Estelle Kilias

Protist distribution in the Western Fram Strait in summer 2010 based on 454-pyrosequencing of 18S rDNA.

In this study, we present the first comprehensive analyses of the diversity and distribution of marine protist (micro‐, nano‐, and picoeukaryotes) in the Western Fram Strait, using 454‐pyrosequencing and high‐pressure liquid chromatography (HPLC) at five stations in summer 2010. Three stations (T1; T5; T7) were influenced by Polar Water, characterized by cold water with lower salinity (<33) and different extents of ice concentrations. Atlantic Water influenced the other two stations (T6; T9). While T6 was located in the mixed water zone characterized by cold water with intermediate salinity (~33) and high ice concentrations, T9 was located in warm water with high salinity (~35) and no ice‐coverage at all. General trends in community structure according to prevailing environmental settings, observed with both methods, coincided well. At two stations, T1 and T7, characterized by lower ice concentrations, diatoms (Fragilariopsis sp., Porosira sp., Thalassiosira spp.) dominated the protist community. The third station (T5) was ice‐covered, but has been ice‐free for ~4 weeks prior to sampling. At this station, dinoflagellates (Dinophyceae 1, Woloszynskia sp. and Gyrodinium sp.) were dominant, reflecting a post‐bloom situation. At station T6 and T9, the protist communities consisted mainly of picoeukaryotes, e.g., Micromonas spp. Based on our results, 454‐pyrosequencing has proven to be an adequate tool to provide comprehensive information on the composition of protist communities. Furthermore, this study suggests that a snap‐shot of a few, but well‐chosen samples can provide an overview of community structure patterns and succession in a dynamic marine environment.

Regional variability in eukaryotic protist communities in the Amundsen Sea

Abstract We determined the composition and structure of late summer eukaryotic protist assemblages along a west–east transect in the Amundsen Sea. We used state-of-the-art molecular approaches, such as automated ribosomal intergenic spacer analysis (ARISA) and 454-pyrosequencing, combined with pigment measurements via high performance liquid chromatography (HPLC) to study the protist assemblage. We found characteristic offshore and inshore communities. In general, total chlorophyll a and microeukaryotic contribution were higher in inshore samples. Diatoms were the dominant group across the entire area, of which Eucampia sp. and Pseudo-nitzschia sp. were dominant inshore and Chaetoceros sp. was dominant offshore. At the most eastern station, the assemblage was dominated by Phaeocystis sp. Under the ice, ciliates showed their highest and haptophytes their lowest abundance. This study delivers a taxon detailed overview of the eukaryotic protist composition in the Amundsen Sea during the summer 2010.

Picoeukaryote Plankton Composition off West Spitsbergen at the Entrance to the Arctic Ocean

Investigation of marine eukaryotic picoplankton composition is limited by missing morphological features for appropriate identification. Consequently, molecular methods are required. In this study, we used 454‐pyrosequencing to study picoplankton communities at four stations in the West Spitsbergen Current (WSC; Fram Strait). High abundances of Micromonas pusilla were detected in the station situated closest to Spitsbergen, as seen in surveys of picoplankton assemblages in the Beaufort Sea. At the other three stations, other phylotypes, affiliating with Phaeocystis pouchetii and Syndiniales in the phylogenetic tree, were present in high numbers, dominating most of them. The picoplankton community structures at three of the stations, all with similar salinity and temperature, were alike. At the fourth station, the influence of the East Spitsbergen Current, transporting cold water from the Barents Sea around Spitsbergen, causes different abiotic parameters that result in a significantly different picoeukaryote community composition, which is dominated by M. pusilla. This observation is particularly interesting with regard to ongoing environmental changes in the Arctic. Ongoing warming of the WSC could convey a new picoplankton assemblage into the Arctic Ocean, which may come to affect the dominance of M. pusilla.

Biogeography and Photosynthetic Biomass of Arctic Marine Pico-Eukaroytes during Summer of the Record Sea Ice Minimum 2012

