Subsurface Bacterioplankton Structure and Diversity in the Strongly-Stratified Water Columns within the Equatorial Eastern Indian Ocean.

The consequences of climate change may directly or indirectly impact the marine biosphere. Although ocean stratification has been recognized as one of the crucial consequences of ocean warming, its impacts on several critical aspects of marine microbes remain largely unknown in the Indian Ocean. Here, we investigate the effects of water stratification, in both surface and subsurface layers, on hydrogeographic parameters and bacterioplankton diversity within the equatorial eastern Indian Ocean (EIO). Strong stratification in the upper 200 m of equatorial EIO was detected with evidential low primary productivity. The vertical bacterioplankton diversity of the whole water columns displayed noticeable variation, with lower diversity occurring in the surface layer than in the subsurface layers. Horizontal heterogeneity of bacterioplankton communities was also in the well-mixed layer among different stations. SAR11 and Prochlorococcus displayed uncharacteristic low abundance in the surface water. Some amplicon sequence variants (ASVs) were identified as potential biomarkers for their specific depths in strongly-stratified water columns. Thus, barriers resulting from stratification are proposed to function as an 'ASV filter' to regulate the vertical bacterioplankton community diversity along the water columns. Overall, our results suggest that the effects of stratification on the structure and diversity of bacterioplankton can extend up to the bathypelagic zone in the strongly-stratified waters of the equatorial EIO. This study provides the first insight into the effect of stratification on the subsurface microbial communities in the equatorial eastern Indian Ocean.

Niche Partitioning of Labyrinthulomycete Protists Across Sharp Coastal Gradients and Their Putative Relationships With Bacteria and Fungi.

While planktonic microbes play key roles in the coastal oceans, our understanding of heterotrophic microeukaryotes' ecology, particularly their spatiotemporal patterns, drivers, and functions, remains incomplete. In this study, we focus on a ubiquitous marine fungus-like protistan group, the Labyrinthulomycetes, whose biomass can exceed that of bacterioplankton in coastal oceans but whose ecology is largely unknown. Using quantitative PCR and amplicon sequencing of their 18S rRNA genes, we examine their community variation in repeated five-station transects across the nearshore-to-offshore surface waters of North Carolina, United States. Their total 18S rRNA gene abundance and phylotype richness decrease significantly from the resource-rich nearshore to the oligotrophic offshore waters, but their Pielou's community evenness appears to increase offshore. Similar to the bacteria and fungi, the Labyrinthulomycete communities are significantly structured by distance from shore, water temperature, and other environmental factors, suggesting potential niche partitioning. Nevertheless, only several Labyrinthulomycete phylotypes, which belong to aplanochytrids, thraustochytrids, or unclassified Labyrinthulomycetes, are prevalent and correlated with cohesive bacterial communities, while more phylotypes are patchy and often co-occur with fungi. Overall, these results complement previous time-series observations that resolve the Labyrinthulomycetes as persistent and short-blooming ecotypes with distinct seasonal preferences, further revealing their partitioning spatial patterns and multifaceted roles in coastal marine microbial food webs.

Patchy Blooms and Multifarious Ecotypes of Labyrinthulomycetes Protists and Their Implication in Vertical Carbon Export in the Pelagic Eastern Indian Ocean.

