Protist Community Composition Research Articles

Structural variability of protist assemblages in surface sediments across Italian Mediterranean marine subregions

Marine sediments host heterogeneous protist communities consisting of both living benthic microorganisms and planktonic resting stages. Despite their key functions in marine ecosystem processes and biogeochemical cycles, their structure and dynamics are largely unknown. In the present study, with a spatially intensive sampling design we investigated benthic protist diversity and function of surface sediment samples from three subregions of the Mediterranean Sea, through an environmental DNA metabarcoding approach targeting the 18S V4 region of rRNA gene. Protists were characterized at the taxonomic level and trophic function, both in terms of alpha diversity and community composition, testing for potential differences among marine subregions and bathymetric groups. Overall, Alveolata and Stramenopiles were the two divisions that dominated the communities. These dominant groups exhibited significant differences among the three Mediterranean subregions in the alpha diversity estimates based on the detected ASVs, for all computed indices (ASV richness, Shannon and Simpson indices). Protist communities were also found to be significantly different in terms of composition at the order rank in the three subregions p-value < 0.01). These differences were mainly driven by Anoecales, Peridiniales, Borokales, Paraliales and Gonyaulacales, which together contributed almost 80% of the average dissimilarity. Anoecales was the dominant order in the Ionian – Central Mediterranean and Adriatic Sea, but with considerably different relative abundances (52% and 36%, respectively), while Borokales was the dominant order in the Western Mediterranean Sea (33%). Similarly, significant differences among the three marine subregions were also highlighted when protist assemblages were examined in terms of trophic function, both in terms of alpha diversity (calculated on the ASVs for each trophic group) and community composition p-value < 0.01. In particular, the Adriatic Sea stood out for having the highest relative abundance of autotrophic/mixotrophic components in the surface sediments analyzed. Conversely, no significant differences in protist assemblages were found among depth groups. This study provided new insights into the taxonomic and trophic composition of benthic protist communities found in Mediterranean surface sediments, revealing geographical differences among regional seas. The results were discussed in relation to the Mediterranean environmental features that could generate the differences among benthic protist communities.

Seasonal changes in the protist communities of Hakodate Bay, southern Hokkaido, from 2020 to 2022

Hakodate Bay at the southern end of Hokkaido experiences physical and chemical environmental changes in the adjacent Tsugaru Strait. Harmful algal blooms have occurred since 2015. However, information on the response of protist community composition, photosynthetic activity to environmental variables or the mechanisms causing harmful blooms in the Hakodate Bay is lacking. We investigated the seasonal changes in the protist community using weekly sampling to reveal the relationship between the protist community and environmental variables in the Hakodate Bay. Based on cluster analysis, six groups were identified: Winter, Spring, Late Spring, Summer, Benthic, and Karenia. High Fv/Fm was seen in the Summer, Benthic and Spring groups supported by high nutrients, warm temperature and sufficient irradiance. From the Winter to Spring group, a species transition from low-temperature-adapting Thalassiosira spp. to Chaetoceros spp. was found. The Summer group comprised high salinity-tolerant species (the diatom Leptocylindrus danicus and dinoflagellate Prorocentrum spp.) because of high fluctuations in salinity due to precipitation. The other groups (Late Spring, Benthic, and Karenia) randomly filled the spaces between the three groups. The dinoflagellate Scrippsiella spp. was dominant in the Late Spring group because of accumulation by wind-driven currents. Because a strong relationship between benthic species and wind speed was exhibited, the Benthic group comprised benthic diatoms released into the water by mixing. The Karenia group was observed only from late August 2021 to early January 2022, when higher ammonium and lower light conditions (i.e., cloudy conditions) were detected. Owing to environmental conditions, diatom growth was restricted by low light intensity, whereas that of Karenia mikimotoi increased by taking advantage of the abundant nutrients. Seasonal changes in the protist community of the Hakodate Bay can generally be explained by temperature; however, in the short term (1–2 weeks), changes were also influenced by meteorological conditions (solar radiation, wind, and precipitation).