Information on recent photosynthetic biomass distribution and biogeography of Arctic marine pico-eukaryotes (0.2–3 μm) is needed to better understand consequences of environmental change for Arctic marine ecosystems. We analysed pico-eukaryote biomass and community composition in Fram Strait and large parts of the Central Arctic Ocean (Nansen Basin, Amundsen Basin) using chlorophyll a (Chl a) measurements, automated ribosomal intergenic spacer analysis (ARISA) and 454-pyrosequencing. Samples were collected during summer 2012, the year with the most recent record sea ice minimum. Chl a concentrations were highest in eastern Fram Strait and pico-plankton accounted for 60–90% of Chl a biomass during the observation period. ARISA-patterns and 454-pyrosequencing revealed that pico-eukaryote distribution is closely related to water mass distribution in the euphotic zone of the Arctic Ocean. Phaeocystaceae, Micromonas sp., Dinophyceae and Syndiniales constitute a high proportion of sequence reads, while sequence abundance of autotrophic Phaeocystaceae and mixotrophic Micromonas sp. was inversely correlated. Highest sequence abundances of Phaeocystaceae were observed in the warm Atlantic Waters in Fram Strait, while Micromonas sp. dominated the abundant biosphere in the arctic halocline. Our results are of particular interest considering existing hypotheses that environmental conditions in Nansen Basin might become more similar to the current conditions in Fram Strait. We propose that in response, biodiversity and biomass of pico-eukaryotes in Nansen Basin could resemble those currently observed in Fram Strait in the future. This would significantly alter biogeochemical cycles in a large part of the Central Arctic Ocean.

Summertime plankton ecology in Fram Strait - a compilation of long- and short-term observations

Between Greenland and Spitsbergen, Fram Strait is a region where cold ice-covered Polar Water exits the Arctic Ocean with the East Greenland Current (EGC) and warm Atlantic Water enters the Arctic Ocean with the West Spitsbergen Current (WSC). In this compilation, we present two different data sets from plankton ecological observations in Fram Strait: (1) long-term measurements of satellite-derived (1998–2012) and in situ chlorophyll a (chl a) measurements (mainly summer cruises, 1991–2012) plus protist compositions (a station in WSC, eight summer cruises, 1998–2011); and (2) short-term measurements of a multidisciplinary approach that includes traditional plankton investigations, remote sensing, zooplankton, microbiological and molecular studies, and biogeochemical analyses carried out during two expeditions in June/July in the years 2010 and 2011. Both summer satellite-derived and in situ chl a concentrations showed slight trends towards higher values in the WSC since 1998 and 1991, respectively. In contrast, no trends were visible in the EGC. The protist composition in the WSC showed differences for the summer months: a dominance of diatoms was replaced by a dominance of Phaeocystis pouchetii and other small pico- and nanoplankton species. The observed differences in eastern Fram Strait were partially due to a warm anomaly in the WSC. Although changes associated with warmer water temperatures were observed, further long-term investigations are needed to distinguish between natural variability and climate change in Fram Strait. Results of two summer studies in 2010 and 2011 revealed the variability in plankton ecology in Fram Strait.

Protist community composition in the Pacific sector of the Southern Ocean during austral summer 2010

Knowledge about the protist diversity of the Pacific sector of the Southern Ocean is scarce. We tested the hypothesis that distinct protist community assemblages characterize large-scale water masses. Therefore, we determined the composition and biogeography of late summer protist assemblages along a transect from the coast of New Zealand to the eastern Ross Sea. We used state of the art molecular approaches, such as automated ribosomal intergenic spacer analysis and 454-pyrosequencing, combined with high-performance liquid chromatography pigment analysis to study the protist assemblage. We found distinct biogeographic patterns defined by the environmental conditions in the particular region. Different water masses harbored different microbial communities. In contrast to the Arctic Ocean, picoeukaryotes had minor importance throughout the investigated transect and showed very low contribution south of the Polar Front. Dinoflagellates, Syndiniales, and small stramenopiles were dominating the sequence assemblage in the Subantarctic Zone, whereas the relative abundance of diatoms increased southwards, in the Polar Frontal Zone and Antarctic Zone. South of the Polar Front, most sequences belonged to haptophytes. This study delivers a comprehensive and taxon detailed overview of the protist composition in the investigated area during the austral summer 2010.

Insight into protist diversity in Arctic sea ice and melt-pond aggregate obtained by pyrosequencing

