Photosynthetic Picoeukaryotes Community Research Articles

Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes.

ABSTRACTAs the key contributor to plankton biomass and nutrient cycling in aquatic ecosystems, photosynthetic picoeukaryotes (PPEs) have been recently investigated in freshwater ecosystems. However, the limited access to remote areas creates challenges for PPE sample preservation before sorting and counting by flow cytometry (FCM) in the laboratory. Here, we explored the effects of different preservation methods on the PPE community by combining FCM sorting and high-throughput sequencing. Our results showed that dimethyl sulfoxide (DMSO) cryoprotection could destroy the fluorescence and cell structure of the PPEs, making the subsequent FCM analysis and sorting difficult. Aldehyde fixation maintained the PPE fluorescence, and the fixed samples were of sufficient quality for abundance analysis and sorting by FCM. However, the sequencing results showed that, after preservation by aldehydes, the proportion of PPEs dramatically decreased to approximately 10%, in comparison to 90% in the fresh samples, and the sequences of Ascomycota significantly increased. In contrast, preservation with Pluronic F68 (F68) not only could maintain the PPE abundance close to the initial value but also could keep the PPE community similar to that in the fresh samples over a storage time of 6 months. Thus, F68 cryopreservation is a suitable preservation method for PPE communities from freshwater lakes.IMPORTANCE PPEs contribute significantly to primary productivity in freshwater ecosystems. The combination of FCM sorting and high-throughput sequencing has been shown to be a powerful approach and can largely improve our view of the PPE diversity. However, the water samples could not be counted and sorted immediately after sampling from many lakes due to the inaccessibility of FCM in the field. Thus, the comparison of different preservation methods that allow subsequent analysis of the community structure by high-throughput sequencing is an urgent need. Our results indicated that F68 cryopreservation could maintain the PPE abundance close to the initial value and keep the community similar to that in the fresh samples over a storage time of 6 months.

Seasonal and Spatial Variations in Synechococcus Abundance and Diversity Throughout the Gullmar Fjord, Swedish Skagerrak.

The picophytoplankton Synechococcus is a globally abundant autotroph that contributes significantly to primary production in the oceans and coastal areas. These cyanobacteria constitute a diverse genus of organisms that have developed independent niche spaces throughout aquatic environments. Here, we use the 16S V3–V4 rRNA gene region and flow cytometry to explore the diversity of Synechococcus within the picophytoplankton community in the Gullmar Fjord, on the west coast of Sweden. We conducted a station-based 1-year time series and two transect studies of the fjord. Our analysis revealed that within the large number of Synechococcus amplicon sequence variants (ASVs; 239 in total), prevalent ASVs phylogenetically clustered with clade representatives in both marine subcluster 5.1 and 5.2. The near-surface composition of ASVs shifted from spring to summer, when a 5.1 subcluster dominated community developed along with elevated Synechococcus abundances up to 9.3 × 104 cells ml–1. This seasonal dominance by subcluster 5.1 was observed over the length of the fjord (25 km), where shifts in community composition were associated with increasing depth. Unexpectedly, the community shift was not associated with changes in salinity. Synechococcus abundance dynamics also differed from that of the photosynthetic picoeukaryote community. These results highlight how seasonal variations in environmental conditions influence the dynamics of Synechococcus clades in a high latitude threshold fjord.

The spatial distribution of the photosynthetic picoeukaryotes community structure in Lake Hongze.

The spatial dynamics of picophytoplankton were investigated by flow cytometry and high-throughput sequencing in Lake Hongze, a large river-connecting lake. Picophytoplankton were mainly composed of phycocyanin-rich picocyanobacteria (PCY) and photosynthetic picoeukaryotes (PPEs). Picocyanobacteria was the dominant picophytoplankton group since the contribution of PPEs to total picophytoplankton was only 11.78%. However, PPEs were highly diverse and were composed of Chlorophyta, Bacillariophyta and Chrysophyceae. Environmental factors showed spatial differences, particularly in total phosphorus (TP), suspended solids (SS) and chemical oxygen demand (CODMn), which showed relatively high concentrations around the river channel. The abundances of PPEs and PCY showed similar spatial patterns, which were relatively low in the river course since they were negatively related to SS and CODMn. The top 10 OTUs contributed 79.18% of the total PPEs reads and affiliated with main species in PPEs. CCA results showed that, PPEs community composition was significantly impacted by temperature and DO at sites away from river channel, while was only influenced by nitrite at sites around the river channel.