Labyrinthulomycetes protists are an important heterotrophic component of microeukaryotes in the world's oceans, but their distribution patterns and ecological roles are poorly understood in pelagic waters. This study employed flow cytometry and high-throughput sequencing to characterize the abundance, diversity, and community structure of Labyrinthulomycetes in the pelagic Eastern Indian Ocean. The total Labyrinthulomycetes abundance varied much more among stations than did the abundance of prokaryotic plankton, reaching over 1,000 cells mL-1 at a few "bloom" stations. The total Labyrinthulomycetes abundance did not decline with depth throughout the whole water column (5 to 2,000 m) like the abundance of prokaryotic plankton did, and the Labyrinthulomycetes average projected biomass over all samples was higher than that of the prokaryotic plankton. However, Labyrinthulomycetes diversity showed obvious vertical variations, with richness, Shannon diversity, and evenness greatest in the upper epipelagic, lower epipelagic, and deep waters, respectively. Many abundant phylotypes were detected across multiple water layers, which aligned with the constant vertical Labyrinthulomycetes biomass, suggesting potential sinking and contribution to the biological pump. Hierarchical clustering revealed distinct ecotypes partitioning by vertical distribution patterns, suggesting their differential roles in the carbon cycle and storage processes. Particularly, most phylotypes showed patchy distributions (occurring in only few samples) as previously found in the coastal waters, but they were less associated with the Labyrinthulomycetes blooms than the prevalent phylotypes. Overall, this study revealed distinct patterns of Labyrinthulomycetes ecotypes and shed light on their importance in the pelagic ocean carbon cycling and sequestration relative to that of the prokaryotic plankton. IMPORTANCE While prokaryotic heterotrophic plankton are well accepted as major players in oceanic carbon cycling, the ecological distributions and functions of their microeukaryotic counterparts in the pelagic ocean remain largely unknown. This study focused on an important group of heterotrophic (mainly osmotrophic) protistan microbes, the Labyrinthulomycetes, whose biomass can surpass that of the prokaryotic plankton in many marine ecosystems, including the bathypelagic ocean. We found patchy horizontal but persistent vertical abundance profiles of the Labyrinthulomycetes protists in the pelagic waters of the Eastern Indian Ocean, which were distinct from the spatial patterns of the prokaryotic plankton. Moreover, multiple Labyrinthulomycetes ecotypes with distinct vertical patterns were detected and, based on the physiologic, metabolic, and genomic understanding of their cultivated relatives, were inferred to play multifaceted key roles in the carbon cycle and sequestration, particularly as contributors to the vertical carbon export from the surface to the dark ocean, i.e., the biological pump.

Vertical community patterns of Labyrinthulomycetes protists reveal their potential importance in the oceanic biological pump.

The biological pump plays a vital role in exporting organic particles into the deep ocean for long-term carbon sequestration. However, much remains unknown about some of its key microbial players. In this study, Labyrinthulomycetes protists (LP) were used to understand the significance of heterotrophic microeukaryotes in the transport of particulate organic matter from the surface to the dark ocean. Unlike the sharp vertical decrease of prokaryotic biomass, the LP biomass only slightly decreased with depth and eventually exceeded prokaryotic biomass in the bathypelagic layer. Sequencing identified high diversity of the LP communities with a dominance of Aplanochytrium at all depths. Notably, ASVs that were observed in the surface layer comprised ~20% of ASVs and ~60% of sequences in each of the deeper (including bathypelagic) layers, suggesting potential vertical export of the LP populations to the deep ocean. Further analyses of the vertical patterns of the 50 most abundant ASVs revealed niche partitioning of LP phylotypes in the pelagic ocean, including those that could decompose organic detritus and/or facilitate the formation of fast-sinking particles. Overall, this study presents several lines of evidence that the LP can be an important component of the biological pump through their multiple ecotypes in the pelagic ocean.

Patchy Distributions and Distinct Niche Partitioning of Mycoplankton Populations across a Nearshore to Open Ocean Gradient.

Evidence increasingly suggests planktonic fungi (or mycoplankton) play an important role in marine food webs and biogeochemical cycles. In order to better understand their ecological role and how oceanographic gradients from the coastal to open ocean shape the mycoplankton community, molecular approaches were used to study fungal dynamics along a repeatedly sampled, five-station transect beginning at the mouth of an estuary and continuing 87 km across the continental shelf to the oligotrophic waters at the boundary of the Sargasso Sea. Similar to patterns in chlorophyll a, fungal 18S rRNA gene abundance showed a sharp decrease from nearshore to offshore stations. While Shannon's diversity was not statistically different across the transect, nonmetric multidimensional scaling (NMDS) ordination revealed that fungal communities at the nearshore station were significantly different from those at other stations. Even though spatial gradients were consistently strong, the shelf mycoplankton were more similar to those of the offshore communities when temperature was high (>20°C) and while they shifted toward the nearshore communities when temperature was low (<19°C), suggesting a role for additional seasonal factors (such as temperature) in shaping mycoplankton distributions. However, overall phylotype distributions were patchy with few taxa observed at all stations and the majority observed at a single station with the nearshore station exhibiting the largest number of exclusive phylotypes. Overall, our findings revealed the patchy spatial distributions and distinct niche partitioning of mycoplankton populations across a nearshore to open ocean gradient, which improved our understanding of fungal ecology in coastal waters. IMPORTANCE Fungi are an important, but understudied, group of heterotrophic microbes in marine environments. Traditionally, fungi in the coastal ocean were largely assumed to be derived from terrestrial inputs. Yet here we find many fungal taxa are endemic to the open ocean environment but are rare or absent in nearshore waters, suggesting they are not washed into the ocean from the land. As observed for the bacterioplankton, coastal oceanographic gradients can function as habitat barriers to partition fungal communities. Compared to the bacterioplankton, however, the mycoplankton exhibit a much patchier distribution pattern, suggesting differential drivers and the potential for spatially/temporally limited habitats or strong density-dependent selection. Therefore, our results show that mycoplankton in the coastal ocean may play a significant but complementary role to that of the bacterioplankton.

