Toward Modern Classification of Eustigmatophytes, Including the Description of Neomonodaceae Fam. Nov. and Three New Genera1.

The class Eustigmatophyceae includes mostly coccoid, freshwater algae, although some genera are common in terrestrial habitats and two are primarily marine. The formal classification of the class, developed decades ago, does not fit the diversity and phylogeny of the group as presently known and is in urgent need of revision. This study concerns a clade informally known as the Pseudellipsoidion group of the order Eustigmatales, which was initially known to comprise seven strains with oval to ellipsoidal cells, some bearing a stipe. We examined those strains as well as 10 new ones and obtained 18S rDNA and rbcL gene sequences. The results from phylogenetic analyses of the sequence data were integrated with morphological data of vegetative and motile cells. Monophyly of the Pseudellipsoidion group is supported in both 18S rDNA and rbcL trees. The group is formalized as the new family Neomonodaceae comprising, in addition to Pseudellipsoidion, three newly erected genera. By establishing Neomonodus gen. nov. (with type species Neomonodus ovalis comb. nov.), we finally resolve the intricate taxonomic history of a species originally described as Monodus ovalis and later moved to the genera Characiopsis and Pseudocharaciopsis. Characiopsiella gen. nov. (with the type species Characiopsiella minima comb. nov.) and Munda gen. nov. (with the type species Munda aquilonaris) are established to accommodate additional representatives of the polyphyletic genus Characiopsis. A morphological feature common to all examined Neomonodaceae is the absence of a pyrenoid in the chloroplasts, which discriminates them from other morphologically similar yet unrelated eustigmatophytes (including other Characiopsis-like species).

Intraspecific Variation in Protists: Clues for Microevolution from Poteriospumella lacustris (Chrysophyceae).

Species delimitation in protists is still a challenge, attributable to the fact that protists are small, difficult to observe and many taxa are poor in morphological characters, whereas most current phylogenetic approaches only use few marker genes to measure genetic diversity. To address this problem, we assess genome-level divergence and microevolution in strains of the protist Poteriospumella lacustris, one of the first free-living, nonmodel organisms to study genome-wide intraspecific variation. Poteriospumella lacustris is a freshwater protist belonging to the Chrysophyceae with an assumed worldwide distribution. We examined three strains from different geographic regions (New Zealand, China, and Austria) by sequencing their genomes with the Illumina and PacBio platforms. The assembled genomes were small with 49-55 Mb but gene-rich with 16,000-19,000 genes, of which ∼8,000 genes could be assigned to functional categories. At least 68% of these genes were shared by all three species. Genetic variation occurred predominantly in genes presumably involved in ecological niche adaptation. Most surprisingly, we detected differences in genome ploidy between the strains (diploidy, triploidy, and tetraploidy). In analyzing intraspecific variation, several mechanisms of diversification were identified including SNPs, change of ploidy and genome size reduction.

Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes.

The division of life into producers and consumers is blurred by evolution. For example, eukaryotic phototrophs can lose the capacity to photosynthesize, although they may retain vestigial plastids that perform other essential cellular functions. Chrysophyte algae have undergone a particularly large number of photosynthesis losses. Here, we present a plastid genome sequence from a nonphotosynthetic chrysophyte, "Spumella" sp. NIES-1846, and show that it has retained a nearly identical set of plastid-encoded functions as apicomplexan parasites. Our transcriptomic analysis of 12 different photosynthetic and nonphotosynthetic chrysophyte lineages reveals remarkable convergence in the functions of these nonphotosynthetic plastids, along with informative lineage-specific retentions and losses. At one extreme, Cornospumella fuschlensis retains many photosynthesis-associated proteins, although it appears to have lost the reductive pentose phosphate pathway and most plastid amino acid metabolism pathways. At the other extreme, Paraphysomonas lacks plastid-targeted proteins associated with gene expression and all metabolic pathways that require plastid-encoded partners, indicating a complete loss of plastid DNA in this genus. Intriguingly, some of the nucleus-encoded proteins that once functioned in the expression of the Paraphysomonas plastid genome have been retained. These proteins were likely to have been dual targeted to the plastid and mitochondria of the chrysophyte ancestor, and are uniquely targeted to the mitochondria in Paraphysomonas Our comparative analyses provide insights into the process of functional reduction in nonphotosynthetic plastids.