Comparing the Environmental Influences and Community Assembly of Protist Communities in Two Anthropogenic Coastal Areas.

Anthropogenic stresses are intensively affecting the structure and function of microbial communities in coastal ecosystems. Despite being essential components of coastal ecosystems, the environmental influences and assembly processes of protist communities remain largely unknown in areas with severe disturbance. Here, we used 18S rRNA gene high-throughput sequencing to compare the composition, assembly process, and functional structure of the protist communities from the coastal areas of the Northern Yellow Sea (NYS) and the Eastern Bohai Sea (EBS). These two areas are separated by the Liaodong Peninsula and experience different anthropogenic stresses due to varying degrees of urbanization. We detected significant differences between the protist communities of the two areas. Environmental and geographic factors both influenced the composition of protist communities, with environmental factors playing a greater role. The neutral community model indicated that the assembly of protist communities was governed by deterministic processes, with stochastic processes having a stronger influence in the EBS area compared to the NYS area. The phototrophic and consumer communities, influenced by different environmental factors, differed significantly between the two areas. Our results provide insights into the biogeography and assembly of protist communities in estuaries under anthropogenic stresses, which may inform future coastal management.

Seasonality in phytoplankton communities and production in three Arctic fjords across a climate gradient

Phytoplankton communities and production in Arctic fjords undergo strong seasonal variations. Phytoplankton blooms are periods with high primary production, leading to elevated algal biomass fueling higher trophic levels. Blooms are typically driven bottom-up by light and nutrient availability but may also be top-down controlled by grazing. While phytoplankton spring blooms are common across all Arctic systems, summer and autumn blooms and their drivers are less predictable. Here we compare the long-term (≥4 years) bloom phenology and protist community composition in three Arctic fjords: Nuup Kangerlua in western Greenland, Ramfjorden in northern Norway, and Adventfjorden in western Svalbard. While Nuup Kangerlua is impacted by tidewater glaciers, Ramfjorden and Adventfjorden are impacted by river-runoff. We discuss and contrast the presence and predictability of spring, summer, and autumn blooms in these fjords and the main physical, chemical, and biological drivers. Spring blooms occurred in all three fjords in April/May as soon as sufficient sunlight was available and typically terminated when nutrients were depleted. Chain-forming diatoms together with the haptophyte Phaeocystis pouchetii were key spring bloom taxa in all three fjords. Summer blooms were found in Nuup Kangerlua and Ramfjorden but were not common in Adventfjorden. In Nuup Kangerlua nutrient supply via subglacial upwelling was the key driver of a diatom-dominated summer bloom. This summer bloom extended far into autumn with strong winds resupplying nutrients to the surface later in the season. In Ramfjorden runoff from a vegetated catchment provided organic nutrients for a flagellate-dominated summer bloom in 2019. A late autumn bloom dominated by Skeletonema spp. and other chain-forming diatoms was present after nutrients were resupplied by wind mixing. In Adventfjorden, we observed only minor summer blooms in 2 of the 8 years, while autumn blooms were never observed. With global warming, we suggest that summer blooms will be negatively impacted in fjords where tidewater glaciers retreat and become land terminating. In fjords with rich vegetated catchments, harmful algal blooms may occur more frequently as summers and autumns become warmer and wetter. However, for fjords in high-Arctic latitudes (>78 N), the day length will continue to restrict the potential for autumn blooms.

Earthworms alleviate microplastics stress on soil microbial and protist communities