Composition change and decreased diversity of microbial eukaryotes in the coastal upwelling waters of South China Sea.

Upwelling plays an important role in marine ecosystems and potentially reshapes microbial communities by enhanced dispersal and distinct environmental drivers. Relative to that of bacterioplankton, however, the response of eukaryotic microbes to upwelling is largely unknown. Here, we investigated the influence of coastal upwelling in South China Sea on the microbial eukaryotic communities. Unlike several folds of increase in the cell abundance of bacterioplankton in upwelling than non-upwelling stations at corresponding water layers, no significant difference was detected for the total microbial eukaryotic 18S rRNA gene abundance. Moreover, the microbial eukaryotes in the upwelling stations exhibited increasing 18S rRNA gene abundance from the surface to the deep, contrasting the vertical cell abundance pattern of the bacterioplankton; but their vertical abundance patterns were similar in non-upwelling stations. Importantly, the coastal upwelling significantly reduced the community evenness of the microbial eukaryotes and slightly reduced their Shannon diversity. Their community composition also varied obviously especially between the surface waters of upwelling and non-upwelling stations. Among the dominant supergroups, Alveolata was found to be less abundant while Stramenopiles, particularly thraustochytrids and diatoms, to be more abundant in the surface water of upwelling than non-upwelling stations. Temperature was identified as the most important factor of the microbial eukaryotic community composition, suggesting potential effects of the cold upwelling water masses on specific taxa. Overall, our results reveal significant and distinct impacts of coastal upwelling on the abundance, diversity, and community structure of microbial eukaryotes, filling the knowledge gap about the microbial responses to this important marine phenomenon.

Annual Partitioning Patterns of Labyrinthulomycetes Protists Reveal Their Multifaceted Role in Marine Microbial Food Webs.

Heterotrophic microbes play a key role in remineralizing organic material in the coastal ocean. While there is a significant body of literature examining heterotrophic bacterioplankton and phytoplankton communities, much less is known about the diversity, dynamics, and ecology of eukaryotic heterotrophs. Here, we focus on the Labyrinthulomycetes, a fungus-like protistan group whose biomass can exceed that of the bacterioplankton in coastal waters. We examined their diversity and community structure in a weekly temperate coastal ocean time series. Their seasonal community patterns were related to temperature, insolation, dissolved inorganic carbon, fungal abundance, ammonia, chlorophyll a, pH, and other environmental variables. Similar to the bacterioplankton, annual community patterns of the Labyrinthulomycetes were dominated by a few persistent taxa with summer or winter preferences. However, like the patterns of fungi at this site, the majority of the Labyrinthulomycetes phylotypes occurred mostly as short, reoccurring, season-specific blooms. Furthermore, some specific phylotypes of Labyrinthulomycetes displayed time-lagged correlations or cooccurrences with bacterial, algal, or fungal phylotypes, suggesting their potentially multifaceted involvement in the marine food webs. Overall, this study reports niche partitioning between closely related Labyrinthulomycetes and identifies distinct ecotypes and temporal patterns compared to bacterioplankton and fungi.IMPORTANCE Increasing evidence has shown that heterotrophic microeukaryotes are an important component in global marine ecosystems, while their diversity and ecological functions remain largely unknown. Without appropriately incorporating these organisms into the food web models, our current understanding of marine microbial community ecology is incomplete, which may further hamper broader studies of biogeochemistry and climate change. This study focuses on a major group of unicellular fungus-like protists (Labyrinthulomycetes) and reveals their distinct annual community patterns relative to fungi and bacteria. Results of our observations provide new information on the community structure and ecology of this protistan group and shed light on the intricate ecological roles of unicellular heterotrophic eukaryotes in the coastal oceans.

