footprint. Notably, this pattern holds true across diverse organisms and shows no dependence on either lifestyle or mitochondrial status. Finally, we observed that unicellular organisms harbour proteins that are tenfold longer than their human homologues, with the extra amino acids forming interdomain regions with a clearly novel albeit undetermined function.

How two species engage in stable endosymbiosis is a biological quandary. The study of facultative endosymbiotic interactions has emerged as a useful approach to understand how endosymbiotic functions can arise. The ciliate protist Paramecium bursaria hosts green algae of the order Chlorellales in a facultative photo-endosymbiosis. We have recently reported RNAi as a tool for understanding gene function in P. bursaria 186b (CCAP strain 1660/18). To complement this work, here we report a near complete host genome and transcriptome sequence dataset, using both Illumina and PacBio sequencing methods, in order to aid genome analysis and to enable the design of RNAi experiments. Our analyses demonstrate P. bursaria 186b, like other ciliates such as diverse species of Paramecia, possess numerous tiny introns. These data patterns, combined with the alternative genetic code common to ciliates, make gene identification and annotation challenging; as such, we identify gene models using Iso-Seq methodologies. These data will aid the investigation of genome evolution in the Paramecia and provide additional source data for the exploration of endosymbiotic functions.

How two species engage in stable endosymbiosis is a biological quandary. The study of facultative endosymbiotic interactions has emerged as a useful approach to understand how endosymbiotic functions can arise. The ciliate protist Paramecium bursaria hosts green algae of the order Chlorellales in a facultative photo-endosymbiosis. We have recently reported RNAi as a tool for understanding gene function in Paramecium bursaria 186b, CCAP strain 1660/18 [1]. To complement this work, here we report a highly complete host genome and transcriptome sequence dataset, using both Illumina and PacBio sequencing methods to aid genome analysis and to enable the design of RNAi experiments. Our analyses demonstrate Paramecium bursaria, like other ciliates such as diverse species of Paramecia, possess numerous tiny introns. These data, combined with the alternative genetic code common to ciliates, makes gene identification and annotation challenging. To explore intron evolutionary dynamics further we show that alternative splicing leading to intron retention occurs at a higher frequency among the smaller number of longer introns, identifying a source of selection against longer introns. These data will aid the investigation of genome evolution in the Paramecia and provide additional source data for the exploration of endosymbiotic functions. ### Competing Interest Statement The authors have declared no competing interest.

Understanding the origin of eukaryotic cells is one of the most difficult problems in all of biology. A key challenge relevant to the question of eukaryogenesis is reconstructing the gene repertoire of the last eukaryotic common ancestor (LECA). As data sets grow, sketching an accurate genomics-informed picture of early eukaryotic cellular complexity requires provision of analytical resources and a commitment to data sharing. Here, we summarise progress towards understanding the biology of LECA and outline a community approach to inferring its wider gene repertoire. Once assembled, a robust LECA gene set will be a useful tool for evaluating alternative hypotheses about the origin of eukaryotes and understanding the evolution of traits in all descendant lineages, with relevance in diverse fields such as cell biology, microbial ecology, biotechnology, agriculture, and medicine. In this Consensus View, we put forth the status quo and an agreed path forward to reconstruct LECA’s gene content.

