Protists regulate microbially mediated organic carbon turnover in soil aggregates.

Soil protists, the major predator of bacteria and fungi, shape the taxonomic and functional structure of soil microbiome via trophic regulation. However, how trophic interactions between protists and their prey influence microbially mediated soil organic carbon turnover remains largely unknown. Here, we investigated the protistan communities and microbial trophic interactions across different aggregates-size fractions in agricultural soil with long-term fertilization regimes. Our results showed that aggregate sizes significantly influenced the protistan community and microbial hierarchical interactions. Bacterivores were the predominant protistan functional group and were more abundant in macroaggregates and silt + clay than in microaggregates, while omnivores showed an opposite distribution pattern. Furthermore, partial least square path modeling revealed positive impacts of omnivores on the C-decomposition genes and soil organic matter (SOM) contents, while bacterivores displayed negative impacts. Microbial trophic interactions were intensive in macroaggregates and silt + clay but were restricted in microaggregates, as indicated by the intensity of protistan-bacterial associations and network complexity and connectivity. Cercozoan taxa were consistently identified as the keystone species in SOM degradation-related ecological clusters in macroaggregates and silt + clay, indicating the critical roles of protists in SOM degradation by regulating bacterial and fungal taxa. Chemical fertilization had a positive effect on soil C sequestration through suppressing SOM degradation-related ecological clusters in macroaggregate and silt + clay. Conversely, the associations between the trophic interactions and SOM contents were decoupled in microaggregates, suggesting limited microbial contributions to SOM turnovers. Our study demonstrates the importance of protists-driven trophic interactions on soil C cycling in agricultural ecosystems.

Multi-scale beta-diversity patterns in testate amoeba communities: species turnover and nestedness along a latitudinal gradient.

The relationship between species diversity and spatial scale is a central topic in spatial community ecology. Latitudinal gradient is among the core mechanisms driving biodiversity distribution on most scales. Patterns of ß-diversity along latitudinal gradient have been well studied for aboveground terrestrial and marine communities, whereas soil organisms remain poorly investigated in this regard. The West Siberian Plain is a good model to address diversity scale-dependence since the latitudinal gradient does not overlap with other possible factors such as elevational or maritime. Here, we collected 111 samples following hierarchical sampling (sub-zones, ecosystem types, microhabitat and replicate samples) and performed multi-scale partitioning of ß-diversity of testate amoeba assemblages as a model of study. We found that among-ecosystem ß-diversity is a leading scale in testate amoeba assemblages variation. Rare species determine ß-diversity at all scale levels especially in the northern regions, where rare taxa almost exclusively accounted for the diversity at the ecosystem level. ß-Diversity is generally dominated by the turnover component at all scales in lower latitudes, whereas nestedness prevailed at among-ecosystem scale in higher latitudes. These findings indicate that microbial assemblages in northern latitudes are spatially homogeneous and constrained by historical drivers at larger scales, whereas in southern regions, it is dominated by the turnover component both at the microhabitat and ecosystem scales and therefore determined by recent vegetation and environmental heterogeneity. Overall, we have provided the evidence for the existence of negative latitudinal gradient for among-ecosystem ß-diversity but not for among-microhabitat and among-sample ß-diversity for terrestrial testate amoeba communities.

Soil Testate Amoebae and Diatoms as Bioindicators of an Old Heavy Metal Contaminated Floodplain in Japan.

