DNA metabarcoding reveals that 200‐μm‐size‐fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes

Microeukaryotic plankton (0.2–200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High‐throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size‐fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore‐size sieve –by using SSU rDNA‐based high‐throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore‐size sieve. We further found that both >0.2 μm and 0.2–200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size‐fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.     

Diversity and dynamics of Antarctic marine microbial eukaryotes under manipulated environmental UV radiation

In the light of the predicted global climate change, it is essential that the status and diversity of polar microbial communities is described and understood. In the present study, molecular tools were used to investigate the marine eukaryotic communities of Prydz Bay, Eastern Antarctica, from November 2002 to January 2003. Additionally, we conducted four series of minicosm experiments, where natural Prydz Bay communities were incubated under six different irradiation regimes, in order to investigate the effects of natural UV radiation on marine microbial eukaryotes. Denaturing gradient gel electrophoresis (DGGE) and 18S rRNA gene sequencing revealed a eukaryotic Shannon diversity index averaging 2.26 and 2.12, respectively. Phylogenetic analysis of 472 sequenced clones revealed 47 phylotypes, belonging to the Dinophyceae, Stramenopiles, Choanoflagellidae, Ciliophora, Cercozoa and Metazoa. Throughout the studied period, three communities were distinguished: a postwinter/early spring community comprising dinoflagellates, ciliates, cercozoans, stramenopiles, viridiplantae, haptophytes and metazoans; a dinoflagellate-dominated community; and a diatom-dominated community that developed after sea ice breakup. DGGE analysis showed that size fraction and time had a strong shaping effect on the community composition; however, a significant contribution of natural UV irradiance towards microeukaryotic community composition could not be detected. Overall, dinoflagellates dominated our samples and their diversity suggests that they fulfill an important role in Antarctic coastal marine ecosystems preceding ice breakup as well as between phytoplankton bloom events.     

Oligotrophic waters of the Northwest Atlantic support taxonomically diverse diatom communities that are distinct from coastal waters

Diatoms are important components of the marine food web and one of the most species-rich groups of phytoplankton. The diversity and composition of diatoms in eutrophic nearshore habitats have been well documented due to the outsized influence of diatoms on coastal ecosystem functioning. In contrast, patterns of both diatom diversity and community composition in offshore oligotrophic regions where diatom biomass is low have been poorly resolved. To compare the diatom diversity and community composition in oligotrophic and eutrophic waters, diatom communities were sampled along a 1,250 km transect from the oligotrophic Sargasso Sea to the coastal waters of the northeast US shelf. Diatom community composition was determined by amplifying and sequencing the 18S rDNA V4 region. Of the 301 amplicon sequence variants (ASVs) identified along the transect, the majority (70%) were sampled exclusively from oligotrophic waters of the Gulf Stream and Sargasso Sea and included the genera Bacteriastrum, Haslea, Hemiaulus, Pseudo-nitzschia, and Nitzschia. Diatom ASV richness did not vary along the transect, indicating that the oligotrophic Sargasso Sea and Gulf Stream are occupied by a diverse diatom community. Although ASV richness was similar between oligotrophic and coastal waters, diatom community composition in these regions differed significantly and was correlated with temperature and phosphate, two environmental variables known to influence diatom metabolism and geographic distribution. In sum, oligotrophic waters of the western North Atlantic harbor diverse diatom assemblages that are distinct from coastal regions, and these open ocean diatoms warrant additional study, as they may play critical roles in oligotrophic ecosystems.     

Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity

Biodiversity is fundamental to both eukaryote and prokaryote ecology, yet investigations of diversity often differ markedly between the two disciplines. Host specificity - the association of organisms with only a few (specialism) or many (generalism) host species - is recognized within eukaryote ecology as a key determinant of diversity. In contrast, its implications for microbial diversity have received relatively little attention. Here we explore the relationship between microbial diversity and host specificity using marine sponge-bacteria associations. We used a replicated, hierarchical sampling design and both 16S rDNA- and rpoB-based denaturing gradient gel electrophoresis (DGGE) to examine whether three co-occurring sponges from temperate Australia -Cymbastela concentrica, Callyspongia sp. and Stylinos sp. - contained unique, specialized communities of microbes. Microbial communities varied little within each species of sponge, but variability among species was substantial. Over five seasons, the microbial community in C. concentrica differed significantly from other sponges, which were more similar to seawater. Overall, three types of sponge-associated bacteria were identified via 16S rDNA sequencing of excised DGGE bands: 'specialists'- found on only one host species, 'sponge associates'- found on multiple hosts but not in seawater, and 'generalists' from multiple hosts and seawater. Analogous to other high diversity systems, the degree of specificity of prokaryotes to host eukaryotes could have a potentially significant effect on estimates of marine microbial diversity.     

High‐throughput sequencing reveals neustonic and planktonic microbial eukaryote diversity in coastal waters

Neustonic organisms inhabit the sea surface microlayer (SML) and have important roles in marine ecosystem functioning. Here, we use high‐throughput 18S rRNA gene sequencing to characterize protist and fungal diversity in the SML at a coastal time‐series station and compare with underlying plankton assemblages. Protist diversity was higher in February (pre‐bloom) compared to April (spring bloom), and was lower in the neuston than in the plankton. Major protist groups, including Stramenopiles and Alveolata, dominated both neuston and plankton assemblages. Chrysophytes and diatoms were enriched in the neuston in April, with diatoms showing distinct changes in community composition between the sampling periods. Pezizomycetes dominated planktonic fungi assemblages, whereas fungal diversity in the neuston was more varied. This is the first study to utilize a molecular‐based approach to characterize neustonic protist and fungal assemblages, and provides the most comprehensive diversity assessment to date of this ecosystem. Variability in the SML microeukaryote assemblage structure has potential implications for biogeochemical and food web processes at the air‐sea interface.     

Spatio‐temporal Variations in the Molecular Diversity of Microeukaryotes in Particular Ciliates in Soil of the Yellow River Delta,China

The Yellow River delta in China is one of the most active regions of land–ocean interaction. It has suffered serious salinization due to drying‐up of the Yellow River, rising sea level, and seashore erosion, and thus represents a special and extreme environment. We evaluated the microeukaryotic molecular diversity and its response to change of seasons and environmental variables, in particular salinity in the soil of the Yellow River delta, by denaturing gradient gel electrophoresis (DGGE) and gene sequencing. The sequencing of the microeukaryotic DGGE bands revealed the presence of diverse groups dominated by protists in particular ciliates. We further recovered a high diversity of marine and soil ciliates inhabiting in coastal soil using the ciliate‐specific DGGE. The neighbor‐joining tree indicated that the ciliate 18S rDNA sequences from high‐salinity soil were affiliated to Colpodea, Spirotrichea, Litostomatea, and Oligohymenophorea, while all the sequences unique to the low‐salinity soil were affiliated to Colpodea. Statistical analysis indicated that the microeukaryotic molecular diversity was significantly different among sites, while statistically indistinguishable among seasons. Soil salinity might be the main factor regulating the distribution of microeukaryotes in the soil from the Yellow River delta.     

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.     

Damming river shapes distinct patterns and processes of planktonic bacterial and microeukaryotic communities

Planktonic bacterial and microeukaryotic communities play important roles in biogeochemical cycles, but their biogeographic patterns and community assembly processes in large damming rivers still remain unclear. In this study, 16S rRNA and 18S rRNA coding genes were used for sample sequencing analysis of planktonic bacterial and microeukaryotic communities in the upper Yangtze River. The upper Yangtze River was divided into dam-affected zones and river zones based on the influence of dams. The results showed that there were significant differences in the bacterial and microeukaryotic communities between the two zones and that dams significantly reduced the α-diversity of the bacterial communities. Co-occurrence network analysis indicated that networks in the river zone were denser than those in the dam-affected zone. The relationships among species in bacterial networks were more complex than those in microeukaryotic networks. Dispersal limitation and ecological drift were the main processes influencing planktonic bacterial and microeukaryotic communities in the dam-affected zone respectively, whereas the role of deterministic processes increased in the river zone. Anthropogenic activities and hydraulic conditions affected suspended sediment and controlled microbial diversity in the river zone. These results suggest that dams impact planktonic bacteria more strongly than planktonic microeukaryotes, indicating that the distribution patterns and processes of the bacterial and microeukaryotic communities in large rivers are significantly different.     