A High-Resolution Time Series Reveals Distinct Seasonal Patterns of Planktonic Fungi at a Temperate Coastal Ocean Site (Beaufort, North Carolina, USA).

There is a growing awareness of the ecological and biogeochemical importance of fungi in coastal marine systems. While highly diverse fungi have been discovered in these marine systems, still, little is known about their seasonality and associated drivers in coastal waters. Here, we examined fungal communities over 3 years of weekly sampling at a dynamic, temperate coastal site (Pivers Island Coastal Observatory [PICO], Beaufort, NC, USA). Fungal 18S rRNA gene abundance, operational taxonomic unit (OTU) richness, and Shannon's diversity index values exhibited prominent seasonality. Fungal 18S rRNA gene copies peaked in abundance during the summer and fall, with positive correlations with chlorophyll a, SiO4, and oxygen saturation. Diversity (measured using internal transcribed spacer [ITS] libraries) was highest during winter and lowest during summer; it was linked to temperature, pH, chlorophyll a, insolation, salinity, and dissolved inorganic carbon (DIC). Fungal communities derived from ITS libraries were dominated throughout the year by Ascomycota, with contributions from Basidiomycota, Chytridiomycota, and Mucoromycotina, and their seasonal patterns linked to water temperature, light, and the carbonate system. Network analysis revealed that while cooccurrence and exclusion existed within fungus networks, exclusion dominated the fungus-and-phytoplankton network, in contrast with reported pathogenic and nutritional interactions between marine phytoplankton and fungi. Compared with the seasonality of bacterial communities in the same samples, the timing, extent, and associated environmental variables for fungi community are unique. These results highlight the fungal seasonal dynamics in coastal water and improve our understanding of the ecology of planktonic fungi.IMPORTANCE Coastal fungal dynamics were long assumed to be due to terrestrial inputs; here, a high-resolution time series reveals strong, repeating annual patterns linked to in situ environmental conditions, arguing for a resident coastal fungal community shaped by environmental factors. These seasonal patterns do, however, differ from those observed in the bacterioplankton at the same site; e.g., fungal diversity peaks in winter, whereas bacterial diversity maxima occur in the spring and fall. While the dynamics of these communities are linked to water temperature and insolation, fungi are also influenced by the carbonate system (pH and DIC). As both fungi and heterotrophic bacteria are thought to be key organic-material metabolizers, differences in their environmental drivers may offer clues as to which group dominates secondary production at this dynamic site. Overall, this study suggests the unique ecological roles of mycoplankton and their potentially broad niche complementarities to other microbial groups in the coastal ocean.

High phylogenetic diversity and abundance pattern of Labyrinthulomycete protists in the coastal waters of the Bohai Sea.

The unicellular Labyrinthulomycete protists have long been considered to play a significant role in ocean carbon cycling. However, their distribution and biogeochemical function remain poorly understood. We present a large-scale study of their spatiotemporal abundance and diversity in the coastal waters of Bohai Sea using flow cytometry and high-throughput sequencing. These protists display niche preferences and episodic higher biomass than that of bacterioplankton with much phylogenetic diversity (> 4000 OTUs) ever reported. They were ubiquitous with a typical abundance range of 100-1000 cells ml-1 and biomass range of 0.06-574.59 µg C L-1 . The observed spatiotemporal abundance variations support the current 'left-over scavengers' nutritional model and highlight these protists as a significant component of the marine microbial loop. The higher average abundance and phylogenetic diversity in the nearshore compared with those in the offshore reveal their predominant role in the terrigenous matter decomposition. Furthermore, the differential relationship of the protist genera to environmental conditions together with their co-occurrence network suggests their unique substrate preferences and niche partitioning. With few subnetworks and possible keystone species, their network topology indicates community resilience and high connectance level of few operational taxonomic units (OTUs). We demonstrate the significant contribution of these protists to the secondary production and nutrient cycling in the coastal waters. As secondary producers, their role will become more important with increasingly coastal eutrophication.