The daily effect is more important than the diurnal effect when shaping photosynthetic picoeukaryotes (PPEs) communities in Lake Taihu at a small temporal scale.

As major primary producers in aquatic systems, the diversity and community composition dynamics of photosynthetic picoeukaryotes (PPEs) have been investigated in recent years. Here, we explored the 3-day diurnal succession of the PPE community in a highly eutrophic lake in early spring using a combination of flow cytometric sorting and high-throughput sequencing. Our results showed that the PPEs were the dominant group of autotrophic picophytoplankton, although they had relatively low diversity and were dominated by the Stephanodiscaceae family in early spring in Lake Taihu. Furthermore, PPE abundance was significantly higher at night than during the day, probably due to their high specific rate of carbon uptake during the day and rapid proliferation at night. Several environmental variables had a significant influence on the PPE community composition, and the daily effect was more important than the diurnal effect when shaping the PPE community in Lake Taihu at a temporal scale of 3 days. Furthermore, based on the variation partitioning analysis (VPA), the relative importance of abiotic factors (deterministic processes) to short-term succession was low, explaining only 20.44% of the PPE community variation. We therefore conclude that stochastic processes determined PPE community assembly over a short temporal scale based on a neutral community model (NCM).

Temporal patterns in the interaction between photosynthetic picoeukaryotes and their attached fungi in Lake Chaohu.

The combination of flow cytometric sorting and high-throughput sequencing revealed the broad existence of photosynthetic picoeukaryote attached fungi (PPE-attached fungi) in Lake Chaohu. The relative sequence abundance of attached fungi was negatively correlated with that of the photosynthetic picoeukaryotes (PPEs). PPE-attached fungal communities were mainly composed of Basidiomycota, Chytridiomycota and Ascomycota. Temperature, Si and PPE community structure are the most important driving factors for the temporal succession of PPE-attached fungal communities. In particular, PPE-attached fungi can be divided into three groups from high to low temperatures. Phylogenetic molecular ecological network results indicated that the connectivity and the total number of links in the network of the high-temperature group (> 21.82°C) are higher than those in the other two temperature groups (between 9.67 and 21.82°C, and < 9.67°C, respectively). Moreover, the interaction between PPE-attached fungi and the PPEs changed from antagonistic to cooperative, with the decline in temperature. The most abundant operational taxonomic units of PPE-attached fungi were affiliated with the Cladosporium, the most common saprophytic fungus, whereas most fungal hub taxa were Chytridiomycota, the main parasite fungi of phytoplankton.

Temperature mainly determines the temporal succession of the photosynthetic picoeukaryote community in Lake Chaohu, a highly eutrophic shallow lake

Photosynthetic picoeukaryotes (PPEs) are key players in aquatic systems, while their diversity and community composition dynamics remain poorly understood. The monthly composition of PPEs in Lake Chaohu was investigated using a combination of flow cytometry sorting and high throughput sequencing. Results indicated that temperature is the most important factor shaping PPEs community structure. The PPEs community can be categorized into three groups that are dominant at different temperature ranges: high temperature (>21.8 °C), intermediate temperature (between 9.8 °C and 21.8 °C) and low temperature (<9.8 °C). At the supergroup level, Cryptophyta were dominant at the intermediate temperature level, and Bacillariophyta were prevalent at low temperatures. In comparison, Chlorophyta PPEs were sensitive to temperature at the order level. Molecular network analysis using 18S rDNA sequencing results from sorted samples revealed that the Operational Taxonomic Units (OTUs) of PPE from the same taxonomic groups were predominantly positive, implying that they were occupying similar niches. The cooccurrence patterns between PPEs and fungi were mostly negative. In particular, OTU101, which was associated with Chytridiomycota, was negatively related to many OTUs belonging to Chlorophyta and Diatom, indicating that their potential parasitic associations may be not species-specific.

Spatial distribution characteristics of photosynthetic picoeukaryotes community structure and its impacting factors in Lake Baiyangdian

Spatial distribution characteristics of photosynthetic picoeukaryotes community structure and its impacting factors in Lake Baiyangdian

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.

Temporal and spatial variations in the composition of freshwater photosynthetic picoeukaryotes revealed by MiSeq sequencing from flow cytometry sorted samples.