Microplastic (MP) pollution can exert significant pressure on soil ecosystems, however, the interactive effects of MPs on soil bacterial, fungal and protist communities remains poorly understood. Soil macrofauna, such as earthworms, can be directly affected by MPs, potentially leading to a range of feedbacks on the soil microbial community. To address this, we conducted a microcosm experiment to examine the effects of conventional (i.e., polyethylene, polystyrene) and biodegradable MPs (i.e. PBAT, polylactic acid) on the structure of the soil bacterial, fungal, and protist communities in the presence or absence of earthworms. We found that MP contamination negatively affected the diversity and composition of soil microbial and protist communities, with smaller-sized conventional MPs having the most pronounced effects. For example, compared with the unamended control, small-sized polyethylene MPs both significantly reduced the Shannon diversity of soil bacteria, fungi, and protist by 4.3 %, 37.0 %, and 9.1 %, respectively. Biodegradable MPs increased negative correlations among bacteria, fungi, and protists. However, earthworms mitigated these effects, enhancing the diversity and altering the composition of these communities. They also increased the niche width and stability of the soil microbial food web network. Our study indicated that earthworms help attenuate the response of soil microorganisms to MPs stress by influencing the diversity and composition of soil microorganisms and soil physicochemical properties and underscores the importance of considering macrofauna in MPs research.

Organic fertilisation enhances network complexity among bacteria, fungi, and protists by improving organic matter and phosphorus in acidic agricultural soils

Research has explored the impact of organic fertilisation on improving agroecosystem productivity and resilience, highlighting the significant contributions of protists in addition to bacteria and fungi. However, the interactions among bacteria, fungi and protists in organically fertilised soils remain largely unknown. In this study, soil samples were collected from four long-term fertilisation treatments: no fertilisation (Control), inorganic fertilisation (NPK), organic fertilisation (OM), and combined inorganic and organic fertilisation (NPKOM). The abundance and composition of bacteria, fungi, and protist communities, as well as co-occurrence networks, were analysed under different fertilisation treatments. Our results showed that the total abundance of bacteria, fungi, and protists increased by a minimum of 2.95, 3.47, and 0.66 times, respectively, after organic fertiliser application. Moreover, the application of organic fertilisers significantly altered the structures of soil microbial communities by enriching bacterial Proteobacteria and Actinobacteria, fungal Ascomycota, and protist Conosa. Changes in the total abundance of bacteria, fungi, and protists, and their community structures in soils with organic fertilisers were associated with increases in soil organic carbon and phosphorous. Additionally, microbial networks exhibited greater complexity in organically fertilised soils than in non-organically fertilised soils by possessing higher linkage density. The increased complexity may be attributed to potential interkingdom associations among bacteria, fungi, and protists in high soil organic carbon and phosphorus. These results highlight that the application of organic fertilisers has the potential to enhance the complexity of microbial coexistence in acidic agricultural soils.

The impact of fungi on soil protist communities in European cereal croplands.

Protists, a crucial part of the soil food web, are increasingly acknowledged as significant influencers of nutrient cycling and plant performance in farmlands. While topographical and climatic factors are often considered to drive microbial communities on a continental scale, higher trophic levels like heterotrophic protists also rely on their food sources. In this context, bacterivores have received more attention than fungivores. Our study explored the connection between the community composition of protists (specifically Rhizaria and Cercozoa) and fungi across 156 cereal fields in Europe, spanning a latitudinal gradient of 3000 km. We employed a machine-learning approach to measure the significance of fungal communities in comparison to bacterial communities, soil abiotic factors, and climate as determinants of the Cercozoa community composition. Our findings indicate that climatic variables and fungal communities are the primary drivers of cercozoan communities, accounting for 70% of their community composition. Structural equation modelling (SEM) unveiled indirect climatic effects on the cercozoan communities through a change in the composition of the fungal communities. Our data also imply that fungivory might be more prevalent among protists than generally believed. This study uncovers a hidden facet of the soil food web, suggesting that the benefits of microbial diversity could be more effectively integrated into sustainable agriculture practices.

Transition from a mixotrophic/heterotrophic protist community during the dark winter to a photoautotrophic spring community in surface waters of Disko Bay, Greenland.

