Comparative genomics reveals new functional insights in uncultured MAST species

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

Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles

Molecular surveys in planktonic marine systems have unveiled a large novel diversity of small protists. A large part of this diversity belongs to basal heterotrophic stramenopiles and is distributed in a set of polyphyletic ribogroups (described from rDNA sequences) collectively named as MAST (MArine STramenopiles). In the few groups investigated, MAST cells are globally distributed and abundant bacterial grazers, therefore having a putatively large impact on marine ecosystem functioning. The main aim of this study is to reevaluate the MAST ribogroups described so far and to determine whether additional groups can be found. For this purpose, we used traditional and state-of-the-art molecular tools, combining 18S rDNA sequences from publicly available clone libraries, single amplified genomes (SAGs) of planktonic protists, and a pyrosequencing survey from coastal waters and sediments. Our analysis indicated a final set of 18 MAST groups plus 5 new ribogroups within Ochrophyta (named as MOCH). The MAST ribogroups were then analyzed in more detail. Seven were typical of anoxic systems and one of oxic sediments. The rest were clearly members of oxic marine picoplankton. We characterized the genetic diversity within each MAST group and defined subclades for the more diverse (46 subclades in 8 groups). The analyses of sequences within subclades revealed further ecological specializations. Our data provide a renovated framework for phylogenetic classification of the numerous MAST ribogroups and support the notion of a tight link between phylogeny and ecological distribution. These diverse and largely uncultured protists are widespread and ecologically relevant members of marine microbial assemblages.     

Biogeography of Heterotrophic Flagellate Populations Indicates the Presence of Generalist and Specialist Taxa in the Arctic Ocean

Heterotrophic marine flagellates (HF) are ubiquitous in the world''s oceans and represented in nearly all branches of the domain Eukaryota. However, the factors determining distributions of major taxonomic groups are poorly known. The Arctic Ocean is a good model environment for examining the distribution of functionally similar but phylogenetically diverse HF because the physical oceanography and annual ice cycles result in distinct environments that could select for microbial communities or favor specific taxa. We reanalyzed new and previously published high-throughput sequencing data from multiple studies in the Arctic Ocean to identify broad patterns in the distribution of individual taxa. HF accounted for fewer than 2% to over one-half of the reads from the water column and for up to 60% of reads from ice, which was dominated by Cryothecomonas. In the water column, many HF phylotypes belonging to Telonemia and Picozoa, uncultured marine stramenopiles (MAST), and choanoflagellates were geographically widely distributed. However, for two groups in particular, Telonemia and Cryothecomonas, some species level taxa showed more restricted distributions. For example, several phylotypes of Telonemia favored open waters with lower nutrients such as the Canada Basin and offshore of the Mackenzie Shelf. In summary, we found that while some Arctic HF were successful over a range of conditions, others could be specialists that occur under particular conditions. We conclude that tracking species level diversity in HF not only is feasible but also provides a potential tool for understanding the responses of marine microbial ecosystems to rapidly changing ice regimes.     

Distribution patterns and phylogeny of marine stramenopiles in the north pacific ocean

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml(-1), respectively. MAST-4 and Synechococcus abundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.     

Distribution and abundance of uncultured heterotrophic flagellates in the world oceans

Heterotrophic flagellates play fundamental roles in marine ecosystems as picoplankton grazers. This recognized importance contrasts with our ignorance of the taxonomic composition of this functional group, which remains mostly unidentified by microscopical and culturing approaches. Recent molecular marine surveys based on 18S rDNA genes have retrieved many sequences unrelated to cultured organisms and marine stramenopiles were among the first reported uncultured eukaryotes. However, little is known about the organisms corresponding to these sequences. Here we determine the abundance of several marine stramenopile lineages in surface marine waters using molecular probes and fluorescent in situ hybridization. We show that these protists are free-living bacterivorous heterotrophic flagellates. They were widely distributed, occurring in the five world oceans, and accounted for a significant fraction (up to 35%) of heterotrophic flagellates in diverse geographic regions. A single group, MAST-4, represented 9% of cells within this functional assemblage, with the intriguing exception of polar waters where it was absent. MAST-4 cells likely contribute substantially to picoplankton grazing and nutrient re-mineralization in vast areas of the oceans and represent a key eukaryotic group in marine food webs.     