The diversity and composition of photosynthetic picoeukaryotes (PPEs) in two large shallow lakes in China (Lake Taihu and Lake Chaohu) were investigated from flow cytometry sorted samples using Miseq high-throughput sequencing. We collected 65 samples covering different regions of the two lakes over four seasons to unveil spatial and temporal patterns of PPEs community composition. The use of flow cytometry sorting largely improved the efficiency of detecting PPEs sequences and over 70% of the retrieved reads belonged to PPEs. Chlorophyta and Bacillariophyta dominated PPEs in most of the samples. A distinct but complex seasonality of PPEs composition emerged at the OTUs level. NGS-based Miseq sequencing facilitates an in-depth view of numerous rare OTUs. Nearly 80% of the PPEs OTUs were rare and lots of them were detected only in one season, whereas most of the abundant OTUs were frequently detected in all seasons but only changed in relative abundances. Besides, a close relative of the marine PPEs species Ostreococcus sp. (OTU_1144, 99% identity) was discovered in freshwater systems for the first time and was abundant especially in winter. The diversity and community composition of PPEs were more dependent on season rather than sampling sites. Temperature, phycocyanin and NO3 N concentrations in Lake Taihu explained the PPE composition variations, whereas in Lake Chaohu TN/TP ratios, temperature, pH and nephelometric turbidity units (NTU) seemed to be the most important factors. In addition, a great number of OTUs belong to nonpigmented picoeukaryotes, especially Chytridiomycota, Perkinsozoa, Ciliophora and Cercozoa, which are known to include algae parasites as well as predators. The results of mantel test also showed that the community of photosynthetic and nonpigmented picoeukaryotes were significantly correlated in both lakes.

Application of cryopreservation to genetic analyses of a photosynthetic picoeukaryote community

Cryopreservation is useful for long-term maintenance of living strains in microbial culture collections. We applied this technique to environmental specimens from two monitoring sites at Sendai Bay, Japan and compared the microbial diversity of photosynthetic picoeukaryotes in samples before and after cryopreservation. Flow cytometry (FCM) showed no considerable differences between specimens. We used 2500 cells sorted with FCM for next-generation sequencing of 18S rRNA gene amplicons and after removing low-quality sequences obtained 10,088–37,454 reads. Cluster analysis and comparative correlation analysis of observed high-level operational taxonomic units indicated similarity between specimens before and after cryopreservation. The effects of cryopreservation on cells were assessed with representative culture strains, including fragile cryptophyte cells. We confirmed the usefulness of cryopreservation for genetic studies on environmental specimens, and found that small changes in FCM cytograms after cryopreservation may affect biodiversity estimation.

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 β-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.

Composition of the summer photosynthetic pico and nanoplankton communities in the Beaufort Sea assessed by T-RFLP and sequences of the 18S rRNA gene from flow cytometry sorted samples

The composition of photosynthetic pico and nanoeukaryotes was investigated in the North East Pacific and the Arctic Ocean with special emphasis on the Beaufort Sea during the MALINA cruise in summer 2009. Photosynthetic populations were sorted using flow cytometry based on their size and pigment fluorescence. Diversity of the sorted photosynthetic eukaryotes was determined using terminal-restriction fragment length polymorphism analysis and cloning/sequencing of the 18S ribosomal RNA gene. Picoplankton was dominated by Mamiellophyceae, a class of small green algae previously included in the prasinophytes: in the North East Pacific, the contribution of an Arctic Micromonas ecotype increased steadily northward becoming the only taxon occurring at most stations throughout the Beaufort Sea. In contrast, nanoplankton was more diverse: North Pacific stations were dominated by Pseudo-nitzschia sp. whereas those in the Beaufort Sea were dominated by two distinct Chaetoceros species as well as by Chrysophyceae, Pelagophyceae and Chrysochromulina spp.. This study confirms the importance of Arctic Micromonas within picoplankton throughout the Beaufort Sea and demonstrates that the photosynthetic picoeukaryote community in the Arctic is much less diverse than at lower latitudes. Moreover, in contrast to what occurs in warmer waters, most of the key pico- and nanoplankton species found in the Beaufort Sea could be successfully established in culture.

Structure and seasonal dynamics of the eukaryotic picophytoplankton community in a wind‐driven coastal upwelling ecosystem

