Microbial dynamics of elevated carbon flux in the open ocean's abyss.

In the open ocean, elevated carbon flux (ECF) events increase the delivery of particulate carbon from surface waters to the seafloor by severalfold compared to other times of year. Since microbes play central roles in primary production and sinking particle formation, they contribute greatly to carbon export to the deep sea. Few studies, however, have quantitatively linked ECF events with the specific microbial assemblages that drive them. Here, we identify key microbial taxa and functional traits on deep-sea sinking particles that correlate positively with ECF events. Microbes enriched on sinking particles in summer ECF events included symbiotic and free-living diazotrophic cyanobacteria, rhizosolenid diatoms, phototrophic and heterotrophic protists, and photoheterotrophic and copiotrophic bacteria. Particle-attached bacteria reaching the abyss during summer ECF events encoded metabolic pathways reflecting their surface water origins, including oxygenic and aerobic anoxygenic photosynthesis, nitrogen fixation, and proteorhodopsin-based photoheterotrophy. The abundances of some deep-sea bacteria also correlated positively with summer ECF events, suggesting rapid bathypelagic responses to elevated organic matter inputs. Biota enriched on sinking particles during a spring ECF event were distinct from those found in summer, and included rhizaria, copepods, fungi, and different bacterial taxa. At other times over our 3-y study, mid- and deep-water particle colonization, predation, degradation, and repackaging (by deep-sea bacteria, protists, and animals) appeared to shape the biotic composition of particles reaching the abyss. Our analyses reveal key microbial players and biological processes involved in particle formation, rapid export, and consumption, that may influence the ocean's biological pump and help sustain deep-sea ecosystems.

In situ imaging reveals the biomass of giant protists in the global ocean.

Planktonic organisms play crucial roles in oceanic food webs and global biogeochemical cycles. Most of our knowledge about the ecological impact of large zooplankton stems from research on abundant and robust crustaceans, and in particular copepods. A number of the other organisms that comprise planktonic communities are fragile, and therefore hard to sample and quantify, meaning that their abundances and effects on oceanic ecosystems are poorly understood. Here, using data from a worldwide in situ imaging survey of plankton larger than 600 µm, we show that a substantial part of the biomass of this size fraction consists of giant protists belonging to the Rhizaria, a super-group of mostly fragile unicellular marine organisms that includes the taxa Phaeodaria and Radiolaria (for example, orders Collodaria and Acantharia). Globally, we estimate that rhizarians in the top 200 m of world oceans represent a standing stock of 0.089 Pg carbon, equivalent to 5.2% of the total oceanic biota carbon reservoir. In the vast oligotrophic intertropical open oceans, rhizarian biomass is estimated to be equivalent to that of all other mesozooplankton (plankton in the size range 0.2-20 mm). The photosymbiotic association of many rhizarians with microalgae may be an important factor in explaining their distribution. The previously overlooked importance of these giant protists across the widest ecosystem on the planet changes our understanding of marine planktonic ecosystems.

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.

Reducing long-branch effects in multi-protein data uncovers a close relationship between Alveolata and Rhizaria.

Rhizaria is a major eukaryotic group of tremendous diversity, including amoebae with spectacular skeletons or tests (Radiolaria and Foraminifera), plasmodial parasites (Plasmodiophorida) and secondary endosymbionts (Chlorarachniophyta). Current phylogeny places Rhizaria in an unresolved trichotomy with Stramenopila and Alveolata (supergroup "SAR"). We assembled a 147-protein data set with extensive rhizarian coverage (M147), including the first transcriptomic data for a euglyphid amoeba. Phylogenetic pre-screening of individual proteins indicated potential problems with radically misplaced sequences due either to contamination of rhizarian sequences amplified from wild collected material and/or extremely long branches (xLBs). Therefore, two data subsets were extracted containing either all proteins consistently recovering rhizarian monophyly (M34) or excluding all proteins with ⩾3 xLBs (defined as ⩾2× the average terminal branch length for the tree). Phylogenetic analyses of M147 give conflicting results depending on the outgroup and method of analysis but strongly support an exclusive Rhizaria+Alveolata (R+A) clade with both data subsets (M34 and M37) regardless of phylogenetic method used. Support for an R+A clade is most consistent when a close outgroup is used and decreases with more distant outgroups, suggesting that support for alternative SAR topologies may reflect a long-branch attraction artifact. A survey of xLB distribution among taxa and protein functional category indicates that small "informational" proteins in particular have highly variable evolutionary rates with no consistent pattern among taxa.

