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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.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

Marine eastern boundary current ecosystems, such as the California Current System (CCS), involve productive, mesotrophic transition zones. The CCS exhibits highly variable primary production (PP), yet factors driving the variability and underlying phytoplankton communities remain poorly understood. We integrated physicochemical and biological data from surface waters sampled during 10 CCS expeditions, spanning 13 yr, and resolved regimes with distinct phytoplankton communities. Additional to an oligotrophic regime (OR), mesotrophic waters beyond the coastal area partitioned into Meso‐High and Meso‐Low regimes, differing in nitrate concentrations and PP. The OR was dominated by Prochlorococcus High‐Light I (HLI), and eukaryotic phytoplankton were largely predatory mixotrophs. Eukaryotes dominated Meso‐Low and Meso‐High phytoplankton biomass. Within the Meso‐Low, Pelagomonas calceolata was important, and Prochlorococcus Low‐Light I (LLI) rose in prominence. In the Meso‐High, the picoprasinophyte Ostreococcus lucimarinus was abundant, and Synechococcus Clade IV was notable. The Meso‐High exhibited the highest PP (38 ± 16 mg C m−3 d−1; p < 0.01) and higher growth rates for photosynthetic eukaryotes (0.84 ± 0.02 d−1) than for Prochlorococcus (0.61 ± 0.01 d−1) and Synechococcus (0.31 ± 0.05 d−1). An experiment simulating seasonal oligotrophic seawater intrusion into the Meso‐High resulted in growth rates reaching 1.18 ± 0.10 d−1 (O. lucimarinus), 0.75 ± 0.21 d−1 (Prochlorococcus LLI), and 0.50 ± 0.04 d−1 (Synechococcus EPC2). Thus, variable PP is underpinned by distinct phytoplankton communities across CCS mesotrophic regimes, and their dynamic nature is influenced by the rapidity with which specific taxa respond to changing environmental conditions or possibly transient nutrient release from viral encounters. Future work should assess whether these dynamics are consistent across eastern boundary current ecosystems and over temporal variations. [ABSTRACT FROM AUTHOR]

BackgroundGenetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets.ResultsHere we explore the role of recombination in both maintaining and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra- and inter- lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations.ConclusionsThese findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.

BackgroundPrasinophytes are widespread marine green algae that are related to plants. Cellular abundance of the prasinophyte Micromonas has reportedly increased in the Arctic due to climate-induced changes. Thus, studies of these unicellular eukaryotes are important for marine ecology and for understanding Viridiplantae evolution and diversification.ResultsWe generated evidence-based Micromonas gene models using proteomics and RNA-Seq to improve prasinophyte genomic resources. First, sequences of four chromosomes in the 22 Mb Micromonas pusilla (CCMP1545) genome were finished. Comparison with the finished 21 Mb genome of Micromonas commoda (RCC299; named herein) shows they share ≤8,141 of ~10,000 protein-encoding genes, depending on the analysis method. Unlike RCC299 and other sequenced eukaryotes, CCMP1545 has two abundant repetitive intron types and a high percent (26 %) GC splice donors. Micromonas has more genus-specific protein families (19 %) than other genome sequenced prasinophytes (11 %). Comparative analyses using predicted proteomes from other prasinophytes reveal proteins likely related to scale formation and ancestral photosynthesis. Our studies also indicate that peptidoglycan (PG) biosynthesis enzymes have been lost in multiple independent events in select prasinophytes and plants. However, CCMP1545, polar Micromonas CCMP2099 and prasinophytes from other classes retain the entire PG pathway, like moss and glaucophyte algae. Surprisingly, multiple vascular plants also have the PG pathway, except the Penicillin-Binding Protein, and share a unique bi-domain protein potentially associated with the pathway. Alongside Micromonas experiments using antibiotics that halt bacterial PG biosynthesis, the findings highlight unrecognized phylogenetic complexity in PG-pathway retention and implicate a role in chloroplast structure or division in several extant Viridiplantae lineages.ConclusionsExtensive differences in gene loss and architecture between related prasinophytes underscore their divergence. PG biosynthesis genes from the cyanobacterial endosymbiont that became the plastid, have been selectively retained in multiple plants and algae, implying a biological function. Our studies provide robust genomic resources for emerging model algae, advancing knowledge of marine phytoplankton and plant evolution.