We studied the structure and seasonal dynamics of the photosynthetic picoeukaryote community at a coastal site off central Chile that is strongly affected by seasonal wind‐driven upwelling. We determined the picoeukaryote phylogenetic diversity by cloning and sequencing the 18S ribosomal ribonucleic acid (rRNA) gene amplified with Chloroplastida‐biased primers. Chloroplastida made up 100% (autumn), 61% (spring), and 20% (summer) of the clone libraries. Most sequences belonged to Mamiellophyceae and clustered with Micromonas (clades A.BC.1, B.E.3, C.D.5), Ostreococcus (clade A), and Crustomastix (clade A). Fluorescent in situ hybridization coupled with tyramide signal amplification (FISH–TSA) was used to determine the time variability in abundance of the total picoeukaryotic community, the Chloroplastida, and the genera Micromonas, Ostreococcus, and Bathycoccus over a period of 2 yr with nearly monthly sampling. The abundance of photosynthetic picoeukaryotes was low during the upwelling season (spring and summer) and high during the nonupwelling season (late fall and winter). Chloroplastida made up, on average, 74% of the total picoeukaryotic community, and the sum of the three genera probed accounted for 100% of the Chloroplastida abundances. Ostreococcus dominated the picophytoplanktonic community numerically throughout the year and, thus, appears to be a key component of the upwelling picoplanktonic community in the eastern South Pacific.

Analysis of photosynthetic picoeukaryote community structure along an extended Ellett Line transect in the northern North Atlantic reveals a dominance of novel prymnesiophyte and prasinophyte phylotypes

Photosynthetic picoeukaryotes (PPEs) of a size <3 μm can contribute significantly to primary production. Here, PPE community structure was analysed along an extended Ellett Line transect, an area in the North Atlantic well studied by physical oceanographers but largely neglected in the field of microalgal ecology. Distribution patterns of specific PPE classes were determined using dot-blot hybridization analysis, while the taxonomic composition of specific PPE classes was revealed by phylogenetic analysis of plastid 16S rRNA gene sequences. In addition, we performed fluorescent in situ hybridization (FISH) analysis of seawater samples collected along the transect to provide a PCR-independent survey of class level PPE distribution patterns. We found the PPE community was dominated by members of the Prymnesiophyceae, Prasinophyceae and Mamiellophyceae. Interestingly, phylogenetic analysis revealed several novel Prymnesiophyceae and Prasinophyceae phylotypes (with only 85–96% identity to neighbouring sequences) within lineages for which cultured counterparts are unknown.

Basin‐scale distribution patterns of photosynthetic picoeukaryotes along an Atlantic Meridional Transect

Photosynthetic picoeukaryotes (PPEs) of a size < 3 µm play a crucial role in oceanic primary production. However, little is known of the structure of the PPE community over large spatial scales. Here, we investigated the distribution of various PPE classes along an Atlantic Meridional Transect sampled in boreal autumn 2004 that encompasses a range of ocean provinces (gyres, upwelling, temperate regions), using dot blot hybridization technology targeting plastid 16S rRNA gene amplicons. Two algal classes, Prymnesiophyceae and Chrysophyceae, dominated the PPE community throughout the Atlantic Ocean, over a range of water masses presenting different trophic profiles. However, these classes showed strongly complementary distributions with Chrysophyceae dominating northern temperate waters, the southern gyre and equatorial regions, while prymnesiophytes dominated the northern gyre. Phylogenetic analyses using both plastid and nuclear rRNA genes revealed a high diversity among members of both classes, including sequences contained in lineages with no close cultured counterpart. Other PPE classes were less prevalent along the transect, with members of the Cryptophyceae, Pelagophyceae and Eustigmatophyceae essentially restricted to specific regions. Multivariate statistical analyses revealed strong relationships between the distribution patterns of some of these latter PPE classes and temperature, light intensity and nutrient concentrations. Cryptophyceae, for example, were mostly found in the upwelling region and associated with higher nutrient concentrations. However, the key classes of Prymnesiophyceae and Chrysophyceae were not strongly influenced by the variables measured. Although there appeared to be a positive relationship between Chrysophyceae distribution and light intensity, the complementary distributions of these classes could not be explained by the variables recorded and this requires further explanation.

Photosynthetic picoeukaryote community structure in the South East Pacific Ocean encompassing the most oligotrophic waters on Earth