Ancient DNA complements microfossil record in deep-sea subsurface sediments.

Deep-sea subsurface sediments are the most important archives of marine biodiversity. Until now, these archives were studied mainly using the microfossil record, disregarding large amounts of DNA accumulated on the deep-sea floor. Accessing ancient DNA (aDNA) molecules preserved down-core would offer unique insights into the history of marine biodiversity, including both fossilized and non-fossilized taxa. Here, we recover aDNA of eukaryotic origin across four cores collected at abyssal depths in the South Atlantic, in up to 32.5 thousand-year-old sediment layers. Our study focuses on Foraminifera and Radiolaria, two major groups of marine microfossils also comprising diverse non-fossilized taxa. We describe their assemblages in down-core sediment layers applying both micropalaeontological and environmental DNA sequencing approaches. Short fragments of the foraminiferal and radiolarian small subunit rRNA gene recovered from sedimentary DNA extracts provide evidence that eukaryotic aDNA is preserved in deep-sea sediments encompassing the last glacial maximum. Most aDNA were assigned to non-fossilized taxa that also dominate in molecular studies of modern environments. Our study reveals the potential of aDNA to better document the evolution of past marine ecosystems and opens new horizons for the development of deep-sea palaeogenomics.

Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida.

Benthic foraminifers inhabit a wide range of aquatic environments including open marine, brackish, and freshwater environments. Here we show that several different and diverse foraminiferal groups (miliolids, rotaliids, textulariids) and Gromia, another taxon also belonging to Rhizaria, accumulate and respire nitrates through denitrification. The widespread occurrence among distantly related organisms suggests an ancient origin of the trait. The diverse metabolic capacity of these organisms, which enables them to respire with oxygen and nitrate and to sustain respiratory activity even when electron acceptors are absent from the environment, may be one of the reasons for their successful colonization of diverse marine sediment environments. The contribution of eukaryotes to the removal of fixed nitrogen by respiration may equal the importance of bacterial denitrification in ocean sediments.

Independent evolution of the thioredoxin system in photosynthetic Paulinella species.

Redox regulation allows phytoplankton to monitor and stabilize metabolic pathways under changing conditions1. In plastids, the thioredoxin (TRX) system is linked to photosynthetic electron transport and fine tuning of metabolic pathways to fluctuating light levels. Expansion of the number of redox signal transmitters and their protein targets, as seen in plants, is believed to increase cell robustness2. In this study, we searched for genes related to redox regulation in the photosynthetic amoeba Paulinella micropora KR01 (hereafter, KR01). The genus Paulinella includes testate filose amoebae, in which a single clade acquired a photosynthetic organelle, the chromatophore, from an alpha-cyanobacterial donor3. This independent primary endosymbiosis occurred relatively recently (∼124 million years ago) when compared to Archaeplastida (>1 billion years ago), making photosynthetic Paulinella a valuable model for studying the early stages of primary endosymbiosis4. Our comparative analysis demonstrates that this lineage has evolved a TRX system similar to other algae, relying, however, on genes with diverse phylogenetic origins (including the endosymbiont, host, bacteria, and red algae). One TRX of eukaryotic provenance is targeted to the chromatophore, implicating host-endosymbiont coordination of redox regulation. A chromatophore-targeted glucose-6-phosphate dehydrogenase (G6PDH) of red algal origin suggests that Paulinella exploited the existing redox regulation system in Archaeplastida to foster integration. Our study elucidates the independent evolution of the TRX system in photosynthetic Paulinella, whose parts derive from the existing genetic toolkit in diverse organisms.

High contribution of Rhizaria (Radiolaria) to vertical export in the California Current Ecosystem revealed by DNA metabarcoding.

Passive sinking of particulate organic matter (POM) is the main mechanism through which the biological pump transports surface primary production to the ocean interior. However, the contribution and variability of different biological sources to vertical export is not fully understood. Here, we use DNA metabarcoding of the 18S rRNA gene and particle interceptor traps (PITs) to characterize the taxonomic composition of particles sinking out of the photic layer in the California Current Ecosystem (CCE), a productive system with high export potential. The PITs included formalin-fixed and 'live' traps to investigate eukaryotic communities involved in the export and remineralization of sinking particles. Sequences affiliated with Radiolaria dominated the eukaryotic assemblage in fixed traps (90%), with Dinophyta and Metazoa making minor contributions. The prominence of Radiolaria decreased drastically in live traps, possibly due to selective consumption by copepods, heterotrophic nanoflagellates, and phaeodarians that were heavily enriched in these traps. These patterns were consistent across the water masses surveyed extending from the coast to offshore, despite major differences in productivity and trophic structure of the epipelagic plankton community. Our findings identify Radiolaria as major actors in export fluxes in the CCE.

