Investigating the diversity, community structure, and ecological processes that mediate community assembly of intertidal microeukaryotes can help us to better understand these poorly characterized ecosystems and facilitate the forecasting of possible trends and consequences of community succession. In the present study, HTS on rRNA gene transcript was used to investigate the diversity, community structure, and shaping factors of both planktonic and benthic microeukaryotes collected simultaneously from the intertidal zone of southeast Fujian province, China.
The relative importance of environmental and spatial factors on community variations was quantified using a phylogenetic null model. For planktonic assemblages, pilot studies have shown distinct differentiation among size-fractioned microeukaryotes in marine environments Logares et al. For benthic assemblages of microeukaryote communities in coastal regions, only minor or no seasonal variations were found Gong et al.
It is still unknown whether intertidal planktonic and benthic microeukaryotes follow the same pattern as those in coastal water environments. Therefore, the first question explored here is whether the diversity and community composition of intertidal microeukaryotes are related to organism size and seasonal variation. Secondly, we investigate whether planktonic and benthic microeukaryotes occupying distinct ecological niches and playing different ecological functions are likely to have distinct responses to environmental and spatial variations.
We hypothesize that intertidal microeukaryotes may be structured by different ecological processes between habitats i. All 13 sampling sites located in intertidal zones of southeast Fujian, China, are influenced by semidiurnal tidal cycles Figure 1. The intertidal zones in the study partially located in the tourism city, Xiamen, which is undergoing rapid urbanization and industrialization. Blooms of Akashiwo sanguinea dinoflagellate frequently occur in spring February—May along the shores Yang et al.
In summer, anthropogenic activity is intense due to large numbers of visiting tourists. Therefore, environmental conditions in the intertidal zone in summer are likely to differ significantly from those in spring, for example, possibly resulting in seasonal difference in the benthos. Altogether, 21 samples for the plankton 10 samples for micro-sized fraction and 11 samples for nano-sized fraction and 15 for the benthos 8 samples for spring and 7 samples for summer were collected Supplementary Table S1.
Sediment more than g from the top 1 cm was sampled in both spring February, 26th—March, 5th; Qiong Lin on April, 27th and summer August, 23th—25th of the same year Supplementary Table S1. Figure 1. Sampling locations in intertidal zones of southeast Fujian, China. Planktonic and benthic samples were collected from 13 sampling sites marked by black circles. For the water samples, 2.
To maximize the number of sampling sites visited during ebb tide and transport the samples to the laboratory as soon as possible, parameters such as concentrations of nutrients and chlorophyll a and abundance of bacteria, i. Samples were transported to the laboratory using ice bags to keep them at low temperature and were processed within hours.
Our preliminary test on intertidal samples showed that it was challenging to get amplicons using the extracted RNA from RNAlater fixed sediments. Salinity and pH were measured in the same way as for water samples. In brief, around 0. The digested sediment samples were then centrifuged at 3, g for 15 min; the supernatant liquid was submitted for metal analysis using ICP-MS Tan et al. Metal concentrations in seawater were measured by the diffusive gradients in thin films technique DGT.
The V4 region is the hypervariable region of the 18S rRNA gene, which has been targeted for estimating the environmental diversity of microbial eukaryotes using HTS technologies Stoeck et al. Previous studies have shown that the V4 region can yield similar values of diversity and community composition as would have been estimated using the full-length 18S rRNA gene Dunthorn et al.
To enable comparison between samples, the OTU table was rarefied to the same sequencing depth of 13, reads per sample by randomly resampling, which corresponds to the minimum number of sequences obtained in the samples.
All statistical analyses were conducted with R software version 3. Principal component analysis PCA was used to reveal the distribution of environmental parameters. Mantel and Partial Mantel tests were performed to reveal the potential factors correlating with community variations. To identify OTUs contributing the most to differences between groups under each condition, i.
A phylogenetic null model was applied using an approach based on phylogenetic and taxonomic diversities in order to quantify microeukaryotic community assembly processes, such as selection, dispersal limitation, ecological drift, and homogenizing dispersal Stegen et al. To quantify ecological processes mediating the community assembly of microeukaryotes, two major steps were processed.
Second, Bray-Curtis-based Raup-Crick RC bray for pairwise community comparisons were evaluated by characterizing the magnitude of deviation between observed OTU composition turnover and null distribution of OTU composition turnover Stegen et al. The distribution of environmental parameters was revealed by principal component analysis PCA Supplementary Figure S1.
In general, the dynamics of environmental factors from sedimentary samples were more complex than those of water samples. Planktonic 21 samples and benthic 15 samples datasets yielded 1,, and , clean amplicons in total, respectively. The alpha-diversity of intertidal microeukaryotes differed significantly between habitats, with benthic microeukaryotes harboring significantly higher diversity on average OTUs than their planktonic counterparts on average OTUs.
This distribution pattern was consistent among diversity indices phylogenetic diversity, For the comparison between plankton and benthos, only samples from the spring season are considered.