Oomycetes are heterotrophic protists that share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a separate and distant region of the eukaryotic tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls. This is a key trait in the pathology of many oomycetes, as the plant cell wall represents a primary barrier to pathogen invasion and a rich source of carbohydrates. Many of the HGT gene families identified have undergone multiple rounds of duplication. Using a combination of phylogenomic analysis and functional assays, we investigate the diversification of a horizontally-transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae . Our analyses detect 11 genes retained in P. sojae among a complex pattern of gene duplications and losses. Using a phenotype assay, based on heterologous expression in yeast, we show that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional xyloglucanase variants analysed subtend an ancestral node close to the fungi-oomycetes gene transfer, suggesting the horizontally-transferred gene was a bona fide xyloglucanase. Expression of xyloglucanase paralogs in Nicotiana benthamiana triggers distinct patterns of reactive oxygen species (ROS) generation, demonstrating that enzyme variants differentially stimulate pattern-triggered immunity in plants. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze production of variant breakdown profiles, suggesting that secretion of multiple xyloglucanase variants increases efficiency of xyloglucan breakdown, as well as potentially diversifying the range of Damage-Associated Molecular Patterns (DAMPs) released during pathogen attack. We suggest that such patterns of protein neofunctionalization, and variant host responses, represent an aspect of the Red Queen host-pathogen co-evolutionary dynamic. Significance Statement The oomycetes are a diverse group of eukaryotic microbes that include some of the most devastating pathogens of plants. Oomycetes perceive, invade, and colonize plants in similar ways to fungi, in part because they acquired the genes to attack and feed on plants from fungi. These genes are predicted to be useful to oomycete plant pathogens because they have undergone multiple rounds of gene duplication. One key enzyme for attacking plant cell wall structures is called xyloglucanase. Xyloglucanase in the oomycetes has undergone multiple rounds of gene duplication, leading to variants including an enzyme with a C-terminal extension that increases activity. Some xyloglucanase variants trigger unique patterns of reactive oxygen species (ROS) in planta , and generate different profiles of cell wall breakdown products - such outcomes could act to mystify and increase the workload of the plant immune system, allowing successful pathogens to proliferate.

Endosymbiosis was a key factor in the evolution of eukaryotic cellular complexity. Yet the mechanisms that allow host regulation of intracellular symbionts, a pre-requisite for stable endosymbiosis and subsequent organelle evolution, are largely unknown. Here, we describe an immune-like glycan-sensing/processing network, partly assembled through horizontal gene-transfers (HGTs), that enables Paramecium bursaria to control its green algal endosymbionts. Using phylogenetics, RNA-interference (RNAi), and metabolite exposure experiments, we show that P. bursaria regulates endosymbiont destruction using glycan-sensing/processing – a system that includes a eukaryotic-wide chitin-binding chitinase-like protein ( CLP ) localized to the host phago-lysosome. RNAi of CLP alters expression of eight host glycan-processing genes, including two prokaryote-derived HGTs, during endosymbiont destruction. Furthermore, glycan-sensing/processing dynamically regulates endosymbiont number in P. bursaria , plasticity crucial to maximize host fitness across ecological conditions. CLP is homologous to a human phagocyte-associated innate immune factor, revealing how immune functions can be alternatively adapted and expanded, partly through HGT, enabling endosymbiotic control. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

With the advent of high-throughput genome sequencing, bioinformatics training has become essential for research in evolutionary biology and related fields. However, individual research groups are often not in the position to teach students about the most up-to-date methodology in the field. To fill this gap, extended bioinformatics courses have been developed by various institutions and provide intense training over the course of two or more weeks. Here, we describe our experience with the organization of a course in one of the longest-running extended bioinformatics series of workshops, the Evomics Workshop on Population and Speciation Genomics that takes place biennially in the UNESCO world heritage town of Český Krumlov, Czech Republic. We list the key ingredients that make this workshop successful in our view, explain the routine for workshop organization that we have optimized over the years, and describe the most important lessons that we have learned from it. We report the results of a survey conducted among past workshop participants that quantifies measures of effective teaching and provide examples of how the workshop setting has led to the cross-fertilisation of ideas and ultimately scientific progress. We expect that our account may be useful for other groups aiming to set up their own extended bioinformatics courses.

The chytrid fungus Blastocladiella emersonii produces spores with swimming tails (zoospores); these cells can sense and swim toward light. Interest in this species stems from ongoing efforts to develop B. emersonii as a model for understanding the evolution of phototaxis and the molecular cell biology of the associated optogenetic circuits. Here, we report a highly contiguous genome assembly and gene annotation of the B. emersonii American Type Culture Collection 22665 strain. We integrate a PacBio long-read library with an Illumina paired-end genomic sequence survey leading to an assembly of 21 contigs totaling 34.27 Mb. Using these data, we assess the diversity of sensory system encoding genes. These analyses identify a rich complement of G-protein-coupled receptors, ion transporters, and nucleotide cyclases, all of which have been diversified by domain recombination and tandem duplication. In many cases, these domain combinations have led to the fusion of a protein domain to a transmembrane domain, tying a putative signaling function to the cell membrane. This pattern is consistent with the diversification of the B. emersonii sensory-signaling systems, which likely plays a varied role in the complex life cycle of this fungus.