Speciation in protists: Spatial and ecological divergence processes cause rapid species diversification in a freshwater chrysophyte.

Although eukaryotic microorganisms are extremely numerous, diverse and essential to global ecosystem functioning, they are largely understudied by evolutionary biologists compared to multicellular macroscopic organisms. In particular, very little is known about the speciation mechanisms which may give rise to the diversity of microscopic eukaryotes. It was postulated that the enormous population sizes and ubiquitous distribution of these organisms could lead to a lack of population differentiation and therefore very low speciation rates. However, such assumptions have traditionally been based on morphospecies, which may not accurately reflect the true diversity, missing cryptic taxa. In this study, we aim to articulate the major diversification mechanisms leading to the contemporary molecular diversity by using a colonial freshwater flagellate, Synura sphagnicola, as an example. Phylogenetic analysis of five sequenced loci showed that S. sphagnicola differentiated into two morphologically distinct lineages approximately 15.4 million years ago, which further diverged into several evolutionarily recent haplotypes during the late Pleistocene. The most recent haplotypes are ecologically and biogeographically much more differentiated than the old lineages, presumably because of their persistent differentiation after the allopatric speciation events. Our study shows that in microbial eukaryotes, species diversification via the colonization of new geographical regions or ecological resources occurs much more readily than was previously thought. Consequently, divergence times of microorganisms in some lineages may be equivalent to the estimated times of speciation in plants and animals.

High planktonic diversity in mountain lakes contains similar contributions of autotrophic, heterotrophic and parasitic eukaryotic life forms.

A rich eukaryotic planktonic community exists in high-mountain lakes despite the diluted, oligotrophic and cold, harsh prevailing conditions. Attempts of an overarching appraisal have been traditionally hampered by observational limitations of small, colorless, and soft eukaryotes. We aimed to uncover the regional eukaryotic biodiversity of a mountain lakes district to obtain general conclusions on diversity patterns, dominance, geographic diversification, and food-web players common to oligotrophic worldwide distributed freshwater systems. An unprecedented survey of 227 high-altitude lakes comprising large environmental gradients was carried out using Illumina massive tag sequencing of the 18S rRNA gene. We observed a large Chrysophyceae dominance in richness, abundance and novelty, and unveiled an unexpected richness in heterotrophic phagotrophs and parasites. In particular, Cercozoa and Chytridiomycota showed diversity features similar to the dominant autotrophic groups. The prominent beta-dispersion shown by parasites suggests highly specific interactions and a relevant role in food webs. Interestingly, the freshwater Pyrenean metacommunity contained more diverse specific populations than its closest marine oligotrophic equivalent, with consistently higher beta-diversity. The relevance of unseen groups opens new perspectives for the better understanding of planktonic food webs. Mountain lakes, with remarkable environmental idiosyncrasies, may be suitable environments for the genetic diversification of microscopic eukaryotic life forms.

Community structure of photosynthetic picoeukaryotes differs in lakes with different trophic statuses along the middle-lower reaches of the Yangtze River.