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

Marine fungus-like eukaryotic unicellular protists (thraustochytrids) are considered to play an important role in the marine microbial food web. However, their abundance, distribution, and relative biomass in coastal waters have not yet been examined in detail. By using a flow cytometry method (FCM) for the rapid enumeration of thraustochytrids in nearshore and offshore stations along the Gulf of Bohai, China, we herein expanded current knowledge on their ecological significance. The FCM method allows for the rapid detection and quantification of prokaryotic and eukaryotic cells, but is rarely applied to the enumeration of small eukaryotic protists. Epifluorescence microscopy (EpiM) has been commonly used for the direct detection and enumeration of thraustochytrids; however, this method is time-consuming and inapplicable to a large-scale analysis of complex seawater samples. There is no available FCM method to track the abundance and biomass of thraustochytrids in marine habitats. The FCM enumeration of thraustochytrids in seawater samples ranged between 400 and 4,080 cells mL-1 with a biomass range of 8.15-83.96 µg C L-1. The thraustochytrid biomass contributed 10.9% to 98.1% of the total biomass of the heterotrophic microbial community comprising bacterioplankton and thraustochytrids. Their overall abundance in nearshore stations was significantly different from that in offshore stations (P<0.5). The present results provide an optimized method for the rapid detection and enumeration of thraustochytrids in seawater and facilitate large-scale studies of the ecological role of thraustochytrids in the microbial food web of coastal waters.

Spatiotemporal Distribution and Assemblages of Planktonic Fungi in the Coastal Waters of the Bohai Sea.

Fungi play a critical role in the nutrient cycling and ecological function in terrestrial and freshwater ecosystems. Yet, many ecological aspects of their counterparts in coastal ecosystems remain largely elusive. Using high-throughput sequencing, quantitative PCR, and environmental data analyses, we studied the spatiotemporal changes in the abundance and diversity of planktonic fungi and their abiotic and biotic interactions in the coastal waters of three transects along the Bohai Sea. A total of 4362 ITS OTUs were identified and more than 60% of which were unclassified Fungi. Of the classified OTUs three major fungal phyla, Ascomycota, Basidiomycota, and Chytridiomycota were predominant with episodic low dominance phyla Cryptomycota and Mucoromycota (Mortierellales). The estimated average Fungi-specific 18S rRNA gene qPCR abundances varied within 4.28 × 106 and 1.13 × 107copies/L with significantly (P < 0.05) different abundances among the transects suggesting potential influence of the different riverine inputs. The spatiotemporal changes in the OTU abundance of Ascomycota and Basidiomycota phyla coincided significantly (P < 0.05) with nutrients traced to riverine inputs and phytoplankton detritus. Among the eight major fungal orders, the abundance of Hypocreales varied significantly (P < 0.01) across months while Capnodiales, Pleosporales, Eurotiales, and Sporidiobolales varied significantly (P < 0.05) across transects. In addition, our results likely suggest a tripartite interaction model for the association within members of Cryptomycota (hyperparasites), Chytridiomycota (both parasites and saprotrophs), and phytoplankton in the coastal waters. The fungal network featured several hubs and keystone OTUs besides the display of cooperative and competitive relationship within OTUs. These results support the notion that planktonic fungi, hitherto mostly undescribed, play diverse ecological roles in marine habitats and further outline niche processes, tripartite and co-occurrence interaction as the major drivers of their community structure and spatiotemporal distribution in the coastal water column.

Distinct Seasonal Patterns of Bacterioplankton Abundance and Dominance of Phyla α-Proteobacteria and Cyanobacteria in Qinhuangdao Coastal Waters Off the Bohai Sea.