Photosynthetic picoeukaryotes (PPEs), comprising organisms < 3 mum in size, are important primary producers in marine food webs and include representatives from all known algal lineages. Little is known, however, regarding the composition and distribution of PPE communities, particularly at large spatial scales, or in relation to the underlying biotic and abiotic factors that influence this structure. Here, we analysed PPE community structure along a transect in the South East Pacific Ocean (BIOSOPE cruise) that encompassed a large trophic gradient, including hyper-oligotrophic waters in the South Pacific Gyre (SPG), considered to be some of the 'clearest' natural waters on Earth. Using dot blot hybridizations with 16S rRNA oligonucleotide probes, we established that the PPE community was dominated by members of the classes Prymnesiophyceae and Chrysophyceae throughout the transect. Moreover, clone library construction followed by phylogenetic analysis of sequenced clones revealed several novel 16S rRNA gene lineages, including new clades of prymnesiophytes (designated Prym 16S-III) and prasinophytes (Pras 16S-VIII). Pras 16S-VIII was found at all five stations at which clone libraries were constructed, representing a range of trophic conditions, including the South Pacific Gyre, suggesting members of this clade have a broad distribution in this part of the South East Pacific at least. In contrast, Prym 16S-III sequences were largely restricted to oligotrophic stations of the SPG. Subsequent multivariate statistical analyses showed that, within the measured factors, chemical and biological factors seem to influence PPE community structure more than physical parameters. However, more than 50% of the variation in distribution of PPE classes remained unexplained.

Groups without Cultured Representatives Dominate Eukaryotic Picophytoplankton in the Oligotrophic South East Pacific Ocean

BackgroundPhotosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone.Methodology/Principal FindingsIn order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated.Conclusions/SignificanceOur data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups.

Molecular analysis of photosynthetic picoeukaryote community structure along an Arabian Sea transect

We developed oligonucleotide probes, based on plastid 16S ribosomal DNA (rDNA) sequences, to target 10 different algal classes (Chlorarachniophyceae, Chrysophyceae, Cryptophyceae, Eustigmatophyceae, Pavlovophyceae, Pelagophyceae, Pinguiophyceae, Prasinophyceae [clade VI], Prymnesiophyceae, and Trebouxiophyceae), for use with dot blot hybridization technology. These class‐specific probes were then used to investigate the community structure of photosynthetic picoeukaryotes (≪3 µm) along a transect in the Arabian Sea during September 2001, using hybridization to plastid 16S rDNA polymerase chain reaction amplicons. The transect ranged from oligotrophic open‐ocean waters, just south of the Equator, through mesotrophic and eutrophic waters toward the north coast of Oman, crossing a region of monsoonal upwelling off the Omani northeast coast. Photosynthetic picoeukaryotes in the surface‐mixed layer (SML) and deep chlorophyll maximum ranged from 1.0 x 103 cells mL‐1 in southern oligotrophic waters to almost 30 x 103 cells mL‐1 at the region of upwelling. Chrysophytes were abundant along most of the transect throughout much of the euphotic zone. Prymnesiophytes were abundant in surface waters along much of the transect. By contrast, trebouxiophytes were confined to deeper waters, below the SML. Pelagophytes were found in surface waters that were more mesotrophic, whereas cryptophytes were only detected in the more nutrient‐rich waters at the northern end of the transect, between depths of 20‐30 m. Pinguiophytes were also detected, but only in the warm surface waters off the north coast of Oman. Chlorarachniophytes, eustigmatophytes, pavlovophytes, and clade VI prasinophytes were essentially below detection limits for the entire transect.

Analysis of photosynthetic picoeukaryote diversity at open ocean sites in the Arabian Sea using a PCR biased towards marine algal plastids

Marine photosynthetic picoeukaryotes (PPEs), representing organisms <3 µm in size, are major contributors to global carbon cycling. However, the key members of the PPE community and hence the major routes of carbon fixation, particularly in the open ocean environment, are poorly described. Here, we have accessed PPE community structure using the plastid encoded 16S rRNA gene. Plastid 16S rRNA genes were sequenced from 65 algal cultures, about half being PPEs, representing 14 algal classes. These included sequences from 5 classes where previously no such sequences from cultured representatives had been available (Bolidophyceae, Dictyochophyceae, Eustigmatophyceae, Pelagophyceae and Pinguiophyceae). Sequences were also obtained for 6 of the 7 (according to 18S rRNA gene sequence) prasinophyte clades. Phylogenetic analysis revealed plastids from the same class as clustering together. Using all the obtained sequences, as well as plastid sequences currently in public databases, a non-degenerate marine algal plastid-biased PCR primer, PLA491F, was developed to minimize amplification of picocyanobacteria, which often dominate numerically environmental samples. Clone libraries subsequently constructed from the pico-sized fraction from 2 open ocean sites in the Arabian Sea, revealed an abundance of 16S rRNA gene clones phylogenetically related to chrysophytes, whilst prymnesiophyte, clade II prasinophyte (Ostreococcus-like) and pelagophyte clones were also well represented. The finding of a wealth of novel clones related to the Chrysophyceae highlights the utility of a PCR biased towards marine algal plastids as a valuable complement to 18S rDNA based studies of PPE diversity.