Photosynthetic picoeukaryotes (PPEs) play an important role in aquatic ecosystem functioning. There is still a relative lack of information on freshwater PPEs, especially in eutrophic lakes. We used a combination of flow cytometric sorting and pyrosequencing to investigate the PPEs community structure in more than 20 mesotrophic and eutrophic lakes along the middle-lower reaches of the Yangtze River in China. The abundance of PPEs ranged between 2.04 × 103 and 5.92 × 103 cells mL-1. The contribution of PPEs to total picophytoplankton abundance was generally higher in eutrophic lakes than in mesotrophic lakes. The sequencing results indicated that the Shannon diversity of PPEs was significantly higher in mesotrophic lakes than in eutrophic lakes. At the class level, PPEs were mainly dominated by three taxonomic groups, including Cryptophyceae, Coscinodiscophyceae and Chlorophyceae, and 15 additional known phytoplankton classes, including Synurophyceae, Dinophyceae, Chrysophyceae, Trebouxiophyceae and Prymnesiophyceae, were identified. Coscinodiscophyceae dominated in the most eutrophic lakes, while Chrysophyceae, Dinophyceae and other classes of PPEs were more abundant in the mesotrophic lakes. We also observed several PPEs operational taxonomic units, and those affiliated with Cyclotella atomus, Chlamydomonas sp. and Poterioochromonas malhamensis tended to be more prevalent in the eutrophic lakes. The canonical correspondence analysis and Mantel analysis highlighted the importance of environmental parameters as key drivers of PPEs community composition.

High-throughput sequencing of microbial eukaryotes in Lake Baikal reveals ecologically differentiated communities and novel evolutionary radiations.

We performed high-throughput 18S rDNA V9 region sequencing analyses of microeukaryote (protist) communities at seven sites with depths ranging from 0 to 1450 m in the southern part of Lake Baikal. We show that microeukaryotic diversity differed according to water column depth and sediment depth. Chrysophytes and perkinsids were diverse in subsurface samples, novel radiations of petalomonads and Ichthyobodo relatives were found in benthic samples, and a broad range of divergent OTUs were detected in deep subbenthic samples. Members of clades usually associated with marine habitats were also detected, including syndineans for the first time in freshwater systems. Fungal- and cercozoan-specific c. 1200 bp amplicon clone libraries also revealed many novel lineages in both planktonic and sediment samples at all depths, a novel radiation of aphelids in shallower benthic samples, and partitioning of sarcomonad lineages in shallow vs deep benthic samples. Putative parasitic lineages accounted for 12.4% of overall reads, including a novel radiation of Ichthyobodo (fish parasite) relatives. Micrometazoans were also analysed, including crustaceans, rotifers and nematodes. The deepest (>1000 m) subsurface sediment samples harboured some highly divergent sequence types, including heterotrophic flagellates, parasites, putative metazoans and sequences likely representing organisms originating from higher up in the water column.

Tracking a century of changes in microbial eukaryotic diversity in lakes driven by nutrient enrichment and climate warming.

High-throughput sequencing of sedimentary DNA (sed-DNA) was utilized to reconstruct the temporal dynamics of microbial eukaryotic communities (MECs) at a centennial scale in two re-oligotrophicated lakes that were exposed to different levels of phosphorus enrichment. The temporal changes within the MECs were expressed in terms of richness, composition and community structure to investigate their relationships with two key forcing factors (i.e., nutrient enrichment and climate warming). Various groups, including Apicomplexa, Cercozoa, Chrysophyceae, Ciliophora, Chlorophyceae and Dinophyceae, responded to phosphorus enrichment levels with either positive or negative impacts on their richness and relative abundance. For both lakes, statistical modelling demonstrated that phosphorus concentration ([P]) was a dominant contributor to MECs modifications before the 1980s; after the mid-80s, the contribution of air temperature changes increased and potentially surpassed the contribution of [P]. Co-occurrence network analysis revealed that some clusters of taxa (i.e., modules) composed mainly of Dinophyceae and unclassified Alveolata were strongly correlated to air temperature in both lakes. Overall, our data showed that sed-DNA constitutes a precious archive of information on past biodiversity changes, allowing the study of the dynamics of numerous eukaryotic groups that were not traditionally considered in paleo-reconstructions.

Small but Manifold - Hidden Diversity in "Spumella-like Flagellates".