Unicellular eukaryotic plankton communities (protists) are the major basis of the marine food web. The spring bloom is especially important, because of its high biomass. However, it is poorly described how the protist community composition in Arctic surface waters develops from winter to spring. We show that mixotrophic and parasitic organisms are prominent in the dark winter period. The transition period toward the spring bloom event was characterized by a high relative abundance of mixotrophic dinoflagellates, while centric diatoms and the haptophyte Phaeocystis pouchetii dominated the successive phototrophic spring bloom event during the study. The data shows a continuous community shift from winter to spring, and not just a dormant spring community waiting for the right environmental conditions. The spring bloom initiation commenced while sea ice was still scattering and absorbing the sunlight, inhibiting its penetration into the water column. The initial increase in fluorescence was detected relatively deep in the water column at ~55 m depth at the halocline, at which the photosynthetic cells accumulated, while a thick layer of snow and sea ice was still obstructing sunlight penetration of the surface water. This suggests that water column stratification and a complex interplay of abiotic factors eventually promote the spring bloom initiation.

Transmission dynamics of symbiotic protist communities in the termite gut: association with host adult eclosion and dispersal.

The fidelity of vertical transmission is a critical factor in maintaining mutualistic associations with microorganisms. The obligate mutualism between termites and intestinal protist communities has been maintained for over 130 million years, suggesting the faithful transmission of diverse protist species across host generations. Although a severe bottleneck can occur when alates disperse with gut protists, how protist communities are maintained during this process remains largely unknown. In this study, we examined the dynamics of intestinal protist communities during adult eclosion and alate dispersal in the termite Reticulitermes speratus. We found that the protist community structure in last-instar nymphs differed significantly from that in workers and persisted intact during adult eclosion, whereas all protists disappeared from the gut during moults between worker stages. The number of protists in nymphs and alates was substantially lower than in workers, whereas the proportion of protist species exhibiting low abundance in workers was higher in nymphs and alates. Using a simulation-based approach, we demonstrate that such changes in the protist community composition of nymphs and alates improve the transmission efficiency of whole protist species communities. This study thus provides novel insights into how termites have maintained mutualistic relationships with diverse gut microbiota for generations.

Pioneer plants enhance soil multifunctionality by reshaping underground multitrophic community during natural succession of an abandoned rare earth mine tailing

Spontaneous natural succession in metal mine tailings is fundamental to the rehabilitation of bare tailing. Here, an abandoned rare earth element (REE) mine tailing with spontaneous colonisation by pioneer plants with different functional traits was selected. Soil nutrient cycling, fertility, organic matter decomposition as well as underground organismal communities and their multitrophic networks were investigated. Compared with the bare tailing, the colonisation with Lycopodium japonicum, Miscanthus sinensis, and Dicranopteris dichotoma increased soil multifunction by 222%, 293%, and 525%, respectively. This was accompanied by significant changes in soil bacterial and protistan community composition and increased soil multitrophic network complexity. Rhizospheres of different plant species showed distinct microbial community composition compared to that of bare tailing. Some WPS-2, Chloroflexi, and Chlorophyta were mainly present in the bare tailing, while some Proteobacteria and Cercozoa were predominantly seen in the rhizosphere. Pearson correlation and Random Forest revealed the biotic factors driving soil multifunction. Structural equation modelling further revealed that pioneer plants improved soil multifunction primarily by decreasing the microbial biodiversity and increasing the multitrophic network complexity. Overall, this highlights the importance of subterrestrial organisms in accelerating soil rehabilitation during natural succession and provides options for the ecological restoration of degraded REE mining areas.

Distinct response patterns of soil micro-eukaryotic communities to early-season and late-season precipitation in a semiarid grassland

Semiarid ecosystems are susceptible to changes in precipitation regimes. However, how soil micro-eukaryote communities in semi-arid ecosystems respond to variations in the seasonal distribution of precipitation remains elusive. A 5-year field experiment was performed to investigate the effect of changed precipitation distributions throughout the growing season on soil micro-eukaryotic communities in the temperate steppe. Early-season precipitation did not significantly affect the biomass of arbuscular mycorrhizal fungi (AMF) nor the composition and structure of AMF and protist communities. Late-season precipitation significantly increased the biomass of AMF while significantly altering the relative abundance of dominant genera in AMF and protist communities. There was a clear difference in the community structure of AMF and protists between the late-season precipitation and natural precipitation. In addition, late-season precipitation decreased the amount of consumer protists but enhanced the abundance of parasitic protists. These results indicate that the impact of precipitation limitations in the middle growing season can be weakened by increased precipitation in the early growing seasons, while an increase in precipitation later in the growing season does not improve the impact of a decrease in precipitation in the middle growing season. Our findings highlight the potential impact of intra-annual changes in precipitation in soil micro-eukaryotic communities in arid and semiarid regions.