Pan-oceanic distribution of new highly diverse clades of deep-sea diplonemids

Molecular rRNA gene surveys reveal a considerable diversity of microbial eukaryotes in different environments. Even within a single clade, the number of distinct phylotypes retrieved often goes beyond previous expectations. Here, we have used specific 18S rRNA PCR primers to investigate the diversity of diplonemids, a poorly known group of flagellates with only a few described species. We analysed surface and deep-sea plankton samples from different oceanic regions, including the water-column in the Marmara Sea. We retrieved a large diversity of diplonemid phylotypes, most of which formed two novel distinct clades without cultured representatives. Although most marine diplonemid phylotypes appeared to be cosmopolitan, they showed a marked stratified distribution through the water column, being very scarce or absent in surface waters. The small and specific diplonemid diversity found in surface samples and the fact that most sequences of uncultured diplonemids found in other studies came from deep-sea environments suggest that the two major uncultured diplonemid clades group species preferentially inhabit the deep ocean.     

Composition of Heterotrophic Flagellates in Coastal Waters of Different Trophic Status

Heterotrophic flagellates (HFs) are important members of the aquatic microbial food web. However, information on their spatial patterns in relation to eutrophication is limited. Here, we examined the composition and spatial distributions of HFs (<3 μm) in subtropical coastal waters of different trophic status by re-analyzing two previously published small subunit rDNA pyrosequence datasets using information from the newly launched Protist Ribosomal Reference database (PR²). Whereas the contributions of different major clades composing the Marine Stramenopiles (MASTs), picobiliphytes and Chrysophyceae were found relatively comparable between the stations, contrasting compositions of the Marine Alveolates (MALV) groups I and II were observed. The high and relatively stable contribution of MAST-1, -3 and -7 among the MASTs in both stations suggest their importance as bacterial grazers in coastal waters, irrespective of trophic status. By contrast, the dominance of clades 3, 5 and 14 of MALV II in the eutrophic station implies their importance in regulating the dinoflagellate population at the site. Our study provides insights into the ecological importance of different HF groups in eutrophic coastal ecosystems.     

Distribution of the uncultured protist MAST-4 in the Indian Ocean, Drake Passage and Mediterranean Sea assessed by real-time quantitative PCR

Molecular surveys of marine picoeukaryotes have revealed a large number of sequences unrelated to cultured organisms, such as those forming the marine stramenopile (MAST)-4 clade. Recent FISH (fluorescent in situ hybridization) data have shown that MAST-4 cells are uncultured heterotrophic flagellates of 2–3 μm in size that have a global distribution in non-polar marine waters. However, FISH is time-consuming and hard to apply to the many samples generated during oceanographic cruises, so we developed a real-time quantitative polymerase chain reaction (Q-PCR) protocol to determine rapidly the abundance of this group using environmental DNA. We designed a primer set targeting the 18S rRNA genes (rDNA) of MAST-4 and optimized and calibrated the Q-PCR protocol using a plasmid with the target sequence as insert. The Q-PCR was then applied to quantify MAST-4 rDNA molecules along three marine transects, longitudinal in the Indian Ocean, latitudinal in the Drake Passage and coastal–offshore in the Mediterranean Sea, and to a temporal study in a Mediterranean Sea coastal station. MAST-4 was detected in all samples processed (averaged abundances between 500 and 1000 rDNA molecules ml⁻¹) except in mesopelagic and Antarctic samples, where it was virtually absent. In general, it was more abundant in the coast than offshore and in the deep chlorophyll maximum than at surface. A comparison of Q-PCR and FISH signals in well-controlled microbial incubations indicated that MAST-4 cells have around 30 copies of the rDNA operon. This Q-PCR assay quickly yielded quantitative data of uncultured MAST-4 cells and confirmed their wide distribution and putative ecological importance.     

High Cryptic Diversity across the Global Range of the Migratory Planktonic Copepods Pleuromamma piseki and P. gracilis