Colourless, nonscaled chrysophytes comprise morphologically similar or even indistinguishable flagellates which are important bacterivors in water and soil crucial for ecosystem functioning. However, phylogenetic analyses indicate a multiple origin of such colourless, nonscaled flagellate lineages. These flagellates are often referred to as "Spumella-like flagellates" in ecological and biogeographic studies. Although this denomination reflects an assumed polyphyly, it obscures the phylogenetic and taxonomic diversity of this important flagellate group and, thus, hinders progress in lineage- and taxon-specific ecological surveys. The smallest representatives of colourless chrysophytes have been addressed in very few taxonomic studies although they are among the dominant flagellates in field communities. To overcome the blurred picture and set the field for further investigation in biogeography and ecology of the organisms in question, we studied a set of strains of specifically small, colourless, nonscaled chrysomonad flagellates by means of electron microscopy and molecular analyses. They were isolated by a filtration-acclimatisation approach focusing on flagellates of around 5 µm. We present the phylogenetic position of eight different lineages on both the ordinal and the generic level. Accordingly, we describe the new genera Apoikiospumella, Chromulinospumella, Segregatospumella, Cornospumella and Acrispumella Boenigk et Grossmann n. g. and different species within them.

Metagenomic Analyses of White Sea Picoalgae: First Data.

Picoalgae (defined as cells smaller than 2-3 µm) include members of diverse taxonomic groups. They are an important constituent of marine plankton and ice biota and play a significant ecological role in biogeochemical cycles. Despite their importance, the true extent of their diversity has only recently been uncovered by molecular surveys. The diversity of picoeukaryotes has not yet been studied in the White Sea, which is a unique marine environment combining features of temperate and Arctic seas. Here, we investigated the taxonomic composition of eukaryotic picoalgae in ice and under-ice water at a station located in the Kandalaksha Bay of the White Sea. We applied metagenomic survey using Illumina sequencing. Eight main algae phyla, namely, Chlorophyta, Katablepharidophyta, Haptophyta, Dinophyta, Cercozoa, Bacillariophyta, Cryptophyta, and Ochrophyta were identified. The genera Paraphysomonas and Micromonas and the order Pedinellales were most numerous in plankton; the genera Paraphysomonas, Micromonas, and Metopion were most abundant in ice. The number of "rare" phylotypes was 80 in under-ice water and 112 in ice. Some taxa of nano- and microalgae are identified for the first time in the White Sea phytoplankton. Our data provide a basis for further research of tiny phototrophs in the Russian Arctic.

Heterosides--compatible solutes occurring in prokaryotic and eukaryotic phototrophs.

The acclimation to osmotic and/or salt stress conditions induces an integrated response at different cellular levels. One acclimation strategy relies on the massive accumulation of low molecular mass compounds, so-called compatible solutes, to balance osmotic gradients and to directly protect critical macromolecules. Heterosides are compounds composed of a sugar and a polyol moiety that represent one chemical class of compatible solutes with interesting features. Well-investigated examples are glucosylglycerol, which is found in many cyanobacteria, and galactosylglycerols (floridoside and isofloridoside), which are accumulated by eukaryotic algae under salt stress conditions. Here, we review knowledge on physiology, biochemistry and genetics of heteroside accumulation in pro- and eukaryotic photoautotrophic organisms.

Scale evolution in Paraphysomonadida (Chrysophyceae): Sequence phylogeny and revised taxonomy of Paraphysomonas, new genus Clathromonas, and 25 new species.

Heterotrophic chrysomonads of the genus Paraphysomonas are ubiquitous phagotrophs with diverse silica scale morphology. Over 50 named species have been described by electron microscopy from uncultured environmental samples. Sequence data exist for very few, but the literature reveals misidentification or lumping of most previously sequenced. For critically integrating scale and sequence data, 59 clonal cultures were studied light microscopically, by sequencing 18S ribosomal DNA, and recording scale morphology by transmission electron microscopy. We found strong congruence between variations in scale morphology and rDNA sequences, and unexpectedly deep genetic diversity. We now restrict Paraphysomonas to species with nail-like spine scales, establishing 23 new species and eight subspecies (Paraphysomonadidae). Species having base-plates with dense margins form three distinct subclades; those with a simple margin only two. We move 29 former Paraphysomonas species with basket scales into a new genus, Clathromonas, and describe two new species. Clathromonas belongs to a very distinct rDNA clade (Clathromonadidae fam. n.), possibly distantly sister to Paraphysomonas. Molecular and morphological data are mutually reinforcing; both are needed for evaluating paraphysomonad diversity and confirm excessive past lumping. Former Paraphysomonas species with neither nail-like nor basket scales are here excluded from Paraphysomonas and will be assigned to new genera elsewhere.