The influence of soil factors on protist community dynamics during plant succession in subalpine natural and planted forests

Protists, as the core component of the soil microbiome, may play an important role in forest succession by altering soil nutrient cycling and plant performance. However, knowledge of how protists change with plant succession is extremely limited. To illustrate protistan community dynamics following plant succession in subalpine natural and planted ecosystems, we used high-throughput sequencing to identify protists in rhizosphere and bulk soils at different successional stages, and the effects of soil abiotic and biotic factors on protists were analysed. Overall, the α-diversity and community composition of protists differed significantly between natural and planted ecosystems, but not between rhizosphere and bulk soils. Moving along the successional gradient, the richness and diversity of protists in rhizosphere and bulk soils of natural ecosystems, as well as the richness of functional taxa (i.e., consumers, parasites and phototrophs), gradually increased. In contrast, the richness of protists and functional taxa in the plantations was generally lowest in the old stands (75 years). The community composition of protists in rhizosphere and bulk soils of these two ecosystems varied significantly among successional stages, and their community composition variation was positively correlated with the time interval of successional stages, indicating that community dynamics exhibited directionality along successional gradients. Soil abiotic and biotic factors jointly regulate protistan α-diversity and community composition, and forest succession regulates functional taxa through multiple pathways. However, the key influencing factors and specific regulatory pathways are not consistent across different ecosystems, soil types and functional taxa. Therefore, our study has revealed that protistan community assembly was distinct along two separate successional gradients, indicating potential functional differences. These results provide a basis for the development of future forest management measures.

Nitrogen-based fertilizers differentially affect protist community composition in paddy field soils

Nitrogen-based fertilizers differentially affect protist community composition in paddy field soils

Protist symbionts of termites: diversity, distribution, and coevolution.

The symbiosis between termites and their hindgut protists is mutually obligate and vertically inherited. It was established by the late Jurassic in the cockroach ancestors of termites as they transitioned to wood feeding. Since then, protist symbionts have been transmitted from host generation to host generation by proctodeal trophallaxis (anal feeding). The protists belong to multiple lineages within the eukaryotic superphylum Metamonada. Most of these lineages have evolved large cells with complex morphology, unlike the non-termite-associated Metamonada. The species richness and taxonomic composition of symbiotic protist communities varies widely across termite lineages, especially within the deep-branching clade Teletisoptera. In general, closely related termites tend to harbour closely related protists, and deep-branching termites tend to harbour deep-branching protists, reflecting their broad-scale co-diversification. A closer view, however, reveals a complex distribution of protist lineages across hosts. Some protist taxa are common, some are rare, some are widespread, and some are restricted to a single host family or genus. Some protist taxa can be found in only a few, distantly related, host species. Thus, the long history of co-diversification in this symbiosis has been complicated by lineage-specific loss of symbionts, transfer of symbionts from one host lineage to another, and by independent diversification of the symbionts relative to their hosts. This review aims to introduce the biology of this important symbiosis and serve as a gateway to the diversity and systematics literature for both termites and protists. A searchable database with all termite-protist occurrence records and taxonomic references is provided as a supplementary file to encourage and facilitate new research in this field.

Protistan community composition and metabolism in the North Pacific Subtropical Gyre: Influences of mesoscale eddies and depth.