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.     

福建敖江口表层沉积硅藻空间分布特征

为探讨福建敖江口表层沉积硅藻空间分布特征,2019年7月对敖江口进行表层沉积硅藻的采样调查,并研究了硅藻与环境因子的关系。结果表明,从13个站点共检出硅藻114种,隶属于39属。硅藻丰度具有从河口向外海先减少后增加的变化特征。聚类分析表明,主要硅藻属种可划分为淡水种组合带和半咸水-咸水种组合带。典型对应分析（CCA）表明,表层海水温度和盐度是影响硅藻分布的主要环境变量。河口区盐度相对较低,主要分布淡水种双面曲壳藻、优美曲壳藻和颗粒沟链藻;外海区北部盐度相对较高,主要分布咸水种流水双菱藻;外海区南部盐度相对较低,主要分布淡水种优美曲壳藻和颗粒沟链藻;滩涂的沉积物较粗,主要分布优美曲壳藻。此外,河口区和外海区南部可能存在一定的水体污染。     

Summer zooplankton communities of selected borrow-pit ponds in Northern Illinois,U.S.A.

Zooplankton samples were collected from 7 borrow-pit ponds during the summer months of 1975 in northern Illinois. Species diversity indices and evenness values were used to characterize the ponds zooplankton community structure. Thirty-one plankton species are reported. Older borrow-pit ponds contained more species and had higher mean densities than did the newer ponds. The number of dominant species reported per pond varied from 1 to 4. Species diversity indices are higher in older ponds, and there was a general tendency for species diversity to increase with pond age. No correlations between physicochemical measurements and the structure of the zooplankton communities were found. Factors possibly governing zooplankton community structure in borrow-pits are speculated upon.     

Comparison of CE-SSCP and DGGE for monitoring a complex microbial community remediating mine drainage

Capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) is a promising high-throughput tool for profiling complex bioremediation communities, but has not been well characterized with respect to other methods such as denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to compare CE-SSCP with DGGE with respect to: 1) overall representation of the community in terms of the dominant species identified and corresponding Shannon diversity indices; 2) reproducibility and resolution; and 3) artifacts, using a complex sulfate-reducing community remediating mine drainage as a model system. Some of the dominant microorganisms were detected by both methods, but there were also differences in the reported community compositions, and more phylogenetic groups were detected by CE-SSCP. CE-SSCP Shannon diversity indices were slightly higher than those determined from DGGE data, and differed in terms of the time point at which the community was reported to have the highest diversity. Both methods had high reproducibility, but CE-SSCP resolution was higher in terms of the total number of peaks resolved, reduced co-migration of distinct DNA sequences, and length and legibility of the DNA sequencing data of clones used to identify peaks. Ten double bands in the same lane representing the same species were found by DGGE, whereas only one such artifact was observed by CE-SSCP. Finally, less overall sample preparation and analysis time was required for CE-SSCP than for DGGE. The results suggest that CE-SSCP offers several advantages over DGGE, especially for high-throughput monitoring.     

Eukaryotic plankton communities across reef environments in Bocas del Toro Archipelago,Panamá