Spliced-leader trans-splicing (SLTS) has been described in distantly related eukaryotes and acts to mark mRNAs with a short 5 ' exon, giving different mRNAs identical 5 ' sequence-signatures. The function of these systems is obscure. Perkinsozoa encompasses a diversity of parasitic protists that infect bivalves, toxic-tide dinoflagellates, fish and frog tadpoles. Here, we report considerable sequence variation in the SLTS-system across the Perkinsozoa and find that multiple variant SLTS-systems are encoded in parallel in the ecologically important Perkinsozoa parasite Parvilucifera sinerae. These results demonstrate that the transcriptome of P. sinerae is segregated based on the addition of different spliced-leader (SL) exons. This segregation marks different gene categories, suggesting that SL-segregation relates to functional differentiation of the transcriptome. By contrast, both sets of gene categories are present in the single SL-transcript type sampled from Maranthos, implying that the SL-segregation of the Parvilucifera transcriptome is a recent evolutionary innovation. Furthermore, we show that the SLTS-system marks a subsection of the transcriptome with increased mRNA abundance and includes genes that encode the spliceosome system necessary for SLTS-function. Collectively, these data provide a picture of how the SLTS-systems can vary within a major evolutionary group and identify how additional transcriptional-complexity can be achieved through SL-segregation.

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses-between two fundamentally selfish biological organisms-are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term "RNAi collisions," represents a mechanism that can promote stability in a facultative eukaryote-eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.

Background The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans -splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small ( rns ) and large ( rnl ) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. Results We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11 , all likely on a single chromosome. Conclusions Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

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.

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.

Global warming is rapidly altering physicochemical attributes of Arctic waters. These changes are predicted to alter microbial networks, potentially perturbing wider community functions including parasite infections and saprotrophic recycling of biogeochemical compounds. Specifically, the interaction between autotrophic phytoplankton and heterotrophic fungi e.g. chytrids (fungi with swimming tails) requires further analysis. Here, we investigate the diversity and distribution patterns of fungi in relation to abiotic variables during one record sea ice minimum in 2012 and explore co-occurrence of chytrids with diatoms, key primary producers in these changing environments. We show that chytrid fungi are primarily encountered at sites influenced by sea ice melt. Furthermore, chytrid representation positively correlates with sea ice-associated diatoms such as Fragilariopsis or Nitzschia . Our findings identify a potential future scenario where chytrid representation within these communities increases as a consequence of ice retreat, further altering community structure through perturbation of parasitic or saprotrophic interaction networks.

Marine sediments are one of the largest carbon reservoir on Earth, yet the microbial communities, especially the eukaryotes, that drive these ecosystems are poorly characterised. Here, we report implementation of a sampling system that enables injection of reagents into sediments at depth, allowing for preservation of RNA in situ. Using the RNA templates recovered, we investigate the ‘ribosomally active’ eukaryotic diversity present in sediments close to the water/sediment interface. We demonstrate that in situ preservation leads to recovery of a significantly altered community profile. Using SSU rRNA amplicon sequencing, we investigated the community structure in these environments, demonstrating a wide diversity and high relative abundance of stramenopiles and alveolates, specifically: Bacillariophyta (diatoms), labyrinthulomycetes and ciliates. The identification of abundant diatom rRNA molecules is consistent with microscopy-based studies, but demonstrates that these algae can also be exported to the sediment as active cells as opposed to dead forms. We also observe many groups that include, or branch close to, osmotrophic–saprotrophic protists (e.g. labyrinthulomycetes and Pseudofungi), microbes likely to be important for detrital decomposition. The sequence data also included a diversity of abundant amplicon-types that branch close to the Fonticula slime moulds. Taken together, our data identifies additional roles for eukaryotic microbes in the marine carbon cycle; where putative osmotrophic–saprotrophic protists represent a significant active microbial-constituent of the upper sediment layer.