Evolution and regulation of Bigelowiella natans light-harvesting antenna system.

Bigelowiella natans is a mixotrophic flagellate and member of the chlorarachniophytes (Rhizaria), whose plastid is derived from a green algal endosymbiont. With the completion of the B. natans nuclear genome we are able to begin the analysis of the structure, function and evolution of the photosynthetic apparatus. B. natans has undergone substantial changes in photosystem structure during the evolution of the plastid from a green alga. While Photosystem II (PSII) composition is well conserved, Photosystem I (PSI) composition has undergone a dramatic reduction in accessory protein subunits. Coinciding with these changes, there was a loss of green algal LHCI orthologs while the PSII-like antenna system has the expected green algal-like proteins (encoded by genes Lhcbm1-8, Lhcb4). There are also a collection of LHCX-like proteins, which are commonly associated with stramenopiles and other eukaryotes with red-algal derived plastids, along with two other unique classes of LHCs- LHCY and LHCZ- whose function remains cryptic. To understand the regulation of the LHC gene family as an initial probe of function, we conducted an RNA-seq experiment under a short-term, high-light (HL) and low-light stress. The most abundant LHCII transcript (Lhcbm6) plus two other LHCBM types (Lhcbm1, 2) were down regulated under HL and up-regulated following a shift to very-low light (VL), as is common in antenna specializing in light harvesting. Many of the other LHCII and LHCY genes had a small, but significant increase in HL and most were only moderately affected under VL. The LHCX and LHCZ genes, however, had a strong up-regulation under HL-stress and most declined under VL, suggesting that they primarily have a role in photoprotection. This contrasts to the LHCY family that is only moderately responsive to light and a much higher basal level of expression, despite being within the LHCSR/LHCX clade. The expression of LHCX/Z proteins under HL-stress may be related to the induction of long-term, non-photochemical quenching mechanisms.

Diurnal Transcriptional Regulation of Endosymbiotically Derived Genes in the Chlorarachniophyte Bigelowiella natans.

Chlorarachniophyte algae possess complex plastids acquired by the secondary endosymbiosis of a green alga, and the plastids harbor a relict nucleus of the endosymbiont, the so-called nucleomorph. Due to massive gene transfer from the endosymbiont to the host, many proteins involved in plastid and nucleomorph are encoded by the nuclear genome. Genome sequences have provided a blueprint for the fate of endosymbiotically derived genes; however, transcriptional regulation of these genes remains poorly understood. To gain insight into the evolution of endosymbiotic genes, we performed genome-wide transcript profiling along the cell cycle of the chlorarachniophyte Bigelowiella natans, synchronized by light and dark cycles. Our comparative analyses demonstrated that transcript levels of 7,751 nuclear genes (35.7% of 21,706 genes) significantly oscillated along the diurnal/cell cycles, and those included 780 and 147 genes for putative plastid and nucleomorph-targeted proteins, respectively. Clustering analysis of those genes revealed the existence of transcriptional networks related to specific biological processes such as photosynthesis, carbon metabolism, translation, and DNA replication. Interestingly, transcripts of many plastid-targeted proteins in B. natans were induced before dawn, unlike other photosynthetic organisms. In contrast to nuclear genes, 99% nucleomorph genes were found to be constitutively expressed during the cycles. We also found that the nucleomorph DNA replication would be controlled by a nucleus-encoded viral-like DNA polymerase. The results of this study suggest that nucleomorph genes have lost transcriptional regulation along the diurnal cycles, and nuclear genes exert control over the complex plastid including the nucleomorph.

Phylogenomics of the intracellular parasite Mikrocytos mackini reveals evidence for a mitosome in rhizaria.