Protists in the central Arctic Ocean are adapted to the harsh environmental conditions of its various habitats. During the Polarstern cruise ARK-XXVI/3 in 2011, at one sea-ice station, large aggregates accumulated at the bottom of the melt ponds. In this study, the protist assemblages of the bottom layer of the sea-ice and melt-pond aggregate were investigated using flow cytometry and 454-pyrosequencing. The objective is to provide a first molecular overview of protist biodiversity in these habitats and to consider the overlaps and/or differences in the community compositions. Results of flow cytometry pointed to a cell size distribution that was dominated by 3–10 µm nanoflagellates. The phylogenetic classification of all sequences was conducted at a high taxonomic level, while a selection of abundant (≥1% of total reads) sequences was further classified at a lower level. At a high taxonomic level, both habitats showed very similar community structures, dominated by chrysophytes and chlorophytes. At a lower taxonomic level, dissimilarities in the diversity of both groups were encountered in the abundant biosphere. While sea-ice chlorophytes and chrysophytes were dominated by Chlamydomonas/Chloromonas spp. and Ochromonas spp., the melt-pond aggregate was dominated by Carteria sp., Ochromonas spp. and Dinobryon faculiferum. We suppose that the similarities in richness and community structure are a consequence of melt-pond freshwater seeping through porous sea ice in late summer. Differences in the abundant biosphere nevertheless indicate that environmental conditions in both habitats vary enough to select for different dominant species.

Evaluating the potential of 18S rDNA clone libraries to complement pyrosequencing data of marine protists with near full-length sequence information

Abstract Sequencing of 18S rDNA clone libraries and 454-pyrosequencing are valuable methods used to describe microbial diversity. The massively parallel 454-pyrosequencing generates vast amounts of ribosomal sequence data and has the potential to uncover more organisms, even rare species. However, the relatively short sequence lengths of ∼500 bp are suboptimal for taxonomic annotation and phylogenetic analyses. In this study, we assessed the potential of 18S ribosomal clone libraries to complement corresponding 454-pyrosequencing data with near full-length sequence information. This involved a comparison of protist community compositions in five polar samples suggested by 18S rDNA clone libraries, with the corresponding community compositions suggested by 454-pyrosequencing. The study was conducted with four Arctic water samples, focusing on the eukaryotic picoplankton (0.4–3 µm), and with one sample collected in the Southern Ocean, examining the entire size spectrum (> 0.4 µm). For all individual samples, the protist community compositions suggested by the two different approaches showed significant similarities. Around 70% of the sequences detected by sequencing of clone libraries were also present in the 454-pyrosequencing data set. However, the clone library sequences reflected only ∼20% of the abundant biosphere identified by 454-pyrosequencing and identified ribosomal sequences that were not detected in the 454-pyrosequencing data sets.

Protists in the polar regions: comparing occurrence in the Arctic and Southern oceans using pyrosequencing

In the ongoing discussion of the distribution of protists, whether they are globally distributed or endemic to one or both of the polar regions is the subject of heated debate. In this study, we compared next-generation sequencing data from the Arctic and the Southern oceans to reveal the extent of similarities and dissimilarities between the protist communities in the polar regions. We found a total overlap of operational taxonomic units (OTUs) between the two regions of 11.2%. On closer inspection of different taxonomic groups, the overlap ranged between 5.5% (haptophytes) and 14.5% (alveolates). Within the different groups, the proportion of OTUs occurring in both regions greatly differed between the polar regions. On the one hand, the overlap between these two regions is remarkable, given the geographical distance between them. On the other hand, one could expect a greater overlap of OTUs between these regions on account of the similar environmental conditions. The overlap suggests a connection between the polar regions for at least certain species or that the evolutionary divergence has been slow, relative to the timescales of isolation. The different proportions of common OTUs among the groups or regions may be a result of different life cycle strategies or environmental adaptations.

Impact of sequence processing and taxonomic classification approaches on eukaryotic community structure from environmental samples with emphasis on diatoms

Next‐generation sequencing is a common method for analysing microbial community diversity and composition. Configuring an appropriate sequence processing strategy within the variety of tools and methods is a nontrivial task and can considerably influence the resulting community characteristics. We analysed the V4 region of 18S rRNA gene sequences of marine samples by 454‐pyrosequencing. Along this process, we generated several data sets with QIIME, mothur, and a custom‐made pipeline based on DNAStar and the phylogenetic tree‐based PhyloAssigner. For all processing strategies, default parameter settings and punctual variations were used. Our results revealed strong differences in total number of operational taxonomic units (OTUs), indicating that sequence preprocessing and clustering had a major impact on protist diversity estimates. However, diversity estimates of the abundant biosphere (abundance of ≥1%) were reproducible for all conducted processing pipeline versions. A qualitative comparison of diatom genera emphasized strong differences between the pipelines in which phylogenetic placement of sequences came closest to light microscopy‐based diatom identification. We conclude that diversity studies using different sequence processing strategies are comparable if the focus is on higher taxonomic levels, and if abundance thresholds are used to filter out OTUs of the rare biosphere.