However, no statistical difference was found between organism sizes and seasons Figures 2A — C. Figure 2. A—C boxplots show Richness, PD, and Shannon values among different comparisons, with a vertical dotted line dividing into three parts, which are based on different grouping standards. Ns means that p is not significant. Dashed lines denote the separation of each condition, i.
Planktonic and benthic microeukaryotes showed an inconsistent pattern in correlations with environmental parameters Supplementary Tables S2 , S3.
Alpha diversity of planktonic microeukaryotes failed to correlate with any environmental parameter measured, whereas that of benthic microeukaryotes showed a significant correlation with concentrations of Cd, Cu, and NO x , and grain sizes Supplementary Tables S2 , S3. In planktonic microeukaryotes, the alpha-diversity of microplankton negatively correlated with concentrations of Ni and Mn r values in a range of 0. In benthic microeukaryotes, the alpha-diversity of spring communities showed correlations with salinity, concentration of Pb, and grain size, whereas that of summer communities significantly correlated with pH, concentrations of Cd, Cu, Zn, phosphate, water content and grain size Supplementary Table S3.
The alpha-diversity of intertidal microeukaryotes exhibited a distinct correlation pattern with environmental factors between habitats water and sediment , organism body sizes nano- and microplankton and seasons spring and summer. All sequences were assigned to 28 lineages within seven recognized supergroups, namely Alveolata, Amoebozoa, Archaeplastida, Excavata, Opisthokonta, Rhizaria, and Stramenopiles Figure 3.
Stramenopiles dominated the total microeukaryote community in terms of numbers both of sequences and OTUs average proportion of total sequence abundance and OTU richness about Figure 3. Heatmap showing the distribution and composition of intertidal microeukaryotes. Top row shows types of sample marked with different colors. Microplankton and Nanoplankton refer to size-fractionated water samples collected in spring; Spring and Summer represent sediment sampled in spring and summer, respectively; Plankton includes Microplankton and Nanoplankton, while Benthos includes samples collected in both Spring and Summer.
All represents all samples. Figure 4. The relative abundance of any given group is the proportion of the sequences of that group to the total number of sequences in the sample, while the relative OTUs number richness is the proportion of OTUs assigned to any given group to the total OTUs number of OTUs in the sample.
For planktonic communities, the stramenopiles group Bacillariophyta average abundance about Size-fraction subcommunities of planktonic microeukaryotes i. For OTU richness, the major contributors of planktonic and benthic communities were Bacillariophyta Similarly, subcommunities of microplankton and nanoplankton and of benthic community in spring and summer showed a similar trend Figures 4D , F. Statistical analysis showed that 27 OTUs contributed the most to differences between plankton and benthos as determined by edgeR Supplementary Figure S3A.
About 12 of the 27 OTUs enriched in sediment were assigned to Bacillariophyta e. The most 12 differential OTUs between benthos sampled in spring and summer were all assigned to Bacillariophyta, with 5 of the OTUs enriched in the samples collected in spring Supplementary Figure S3C. Planktonic and benthic microeukaryote communities clustered into two groups, and each exhibited distinct patterns, with benthic communities clustering into spring and summer subgroups and planktonic communities grouping into nano- and micro-size subgroups Figure 5A.
Figure 5. Distribution pattern and assembly mechanism of microeukaryotes in intertidal zones. Quantification of ecological processes shaping microeukaryotes in intertidal zones B. Microplankton and Nanoplankton represent size-fractionated water samples, while Spring and Summer represent sediment sampled in spring and summer, respectively; Plankton includes Microplankton and Nanoplankton, while Benthos includes samples collected in both Spring and Summer.
Table 1. Among the 22 measured environmental factors, the concentration of Cu and geographic distance were found to be important influencing factors on the planktonic community, whereas the concentration of Cd, the water content of sediment, and geographic distance significantly correlated with community variation of benthic microeukaryotes Figure 6.
Figure 6. Heatmaps showing the results of simple and partial Mantel tests for the correlations between environmental factors, spatial variables, and intertidal benthic A and planktonic B microeukaryotes. Benthos represent all 15 benthic samples. Spring 8 samples and Summer 7 samples represent sediment sampled in spring and summer, respectively. Plankton represents all 21 planktonic samples collected in spring. Microplankton 10 samples and Nanoplankton 11 samples refer to size-fractionated water samples collected in spring.
Community variations were based on Bray-Curtis similarity. The phylogenetic null model analysis showed that dispersal limitation prevailed in both planktonic and benthic communities under each condition, i. In planktonic microeukaryotes, dispersal limitation had a larger effect on nanoplankton In benthic microeukaryotes, dispersal limitation In the present study, no replicates for each sampling site were collected; therefore, it is impossible to evaluate the variation within each location.
The following discussion does not take into account intra-site variations. Our results showed that sediment harbored a higher diversity of microbial eukaryotes than water in intertidal zones Figures 2A — C. A similar pattern was observed in coastal regions of Europe Massana et al. This has been attributed to physicochemical microhabitat heterogeneity, niche partitioning, and allopatric speciation Forster et al. Therefore, the most plausible reason for the alpha distribution pattern seen in intertidal zones was the higher microhabitat heterogeneity of intertidal sediments compared to the overlying water Supplementary Figure S2.