Effects of long-term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach.

To understand the fine-scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long-term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae-Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88,706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic- (farmyard manure), mineral- and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co-occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.

Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea.

Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.

Impact of protist grazing on a key bacterial group for biogeochemical cycling in Baltic Sea pelagic oxic/anoxic interfaces.

Barrier zones between oxic and anoxic water masses (redoxclines) host highly active prokaryotic communities with important roles in biogeochemical cycling. In Baltic Sea pelagic redoxclines, Epsilonproteobacteria of the genus Sulfurimonas (subgroup GD17) have been shown to dominate chemoautotrophic denitrification. However, little is known on the loss processes affecting this prokaryotic group. In the present study, the protist grazing impact on the Sulfurimonas subgroup GD17 was determined for suboxic and oxygen/hydrogen sulphide interface depths of Baltic Sea redoxclines, using predator exclusion assays and bacterial amendment with the cultured representative 'Sulfurimonas gotlandica' strain GD1. Additionally, the principal bacterivores were identified by RNA-Stable Isotope Probing (RNA-SIP). The natural Sulfurimonas subgroup GD17 population grew strongly under oxygen/hydrogen sulphide interface conditions (doubling time: 1-1.5 days), but protist grazing could consume the complete new cell production per day. In suboxic samples, little or no growth of Sulfurimonas subgroup GD17 was observed. RNA-SIP identified five active grazers, belonging to typical redoxcline ciliates (Oligohymenophorea, Prostomatea) and globally widespread marine flagellate groups (MAST-4, Chrysophyta, Cercozoa). Overall, we demonstrate for the first time that protist grazing can control the growth, and potentially the vertical distribution, of a chemolithoautotrophic key-player of oxic/anoxic interfaces.

A global perspective on marine photosynthetic picoeukaryote community structure.

A central goal in ecology is to understand the factors affecting the temporal dynamics and spatial distribution of microorganisms and the underlying processes causing differences in community structure and composition. However, little is known in this respect for photosynthetic picoeukaryotes (PPEs), algae that are now recognised as major players in marine CO2 fixation. Here, we analysed dot blot hybridisation and cloning-sequencing data, using the plastid-encoded 16S rRNA gene, from seven research cruises that encompassed all four ocean biomes. We provide insights into global abundance, α- and ß-diversity distribution and the environmental factors shaping PPE community structure and composition. At the class level, the most commonly encountered PPEs were Prymnesiophyceae and Chrysophyceae. These taxa displayed complementary distribution patterns, with peak abundances of Prymnesiophyceae and Chrysophyceae in waters of high (25:1) or low (12:1) nitrogen:phosphorus (N:P) ratio, respectively. Significant differences in phylogenetic composition of PPEs were demonstrated for higher taxonomic levels between ocean basins, using Unifrac analyses of clone library sequence data. Differences in composition were generally greater between basins (interbasins) than within a basin (intrabasin). These differences were primarily linked to taxonomic variation in the composition of Prymnesiophyceae and Prasinophyceae whereas Chrysophyceae were phylogenetically similar in all libraries. These data provide better knowledge of PPE community structure across the world ocean and are crucial in assessing their evolution and contribution to CO2 fixation, especially in the context of global climate change.

Ecological and evolutionary genomics of marine photosynthetic organisms.

Environmental (ecological) genomics aims to understand the genetic basis of relationships between organisms and their abiotic and biotic environments. It is a rapidly progressing field of research largely due to recent advances in the speed and volume of genomic data being produced by next generation sequencing (NGS) technologies. Building on information generated by NGS-based approaches, functional genomic methodologies are being applied to identify and characterize genes and gene systems of both environmental and evolutionary relevance. Marine photosynthetic organisms (MPOs) were poorly represented amongst the early genomic models, but this situation is changing rapidly. Here we provide an overview of the recent advances in the application of ecological genomic approaches to both prokaryotic and eukaryotic MPOs. We describe how these approaches are being used to explore the biology and ecology of marine cyanobacteria and algae, particularly with regard to their functions in a broad range of marine ecosystems. Specifically, we review the ecological and evolutionary insights gained from whole genome and transcriptome sequencing projects applied to MPOs and illustrate how their genomes are yielding information on the specific features of these organisms.