Marine protists and their metabolic activities are intricately tied to the cycling of nutrients and the flow of energy through microbial food webs. Physiochemical changes in the environment, such as those that result from mesoscale eddies, may impact protistan communities, but the effects that such changes have on protists are poorly known. A metatranscriptomic study was conducted to investigate how eddies affected protists at adjacent cyclonic and anticyclonic eddy sites in the oligotrophic ocean at four depths from 25 to 250 m. Eddy polarity impacted protists at all depths sampled, although the effects of eddy polarity were secondary to the impact of depth across the depth range. Eddy-induced vertical shifts in the water column yielded differences in the cyclonic and anticyclonic eddy protistan communities, and these differences were the most pronounced at and just below the deep chlorophyll maximum. An analysis of transcripts associated with protistan nutritional physiology at 150 m revealed that cyclonic eddies may support a more heterotrophic community, while anticyclonic eddies promote a more phototrophic community. The results of this study indicate that eddies alter the metabolism of protists particularly in the lower euphotic zone and may therefore impact carbon export from the euphotic zone.

Urban greenspaces shape soil protist communities in a location-specific manner

The impacts of urbanization on aboveground biodiversity are well studied, and its impact on soil microorganisms are also receiving increased attention. However, the impact of urbanization on the soil protists are hardly investigated. Here, we studied how urbanization and distinct urban greenspaces affect protist communities. We used amplicon sequencing of the18 S rRNA gene of samples from five types of urban greenspaces (parks, greenbelts, industrial areas, residential areas and hospital lawns), neighboring natural forests and agricultural ecosystems in Ningbo, China. We found that urban greenspaces harbored higher protist α-diversity than forests, while protist β-diversity increased from agricultural systems to urban greenspaces to forests. Among the studied driving factors, soil bacterial α- and β-diversity best predicted phagotrophic protist α- and β-diversity in urban greenspaces, while differences in α- and β-diversity of phototrophic protists were best explained by soil carbon-to-nitrogen ratio and fungal β-diversity, respectively. Abiotic factors i.e., total phosphorus and carbon-to-nitrogen ratio, best predicted the α- and β-diversity of protist parasites in urban greenspaces, respectively. The results revealed that the composition and drivers of protist communities vary between functional groups and urban ecosystems. Overall, our findings contribute to a better understanding of drivers of soil protist communities and indicate that soil protist communities and associated soil functions could be managed in predictable ways in urban greenspaces.

Mesoscale oceanographic meanders influence protist community function and structure in the southern Indian Ocean.

The interface between the nutrient-rich Southern Ocean and oligotrophic Indian Ocean creates unique environmental conditions that can strongly influence biological processes. We investigated protist communities across a mesoscale meander of the Subtropical Front within the Southern Indian Ocean. 18S V9 rDNA metabarcoding suggests a diverse protist community in which the dinoflagellates and parasitic Syndiniales were abundant. Diversity was highest in frontal waters of the mesoscale meander, with differences in community structure inside and outside the meander. While the overall community was dominated by mixotrophic taxa, the frontal boundary of the meander had increased abundances of heterotrophic taxa, with potential implications for net atmospheric CO2 drawdown. Pulse amplitude modulated (PAM) fluorimetry revealed significant differences in the photophysiology of phytoplankton communities inside and outside the meander. By using single-cell PAM microscopy, we identified physiological differences between dinoflagellate and coccolithophore taxa, which may have contributed to changes in photophysiology observed at community level. Overall, our results demonstrate that frontal areas have a strong impact on the composition of protist communities in the Southern Ocean with important implications for understanding biological processes in this region.

Seasonality regulates the taxonomic and functional compositions of protists responding to climate warming in forest ecosystems