Variation in light and temperature can influence the genetic diversity and structure of marine plankton communities. While open-ocean plankton communities receive much scientific attention, little is known about how environmental variation affects plankton communities on tropical coral reefs. Here, we characterize eukaryotic plankton communities on coral reefs across the Bocas del Toro Archipelago, Panamá. Temperature loggers were deployed, and midday light levels were measured to quantify environmental differences across reefs at four inshore and four offshore sites (Inshore = Punta Donato, Smithsonian Tropical Research Institute (STRI) Point, Cristobal, Punta Laurel and Offshore = Drago Mar, Bastimentos North, Bastimentos South, and Cayo de Agua). Triplicate vertical plankton tows were collected midday, and high-throughput 18S ribosomal DNA metabarcoding was leveraged to investigate the relationship between eukaryotic plankton community structure and inshore/offshore reef environments. Plankton communities from STRI Point were additionally characterized in the morning (~ 08:00), midday (~ 12:00), and late-day (~ 16:00) to quantify temporal variation within a single site. We found that inshore reefs experienced higher average seawater temperatures, while offshore sites offered higher light levels, presumably associated with reduced water turbidity on reefs further from shore. These significant environmental differences between inshore and offshore reefs corresponded with overall plankton community differences. We also found that temporal variation played a structuring role within these plankton communities, and conclude that time of community sampling is an important consideration for future studies. Follow-up studies focusing on more intensive sampling efforts across space and time, coupled with techniques that can detect more subtle genetic differences between and within communities will more fully capture plankton dynamics in this region and beyond.

     

Estimating Bacterial Diversity for Ecological Studies: Methods,Metrics, and Assumptions

Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques.     

Recruitment of Antarctic marine eukaryotes onto artificial surfaces

Activities related to Antarctic research stations have caused significant local impacts on the marine environment, potentially affecting the recruitment of benthic invertebrates. Herein, we report the community structure of recruiting marine eukaryotes onto artificial substrata using molecular techniques. Slides were deployed at three sites adjacent to McMurdo Station, Scott Base, and Cape Armitage in McMurdo Sound. Denaturing gradient gel electrophoresis (DGGE) analysis revealed complex and diverse eukaryotic communities had established on artificial surfaces deployed at a range of site and depth regimes after 12 months. Analysis of similarity results detected significantly greater variability in community profiles among sites than within sites. The nonmetric multidimensional scaling plot constructed from DGGE banding patterns revealed different benthic communities had established at 12 and 18 m depths. Despite this, the variation in community composition was greater among sites than between depths, especially at Cape Armitage and Scott Base. Sequence analysis of excised DGGE bands revealed a predominance of arthropod and dinoflagellate sequences at Cape Armitage. In contrast, a wide diversity of phyla including cnidaria, bryozoa, protozoa, dinoflagellates, arthropods, platyhelminths, and annelids were present adjacent to the two research stations. The abundance of diatoms detected in Cape Armitage benthic assemblages exceeded the abundance of diatoms from McMurdo Station and Scott Base by almost two orders of magnitude. The discovery that distinct eukaryotic communities recruit at different sites and depths is probably due to complex interactions between multiple factors including water quality, larval supply, and light. The detection of sessile phyla on slides at each of the sites indicates that the pollution profiles present at each site is not an impediment to successful recruitment of these species.     

Effects of operational taxonomic unit inference methods on soil microeukaryote community analysis using long‐read metabarcoding

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large‐scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus''s time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic‐dry part that has a species rich grass‐dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%–40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.     

Distribution patterns of epilithic diatoms along climatic,spatial and physicochemical variables in the Baltic Sea

The species richness and community composition of the diatom communities were studied in the Baltic Sea, Northern Europe, to enhance knowledge about the diversity of these organisms in a brackish water ecosystem. Many organisms in the Baltic Sea have been studied extensively, but studies investigating littoral diatoms are scarce. The goal of this study was to examine the importance of climatic, spatial and water physicochemical variables as drivers of epilithic diatoms in the Gulf of Finland and the Gulf of Bothnia. The variation in species richness was best explained by pH, total phosphorus and total nitrogen. Redundancy Analysis indicated that the most important factors correlating with species composition were air temperature, silicon, total phosphorus, water temperature, salinity and pH. Variation Partitioning showed that the species composition was mostly affected by climatic and spatial variables, whereas physicochemical variables had little impact. However, the strongest factor was the combined influence of climatic, spatial and physicochemical variables. The results suggest that diatom species richness in the northern Baltic Sea is primarily regulated by local factors, while climatic and spatial variables have little impact on richness. Species composition is mostly affected by climatic and spatial variables. We conclude that understanding the distribution patterns of Baltic Sea diatoms requires the inclusion of climatic, spatial and water chemistry variables.