Genetic diversity of planktonic eukaryotes in high mountain lakes (Central Pyrenees, Spain).

The genetic diversity of planktonic eukaryotic microorganisms (size range 3-40 µm) inhabiting 11 alpine lakes of the Central Pyrenees (Spain) was analysed by cloning and sequencing of the 18S rRNA gene. The selected lakes covered a wide range of environmental conditions representative of the regional landscape heterogeneity. Overall, we obtained 953 sequences (averaged length 750 bp) that were grouped in 343 representative OTUs (98% identity). The genetic richness was high, and the 18S rRNA gene sequences spread within nine high-rank taxonomic groups and grouped in 26 eukaryal classes. Most of the sequences affiliated with Stramenopiles (> 55% of total sequences, mostly Chrysophyceae), Cryptophyta and Alveolata (15% each). Three groups had relative abundance < 5%, i.e. Opisthokonta (mostly Fungi), Viridiplantae (mostly Chlorophyceae) and Rhizaria (cercomonads). Finally, minor groups were related to Katablepharidophyta, Euglenozoa and Telonemida. The lakes showed a different community structure being pH, and phosphorous and Chl a concentrations the main environmental drivers. The novelty level was high, and a quarter of the retrieved OTUs were notably divergent (< 97% identity) from any previously known sequence, mainly for Rhizaria and Opisthokonta. More than 50% of the sequences affiliated with clusters exclusively formed by uncultured protists. Cryptophyta and Viridiplantae showed the largest number of sequences closely related to cultured counterparts. This work is the first description of the genetic diversity of eukaryotic assemblages in ultraoligotrophic high mountain lakes, and the study unveils alpine environments as an important reservoir of microbial eukaryotic biodiversity.

Phytoplankton distribution patterns in the northwestern Sargasso Sea revealed by small subunit rRNA genes from plastids.

Phytoplankton species vary in their physiological properties, and are expected to respond differently to seasonal changes in water column conditions. To assess these varying distribution patterns, we used 412 samples collected monthly over 12 years (1991-2004) at the Bermuda Atlantic Time-Series Study site, located in the northwestern Sargasso Sea. We measured plastid 16S ribosomal RNA gene abundances with a terminal restriction fragment length polymorphism approach and identified distribution patterns for members of the Prymnesiophyceae, Pelagophyceae, Chrysophyceae, Cryptophyceae, Bacillariophyceae and Prasinophyceae. The analysis revealed dynamic bloom patterns by these phytoplankton taxa that begin early in the year, when the mixed layer is deep. Previously, unreported open-ocean prasinophyte blooms dominated the plastid gene signal during convective mixing events. Quantitative PCR confirmed the blooms and transitions of Bathycoccus, Micromonas and Ostreococcus populations. In contrast, taxa belonging to the pelagophytes and chrysophytes, as well as cryptophytes, reached annual peaks during mixed layer shoaling, while Bacillariophyceae (diatoms) were observed only episodically in the 12-year record. Prymnesiophytes dominated the integrated plastid gene signal. They were abundant throughout the water column before mixing events, but persisted in the deep chlorophyll maximum during stratified conditions. Various models have been used to describe mechanisms that drive vernal phytoplankton blooms in temperate seas. The range of taxon-specific bloom patterns observed here indicates that different 'spring bloom' models can aptly describe the behavior of different phytoplankton taxa at a single geographical location. These findings provide insight into the subdivision of niche space by phytoplankton and may lead to improved predictions of phytoplankton responses to changes in ocean conditions.

Unveiling in situ interactions between marine protists and bacteria through single cell sequencing.

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator-prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.