Mikrocytos mackini is an intracellular protistan parasite of oysters whose position in the phylogenetic tree of eukaryotes has been a mystery for many years [1,2]. M. mackini is difficult to isolate, has not been cultured, and has no defining morphological feature. Furthermore, its only phylogenetic marker that has been successfully sequenced to date (the small subunit ribosomal RNA) is highly divergent and has failed to resolve its evolutionary position [2]. M. mackini is also one of the few eukaryotes that lacks mitochondria [1], making both its phylogenetic position and comparative analysis of mitochondrial function particularly important. Here, we have obtained transcriptomic data for M. mackini from enriched isolates and constructed a 119-gene phylogenomic data set. M. mackini proved to be among the fastest-evolving eukaryote lineages known to date, but, nevertheless, our analysis robustly placed it within Rhizaria. Searching the transcriptome for genetic evidence of a mitochondrion-related organelle (MRO) revealed only four mitochondrion-derived genes: IscS, IscU, mtHsp70, and FdxR. Interestingly, all four genes are involved in iron-sulfur cluster formation, a biochemical pathway common to other highly reduced "mitosomes" in unrelated MRO-containing lineages [7]. This is the first evidence of MRO in Rhizaria, and it suggests the parallel evolution of mitochondria to mitosomes in this supergroup.

Intra-genomic ribosomal RNA polymorphism and morphological variation in Elphidium macellum suggests inter-specific hybridization in foraminifera.

Elphidium macellum is a benthic foraminifer commonly found in the Patagonian fjords. To test whether its highly variable morphotypes are ecophenotypes or different genotypes, we analysed 70 sequences of the SSU rRNA gene from 25 specimens. Unexpectedly, we identified 11 distinct ribotypes, with up to 5 ribotypes co-occurring within the same specimen. The ribotypes differ by varying blocks of sequence located at the end of stem-loop motifs in the three expansion segments specific to foraminifera. These changes, distinct from typical SNPs and indels, directly affect the structure of the expansion segments. Their mosaic distribution suggests that ribotypes originated by recombination of two or more clusters of ribosomal genes. We propose that this expansion segment polymorphism (ESP) could originate from hybridization of morphologically different populations of Patagonian Elphidium. We speculate that the complex geological history of Patagonia enhanced divergence of coastal foraminiferal species and contributed to increasing genetic and morphological variation.

Possible import routes of proteins into the cyanobacterial endosymbionts/plastids of Paulinella chromatophora.

The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active and deeply integrated cyanobacterial endosymbionts acquired ~60 million years ago. Recent genomic analyses of P. chromatophora have revealed the loss of many essential genes from the endosymbiont's genome, and have identified more than 30 genes that have been transferred to the host cell's nucleus through endosymbiotic gene transfer (EGT). This indicates that, similar to classical primary plastids, Paulinella endosymbionts have evolved a transport system to import their nuclear-encoded proteins. To deduce how these proteins are transported, we searched for potential targeting signals in genes for 10 EGT-derived proteins. Our analyses indicate that five proteins carry potential signal peptides, implying they are targeted via the host endomembrane system. One sequence encodes a mitochondrial-like transit peptide, which suggests an import pathway involving a channel protein residing in the outer membrane of the endosymbiont. No N-terminal targeting signals were identified in the four other genes, but their encoded proteins could utilize non-classical targeting signals contained internally or in C-terminal regions. Several amino acids more often found in the Paulinella EGT-derived proteins than in their ancestral set (proteins still encoded in the endosymbiont genome) could constitute such signals. Characteristic features of the EGT-derived proteins are low molecular weight and nearly neutral charge, which both could be adaptations to enhance passage through the peptidoglycan wall present in the intermembrane space of the endosymbiont's envelope. Our results suggest that Paulinella endosymbionts/plastids have evolved several different import routes, as has been shown in classical primary plastids.

Peroxisome diversity and evolution.

Peroxisomes are organelles bounded by a single membrane that can be found in all major groups of eukaryotes. A single evolutionary origin of this cellular compartment is supported by the presence, in diverse organisms, of a common set of proteins implicated in peroxisome biogenesis and maintenance. Their enzymatic content, however, can vary substantially across species, indicating a high level of evolutionary plasticity. Proteomic analyses have greatly expanded our knowledge on peroxisomes in some model organisms, including plants, mammals and yeasts. However, we still have a limited knowledge about the distribution and functionalities of peroxisomes in the vast majority of groups of microbial eukaryotes. Here, I review recent advances in our understanding of peroxisome diversity and evolution, with a special emphasis on peroxisomes in microbial eukaryotes.