Qinhuangdao coastal waters in northern China are heavily impacted by anthropogenic and natural activities, and we anticipate a direct influence of the impact on the bacterioplankton abundance and diversity inhabiting the adjacent coastal areas. To ascertain the anthropogenic influences, we first evaluated the seasonal abundance patterns and diversity of bacterioplankton in the coastal areas with varied levels of natural and anthropogenic activities and then analyzed the environmental factors which influenced the abundance patterns. Results indicated distinct patterns in bacterioplankton abundance across the warm and cold seasons in all stations. Total bacterial abundance in the stations ranged from 8.67 × 104 to 2.08 × 106 cells/mL and had significant (p < 0.01) positive correlation with total phosphorus (TP), which indicated TP as the key monitoring parameter for anthropogenic impact on nutrients cycling. Proteobacteria and Cyanobacteria were the most abundant phyla in the Qinhuangdao coastal waters. Redundancy analysis revealed significant (p < 0.01) influence of temperature, dissolved oxygen and chlorophyll a on the spatiotemporal abundance pattern of α-Proteobacteria and Cyanobacteria groups. Among the 19 identified bacterioplankton subgroups, α-Proteobacteria (phylum Proteobacteria) was the dominant one followed by Family II (phylum Cyanobacteria), representing 19.1-55.2% and 2.3-54.2% of total sequences, respectively. An inverse relationship (r = -0.82) was observed between the two dominant subgroups, α-Proteobacteria and Family II. A wide range of inverse Simpson index (10.2 to 105) revealed spatial heterogeneity of bacterioplankton diversity likely resulting from the varied anthropogenic and natural influences. Overall, our results suggested that seasonal variations impose substantial influence on shaping bacterioplankton abundance patterns. In addition, the predominance of only a few cosmopolitan species in the Qinhuangdao coastal wasters was probably an indication of their competitive advantage over other bacterioplankton groups in the degradation of anthropogenic inputs. The results provided an evidence of their ecological significance in coastal waters impacted by seasonal inputs of the natural and anthropogenic matter. In conclusion, the findings anticipate future development of effective indicators of coastal health monitoring and subsequent management strategies to control the anthropogenic inputs in the Qinhuangdao coastal waters.

Seasonal influence of scallop culture on nutrient flux, bacterial pathogens and bacterioplankton diversity across estuaries off the Bohai Sea Coast of Northern China.

In this study, we investigated the environmental impacts of scallop culture on two coastal estuaries adjacent the Bohai Sea including developing a quantitative PCR assay to assess the abundance of the bacterial pathogens Escherichia coli and Vibrio parahaemolyticus. Scallop culture resulted in a significant reduction of nitrogen, Chlorophyll a, and phosphorous levels in seawater during summer. The abundance of bacteria including V. parahaemolyticus varied significantly across estuaries and breeding seasons and was influenced by nitrate as well as nutrient ratios (Si/DIN, N/P). Bacterioplankton diversity varied across the two estuaries and seasons, and was dominated by Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes. Overall, this study suggests a significant influence of scallop culture on the ecology of adjacent estuaries and offers a sensitive tool for monitoring scallop contamination.

Abundance and molecular diversity of thraustochytrids in coastal waters of southern China.

Thraustochytrids are unicellular fungi-like (heterotrophic) marine protists that have long been considered to play an important role in the biogeochemical cycles of the coastal oceans. However, the significance of their ecological functions and their diversity in marine ecosystems remain largely unknown. In this report, we examined the spatial and temporal variations of planktonic thraustochytrids, their relationship with other environmental factors, and their diversity in the subtropical coastal waters of China. The abundance of planktonic thraustochytrids ranged from 2.56 × 105 to 17.57 × 105 cells L-1 with highest abundance detected in polluted coastal water in the Spring (March) season. The thraustochytrid biomass was greater than the bacterial biomass in most seawater samples, ranging from 32.29 to 359.51% that of bacterioplankton. The abundance of thraustochytrids appeared to be largely related to that of bacterioplankton and to chemical oxygen demand in water columns. High-throughput sequencing analyses revealed a total of 105 OTUs (97% similarity), which were members of genera Thraustochytrium, Aplanochytrium, Oblongichytrium, Ulkenia, Labyrinthula and undescribed novel phylotypes. Results of this study indicated unprecedented high diversity of labyrinthulomycetes as well as the presence of novel labyrinthulomycete and thraustochytrid lineages, and also provided new information on the significant role of thraustochytrids in microbial food webs in a coastal marine ecosystem.