This heterogeneity is probably induced by a combination of daily tides, waves, and anthropogenic activities, which create a wider range of ecological niches compared to water, which is a relatively homogeneous environment because it is well mixed and subject to long-distance transportation by tides and winds. By employing high throughput sequencing on the rRNA gene, previous studies on microeukaryotes showed that alpha diversity was about one order of magnitude higher than that in the present study in the same region Chen et al.
We speculate that the difference might be due to any or all of the following reasons. First, data generated from different sources might contribute to the apparent difference in alpha diversity.
Environmental RNA, used in the present study, is thought to exist in living, ribosomally active cells only Blazewicz et al. Secondly, the target region chosen as a marker gene to explore alpha diversity might explain part of the variance. Thirdly, there were inconsistencies in sampling strategies. In the present study, total RNA was extracted from sediments directly without any pre-processing in order to minimize the loss of microorganisms.
Therefore, to make results from different investigations comparable, standard procedures for sampling, sample processing and data analyses should be established. Interestingly, this study showed a much higher percentage of shared OTUs between planktonic and benthic microeukaryotes For example, Chen et al. In a coastal region of Europe, Forster et al. Compared with coastal regions, however, intertidal zones are much more dynamic because of strong perturbation by cyclical fluctuations resulting in stronger mixing between water and sediment.
This mixing might enhance the exchange between planktonic and benthic microeukaryote communities, producing a high percentage of shared OTUs by the two groups in intertidal zones. Also, sediment serving as a sink could accumulate microeukaryotic cells from the water above, which could result in more shared OTUs between plankton and benthos. Finally, methodological differences might contribute to the discrepancies among these studies.
Correlation analyses showed that none of the environmental factors measured was correlated with alpha diversity of planktonic microeukaryotes, whereas benthic microeukaryotes were negatively correlated with concentrations of Cd, Cu, NO x , and grain size Supplementary Tables S2 , S3.
Previous studies have shown that there are no consistent effects of heavy metals on prokaryote or microeukaryote diversity. For example, Gong et al. The diversity of bacteria in sediments in an estuary of southeast Australia and in a coastal region of Italy was found to be negatively correlated with heavy metals Sun et al. The distribution of marine organisms depends on the chemical and physical properties of seawater temperature, salinity, and dissolved nutrients , on ocean currents which carry oxygen to subsurface waters and disperse nutrients, wastes, spores, eggs, larvae, and plankton , and on penetration of light.
Very abundant phytoplankton include the diatoms and dinoflagellates see Dinoflagellata. Heterotrophic plankton zooplankton include such protozoans as the foraminiferans ; they are found at all depths but are more numerous near the surface. Bacteria are abundant in upper waters and in bottom deposits. The scientific study of marine biology dates from the early 19th cent.
Research has been furthered by unmanned and manned craft, such as the submersible Alvin. See R. Meroplankton are temporary members, spending only a part of their life cycle in the plankton.
They include larvae of anemones, barnacles, crabs and even fish, which later in life will join the nekton or the benthos. Meroplankton are very much a feature of the sea, particularly coastal waters, as the often sedentary adult forms of coastal species use their planktonic stage for dispersal.
Nekton are organisms swimming actively in the water, it includes a variety of animals, mostly fish. Benthos comprises organisms on the bed of the water body. Animals attached to or living on the bottom are referred to as epifauna, while those which burrow into soft sediments or live in spaces between sediment particles are described as infauna. Attached multicellular plants and algae are referred to as macrophytes, while single-celled or filamentous algae are called as periphyton or microphytobenthos.
Epiphytic algae are those which grow on macrophytes. Neuston are those organisms associated with the water surface, where they are supported by surface tension.
Most neuston require very still water surface and is therefore very restricted in the sea. Fringing communities are floral communities that occur where the water is shallow enough for plentiful light to reach the bottom, allowing the growth of attached photosynthesisers, which may be entirely submerged or emergent into the air.
Marine communities are composed mostly out of algal seaweeds. Wetlands are composed of this type of vegetation. This is a comprehensive pan-European system to facilitate the harmonized description and collection of data across Europe through the use of criteria for habitat identification; it covers all types of habitats from natural to artificial, from terrestrial to freshwater and marine. A simplified description of a food web: the phytoplankton are the primary producers and are eaten by the zooplankton smallest floating animals.
The zooplankton are eaten by small fish sardines, herring and small fish are eaten by larger fish. At the top of the marine food web are the large predators tuna, seals, sea-birds and some species of whales. Phytoplankton, small zooplankton and large zooplankton, larger animals and top predators all interact in a marine food web. Each species eats and is eaten by several other species at different trophic levels. The interactions in a food web are far more complex than the interactions in a food chain.
Furthermore, the branching structure of food webs leads to fewer top predators compared with the numbers of top predators in a food chain.
In the microbial loop, bacteria consume Dissolved Organic Material DOM that cannot be directly ingested by larger organisms.
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