AbstractIntroductionProtists are unicellular eukaryotes including important predators, parasites and phototrophs, and play pivotal roles in organic matter decomposition, biogeochemical nutrient cycling and various ecosystem functions. Unravelling the impact of climate warming on soil protists is paramount in predicting how these microorganisms will continue to provide essential ecosystem services in the face of changing climatic conditions.Materials and MethodsWe examined the effects of 5‐year simulated climate warming scenario, with temperatures increased by 4°C above ambient levels, on the diversity and community composition of soil protists, as well as their interactions with other microorganisms in both natural and plantation forest ecosystems during three seasons: summer, autumn and winter.ResultsWe found a season‐dependent response of protists to climate warming, with a significant decrease in protist diversity during winter in natural forests. Furthermore, we identified significant alterations in the community compositions of protists during summer in both natural and plantation forests, as well as during winter in both forest types, under warming. Our analysis pinpointed specific functional protist taxa, such as consumers, parasites and phototrophs, which exhibited significant shifts in their relative abundances under warming. Additionally, we found that warming facilitated trophic interactions between protists and bacteria, while also strengthening interactions between bacterial and fungal communities. Warming could induce direct modifications in protist community compositions or indirectly affect them by modifying bacterial and fungal communities, as revealed by structural equation modelling.ConclusionThese findings demonstrate the substantial impact of warming on the taxonomic and functional compositions of protists in forest ecosystems, with the magnitude of these effects varying across seasons. Our study implicates that ongoing climate warming could have significant consequences for the profiles of soil protists, as well as their trophic interactions with bacteria and fungi, highlighting the importance of considering these effects for the sustainable provision of ecosystem functions.

Response of protists to nitrogen addition, arbuscular mycorrhizal fungi manipulation, and mesofauna reduction in a tropical montane rainforest in southern Ecuador.

The tropical Andes are a species-rich and nitrogen-limited system, susceptible to increased nitrogen (N) inputs from the atmosphere. However, our understanding of the impacts of increased N input on belowground systems, in particular on protists and their role in nutrient cycling, remains limited. We explored how increased N affects protists in tropical montane rainforests in Ecuador using high-throughput sequencing (HTS) of environmental DNA from two litter layers. In addition, we manipulated the amount of arbuscular mycorrhizal fungi (AMF) and mesofauna, both playing a significant role in N cycling and interacting in complex ways with protist communities. We found that N strongly affected protist community composition in both layers, while mesofauna reduction had a stronger effect on the lower layer. Changes in concentration of the AMF marker lipid had little effect on protists. In both layers, the addition of N increased phagotrophs and animal parasites and decreased plant parasites, while mixotrophs decreased in the upper layer but increased in the lower layer. In the upper layer with higher AMF concentration, mixotrophs decreased, while in the lower layer, photoautotrophs increased and plant parasites decreased. With reduced mesofauna, phagotrophs increased and animal parasites decreased in both layers, while plant parasites increased only in the upper layer. The findings indicate that to understand the intricate response of protist communities to environmental changes, it is critical to thoroughly analyze these communities across litter and soil layers, and to include HTS.

Phosphorus Fertilization Boosts Mineral-Associated Soil Organic Carbon Formation Associated with Phagotrophic Protists.

Long-term fertilization affects soil organic C accumulation. A growing body of research has revealed critical roles of bacteria in soil organic C accumulation, particularly through mineral-associated organic C (MAOC) formation. Protists are essential components of soil microbiome, but the relationships between MAOC formation and protists under long-term fertilization remain unclear. Here, we used cropland soil from a long-term fertilization field trial and conducted two microcosm experiments with 13C-glucose addition to investigate the effects of N and P fertilizations on MAOC formation and the relationships with protists. The results showed that long-term fertilization (especially P fertilization) significantly (P < 0.05) increased 13C-MAOC content. Compared with P-deficient treatment, P replenishment enriched the number of protists (mainly Amoebozoa and Cercozoa) and bacteria (mainly Acidobacteriota, Bacteroidota, and Gammaproteobacteria), and significantly (P < 0.001) promoted the abundances of bacterial functional genes controlling C, N, P, and S metabolisms. The community composition of phagotrophic protists prominently (P < 0.001) correlated with the bacterial community composition, bacterial functional gene abundance, and 13C-MAOC content. Co-occurrence networks of phagotrophic protists and bacteria were more connected in soil with the N inoculum added than in soil with the NP inoculum added. P replenishment strengthened bacterial 13C assimilation (i.e., 13C-phospholipid fatty acid content), which negatively (P < 0.05) correlated with the number and relative abundance of phagotrophic Cercozoa. Together, these results suggested that P fertilization boosts MAOC formation associated with phagotrophic protists. Our study paves the way for future research to harness the potential of protists to promote belowground C accrual in agroecosystems.