Although holoplankton are ocean drifters and exhibit high dispersal potential, a number of studies on single species are finding highly divergent genetic clades. These cryptic species complexes are important to discover and describe, as identification of common marine species is fundamental to understanding ecosystem dynamics. Here we investigate the global diversity within Pleuromamma piseki and P. gracilis, two dominant members of the migratory zooplankton assemblage in subtropical and tropical waters worldwide. Using DNA sequence data from the mitochondrial gene cytochrome c oxidase subunit II (mtCOII) from 522 specimens collected across the Pacific, Atlantic and Indian Oceans, we discover twelve well-resolved genetically distinct clades in this species complex (Bayesian posterior probabilities >0.7; 6.3–17% genetic divergence between clades). The morphologically described species P. piseki and P. gracilis did not form monophyletic groups, rather they were distributed throughout the phylogeny and sometimes co-occurred within well-resolved clades: this result suggests that morphological characters currently used for taxonomic identification of P. gracilis and P. piseki may be inaccurate as indicators of species’ boundaries. Cryptic clades within the species complex ranged from being common to rare, and from cosmopolitan to highly restricted in distribution across the global ocean. These novel lineages appear to be ecologically divergent, with distinct biogeographic distributions across varied pelagic habitats. We hypothesize that these mtDNA lineages are distinct species and suggest that resolving their systematic status is important, given the ecological significance of the genus Pleuromamma in subtropical-tropical waters worldwide.     

Vertical distribution of picoeukaryotic diversity in the Sargasso Sea

Eukaryotic molecular diversity within the picoplanktonic size-fraction has primarily been studied in marine surface waters. Here, the vertical distribution of picoeukaryotic diversity was investigated in the Sargasso Sea from euphotic to abyssal waters, using size-fractionated samples (< 2 microm). 18S rRNA gene clone libraries were used to generate sequences from euphotic zone samples (deep chlorophyll maximum to the surface); the permanent thermocline (500 m); and the pelagic deep-sea (3000 m). Euphotic zone and deep-sea data contrasted strongly, the former displaying greater diversity at the first-rank taxon level, based on 232 nearly full-length sequences. Deep-sea sequences belonged almost exclusively to the Alveolata and Radiolaria, while surface samples also contained known and putative photosynthetic groups, such as unique Chlorarachniophyta and Chrysophyceae sequences. Phylogenetic analyses placed most Alveolata and Stramenopile sequences within previously reported 'environmental' clades, i.e. clades within the Novel Alveolate groups I and II (NAI and NAII), or the novel Marine Stramenopiles (MAST). However, some deep-sea NAII formed distinct, bootstrap supported clades. Stramenopiles were recovered from the euphotic zone only, although many MAST are reportedly heterotrophic, making the observed distribution a point for further investigation. An unexpectedly high proportion of radiolarian sequences were recovered. From these, five environmental radiolarian clades, RAD-I to RAD-V, were identified. RAD-IV and RAD-V were composed of Taxopodida-like sequences, with the former solely containing Sargasso Sea sequences, although from all depth zones sampled. Our findings highlight the vast diversity of these protists, most of which remain uncultured and of unknown ecological function.     

The Kernel of Maximum Agreement Subtrees

A Maximum Agreement SubTree (MAST) is a largest subtree common to a set of trees and serves as a summary of common substructure in the trees. A single MAST can be misleading, however, since there can be an exponential number of MASTs, and two MASTs for the same tree set do not even necessarily share any leaves. In this paper, we introduce the notion of the Kernel Agreement SubTree (KAST), which is the summary of the common substructure in all MASTs, and show that it can be calculated in polynomial time (for trees with bounded degree). Suppose the input trees represent competing hypotheses for a particular phylogeny. We explore the utility of the KAST as a method to discern the common structure of confidence, and as a measure of how confident we are in a given tree set. We also show the trend of the KAST, as compared to other consensus methods, on the set of all trees visited during a Bayesian analysis of flatworm genomes.     

Sequence diversity and novelty of natural assemblages of picoeukaryotes from the Indian Ocean

Despite the ecological importance of marine pico-size eukaryotes, the study of their in situ diversity using molecular tools started just a few years ago. These studies have revealed that marine picoeukaryotes are very diverse and include many novel taxa. However, the amount and structure of their phylogenetic diversity and the extent of their sequence novelty still remains poorly known, as a systematic analysis has been seldom attempted. In this study, we use a coherent and carefully curated data set of 500 published 18S ribosomal DNA sequences to quantify the diversity and novelty patterns of picoeukaryotes in the Indian Ocean. Our phylogenetic tree showed many distant lineages. We grouped sequences in OTUs (operational taxonomic units) at discrete values delineated by pair-wise Jukes–Cantor (JC) distances and tree patristic distances. At a distance of 0.01, the number of OTUs observed (237/242; using JC or patristic distances, respectively) was half the number of sequences analyzed, indicating the existence of microdiverse clusters of highly related sequences. At this distance level, we estimated 600–800 OTUs using several statistical methods. The number of OTUs observed was still substantial at higher distances (39/82 at 0.20 distance) suggesting a large diversity at high-taxonomic ranks. Most sequences were related to marine clones from other sites and many were distant to cultured organisms, highlighting the huge culturing gap within protists. The novelty analysis indicated the putative presence of pseudogenes and of truly novel high-rank phylogenetic lineages. The identified diversity and novelty patterns among marine picoeukaryotes are of great importance for understanding and interpreting their ecology and evolution.     