Spliceosomal introns are a hallmark of eukaryotic genes that are hypothesized to play important roles in genome evolution but have poorly understood origins. Although most introns lack sequence homology to each other, new families of spliceosomal introns that are repeated hundreds of times in individual genomes have recently been discovered in a few organisms. The prevalence and conservation of these introner elements (IEs) or introner-like elements in other taxa, as well as their evolutionary relationships to regular spliceosomal introns, are still unknown. Here, we systematically investigate introns in the widespread marine green alga Micromonas and report new families of IEs, numerous intron presence-absence polymorphisms, and potential intron insertion hot-spots. The new families enabled identification of conserved IE secondary structure features and establishment of a novel general model for repetitive intron proliferation across genomes. Despite shared secondary structure, the IE families from each Micromonas lineage bear no obvious sequence similarity to those in the other lineages, suggesting that their appearance is intimately linked with the process of speciation. Two of the new IE families come from an Arctic culture (Micromonas Clade E2) isolated from a polar region where abundance of this alga is increasing due to climate induced changes. The same two families were detected in metagenomic data from Antarctica--a system where Micromonas has never before been reported. Strikingly high identity between the Arctic isolate and Antarctic coding sequences that flank the IEs suggests connectivity between populations in the two polar systems that we postulate occurs through deep-sea currents. Recovery of Clade E2 sequences in North Atlantic Deep Waters beneath the Gulf Stream supports this hypothesis. Our research illuminates the dynamic relationships between an unusual class of repetitive introns, genome evolution, speciation, and global distribution of this sentinel marine alga.

Algae with secondary plastids of a red algal origin, such as ochrophytes (photosynthetic stramenopiles), are diverse and ecologically important, yet their evolutionary history remains controversial. We sequenced plastid genomes of two ochrophytes, Ochromonas sp. CCMP1393 (Chrysophyceae) and Trachydiscus minutus (Eustigmatophyceae). A shared split of the clpC gene as well as phylogenomic analyses of concatenated protein sequences demonstrated that chrysophytes and eustigmatophytes form a clade, the Limnista, exhibiting an unexpectedly elevated rate of plastid gene evolution. Our analyses also indicate that the root of the ochrophyte phylogeny falls between the recently redefined Khakista and Phaeista assemblages. Taking advantage of the expanded sampling of plastid genome sequences, we revisited the phylogenetic position of the plastid of Vitrella brassicaformis, a member of Alveolata with the least derived plastid genome known for the whole group. The results varied depending on the dataset and phylogenetic method employed, but suggested that the Vitrella plastids emerged from a deep ochrophyte lineage rather than being derived vertically from a hypothetical plastid-bearing common ancestor of alveolates and stramenopiles. Thus, we hypothesize that the plastid in Vitrella, and potentially in other alveolates, may have been acquired by an endosymbiosis of an early ochrophyte.

Phytochrome photosensors control a vast gene network in streptophyte plants, acting as master regulators of diverse growth and developmental processes throughout the life cycle. In contrast with their absence in known chlorophyte algal genomes and most sequenced prasinophyte algal genomes, a phytochrome is found in Micromonas pusilla, a widely distributed marine picoprasinophyte (

Chapter 1 introduces marine natural products and discusses the symbiosis of bacteria and marine invertebrates as a source of these molecules. Indications for a bacterial origin of compounds isolated from invertebrates are presented and the major types of bacterial natural product biosynthesis pathways are introduced. The study of biosynthesis opens the possibility of solving the supply issue that often prevents the development of drugs from marine natural products. It also yields novel biochemical reactions and allows insight into the evolution of biosynthetic pathways.Chapter 2 explores the biosynthetic genes responsible for the production of the anticancer compound bryostatin. Bryostatin was isolated from a marine bryozoan; here a putative biosynthetic pathway from a symbiotic bacterium via a modular polyketide synthase (PKS) mechanism is presented. Symbiont DNA was enriched using ultracentrifugation techniques and lambda phage library constructed. For homology screening initially a DNA fragment was used specific for a symbiont PKS derived in earlier work by degenerate PCR Subsequently the ends of the isolated lambda fragments were used in a series of screenings. Short gaps were bridged by PCR. This chromosome walking approach yielded the entire 80 kb bry cluster. The cluster contains all the major elements needed for the production of bryostatin. A putative biosynthetic scheme is described.Chapter 3 investigates the phylogeny of Prochloron sp., a cyanobacterial symbiont of ascidians, based on 16S rRNA gene and 16S-23S internally transcribed spacer region sequences as well as denaturing gradient gel electrophoresis. Prochloron diversity is very low and there is no evidence for cospeciation with the ascidian host. This is in contrast to the vertical transmission of symbionts reported for some ascidians. Prochloron is the source of the patellamides, the relationship between phylogenetic and biosynthetic diversity is discussed.Chapter 4 presents a novel cyclic peptide, trichamide, from the free-living cyanobacterium Trichodesmium erythraeum. A gene cluster related to the patellamide biosynthetic genes from Prochloron sp. was identified by homology in T. erythraeum sequence data available in Genbank. The structure of trichamide was proposed based on sequence information and could be verified by Fourier-transfer mass spectromet.

A gene cluster for the biosynthesis of a new small cyclic peptide, dubbed trichamide, was discovered in the genome of the global, bloom-forming cyanobacterium Trichodesmium erythraeum ISM 101 because of striking similarities to the previously characterized patellamide biosynthesis cluster. Based upon detailed sequence analysis, a structure was predicted for trichamide and confirmed by Fourier transform mass spectrometry.