Molecular diversity and temporal variation of picoeukaryotes in two Arctic fjords,Svalbard

Picoeukaryotes (protists <3 μm) form an important component of Arctic marine ecosystems, although knowledge of their diversity and ecosystem functioning is limited. In this study, the molecular diversity and autotrophic biomass contribution of picoeukaryotes from January to June 2009 in two Arctic fjords at Svalbard were examined using 18S environmental cloning and size-fractioned chlorophyll a measurements. A total of 62 putative picoeukaryotic phylotypes were recovered from 337 positive clones. Putative picoeukaryotic autotrophs were mostly limited to one species: Micromonas pusilla, while the putative heterotrophic picoeukaryote assemblage was more diverse and dominated by uncultured marine stramenopiles (MAST) and marine alveolate groups. One MAST-1A phylotype was the only phylotype to be found in all clone libraries. The diversity of picoeukaryotes in general showed an inverse relationship with total autotrophic biomass, suggesting that the conditions dominating during the peak of the spring bloom may have a negative impact on picoeukaryote diversity. Picoplankton could contribute more than half of total autotrophic biomass before and after the spring bloom and benefited from an early onset of the growth season, whereas larger cells dominated the bloom itself.     

Biogeography of the uncultured marine picoeukaryote MAST-4: temperature-driven distribution patterns

The MAST-4 (marine stramenopile group 4) is a widespread uncultured picoeukaryote that makes up an important fraction of marine heterotrophic flagellates. This group has low genetic divergence and is composed of a small number of putative species. We combined ARISA (automated ribosomal intergenic spacer analysis) and ITS (Internal Transcribed Spacer) clone libraries to study the biogeography of this marine protist, examining both spatial and temporal trends in MAST-4 assemblages and associated environmental factors. The most represented MAST-4 clades appeared adapted to different temperature ranges, and their distributions did not suggest clear geographical barriers for dispersal. Distant samples sharing the same temperature had very similar assemblages, especially in cold temperatures, where only one clade, E1, dominated. The most highly represented clades, A and E1, showed very little differentiation between populations from distant geographical regions. Within a single site, temporal variation also followed patterns governed by temperature. Our results contribute to the general discussion on microbial biogeography by showing strong environmental selection for some picoeukaryotes in the marine environment.     

Population Genetic Diversity in the Australian ‘Seascape’: A Bioregion Approach

Genetic diversity within species may promote resilience to environmental change, yet little is known about how such variation is distributed at broad geographic scales. Here we develop a novel Bayesian methodology to analyse multi-species genetic diversity data in order to identify regions of high or low genetic diversity. We apply this method to co-distributed taxa from Australian marine waters. We extracted published summary statistics of population genetic diversity from 118 studies of 101 species and > 1000 populations from the Australian marine economic zone. We analysed these data using two approaches: a linear mixed model for standardised data, and a mixed beta-regression for unstandardised data, within a Bayesian framework. Our beta-regression approach performed better than models using standardised data, based on posterior predictive tests. The best model included region (Integrated Marine and Coastal Regionalisation of Australia (IMCRA) bioregions), latitude and latitude squared. Removing region as an explanatory variable greatly reduced model performance (delta DIC 23.4). Several bioregions were identified as possessing notably high genetic diversity. Genetic diversity increased towards the equator with a ‘hump’ in diversity across the range studied (−9.4 to −43.7°S). Our results suggest that factors correlated with both region and latitude play a role in shaping intra-specific genetic diversity, and that bioregion can be a useful management unit for intra-specific as well as species biodiversity. Our novel statistical model should prove useful for future analyses of within species genetic diversity at broad taxonomic and geographic scales.     

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances

Grazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half-saturation constant of 6.7 × 10⁵ prey ml⁻¹, a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST-4 and MAST-7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.     

Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters

To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.     

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.