Soil protists are rarely included in ecotoxicological investigations, despite their fundamental role in ecological processes. Moreover, testate amoebae and diatoms contribute considerably to silicon fluxes in soils. We investigated the effects of heavy metals on testate amoebae (species and individual densities) and diatoms (individual densities) in aged soils of a floodplain (Watarase retarding basin, Japan) taking soil samples from two unpolluted reference sites and two polluted sites. The total concentrations of Cu, Pb, and Zn in soil were higher at the polluted sites as compared with the reference sites. The available concentrations of Co, Cu, and Zn in CaCl2 extracts were higher at the polluted sites but available Pb was not detectable. Testate amoeba taxonomic richness was higher in the reference sites (45/38 taxa) than in the polluted sites (36/27 taxa). The reference sites had higher diatom and amoeba densities than the polluted sites. There was a significant negative correlation between total testate amoeba density and heavy metal concentration (available Co), while significant negative correlations were found between diatom density and Co, Cu, and Zn (available and total concentration). Densities of Cyclopyxis kahli cyclostoma, Centropyxis spp., and Trinema complanatum were negatively correlated to concentrations of available heavy metals. The observed decrease in individual numbers due to heavy metal pollution resulted in a considerable decline in protozoic (testate amoebae) and protophytic (pennate diatoms) silicon pools. Our data suggest that heavy metal pollution affects biogeochemical cycling in this system.

Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa.

Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into common biases associated with this method are limited to aquatic taxa and samples. We created a mock community of common free-living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and at investigating whether the expected protist community structure is altered by the co-amplification of metazoan-associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly over-represented, while those of most amoebae and flagellates were under-represented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free-living protist communities. Furthermore, we detected a wide range of nonadded protist taxa probably introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co-extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free-living soil protist communities.

Diversity of Cellular Slime Molds (Dictyostelids) in the Fanjing Mountain Nature Reserve and Geographical Distribution Comparisons with Other Representative Nature Reserves in Different Climate Zones of China.

Protected areas are widely considered an essential strategy for biodiversity conservation. Dictyostelids are unique protists known to have important ecological functions in promoting soil and plant health through their top-down regulation of ecosystem processes, such as decomposition, that involve bacterial populations. But the relationship between dictyostelid diversity within protected areas remains poorly understood, especially on a large scale. Herein, we report data on the distribution of dictyostelids, identified with ITS + SSU rRNA molecular and morphology-based taxonomy, from soil samples collected in the Fanjing Mountain protected area of Guizhou Province, Southwest China. We compared the biodiversity data of dictyostelids in Fanjing Mountain with similar data from previously sampled sites in four other protected areas, including Changbai Mountain (CB), Gushan Mountain (GS), Baiyun Mountain (BY), and Qinghai-Tibet Plateau (QT) in China. We identified four species of dictyostelids belonging to three genera (Dictyostelium, Heterostelium, and Polysphondylium) and herein provide information on the taxonomy of these species. Two species (Heterostelium pallidum and Dictyostelium purpureum) are common and widely distributed throughout the world, but one species (Polysphondylium fuscans) was new to China. Our data indicate that there is no distinguishable significant correlation between the dictyostelid species studied and environmental factors. Overall, the similarity index between Baiyun Mountain in Henan Province and Fanjing Mountain in Guizhou Province, located at approximately the same longitude, is the highest, and the Jaccard similarity coefficients (Jaccard index) of family, genus, and species are 100%, 100%, and 12.5%, respectively. From a species perspective, species in the same climate zone are not closely related, but obvious geographical distributions are evident in different climate zones. This preliminary study provided evidence of the ecological adaptation of dictyostelids to different biological niches.

Trophic relationships between protists and bacteria and fungi drive the biogeography of rhizosphere soil microbial community and impact plant physiological and ecological functions.

Rhizosphere microorganisms play a vital role in enhancing plant health, productivity, and the accumulation of secondary metabolites. Currently, there is a limited understanding of the ecological processes that control the assembly of community. To address the role of microbial interactions in assembly and for functioning of the rhizosphere soil microbiota, we collected rhizosphere soil samples from Anisodus tanguticus on the Tibetan Plateau spanning 1500 kilometers, and sequenced the bacteria, fungi, archaea, and protist communities. We observed a significant but weak distance-decay relationship in the microbial communities of rhizosphere soil. Our comprehensive analysis of spatial, abiotic, and biotic factors showed that trophic relationships between protists and bacteria and fungi predominantly influenced the alpha and beta diversity of bacterial, fungal, and protistan communities, while abiotic factors had a greater impact on archaeal communities, including soil pH, available phosphorus, total phosphorus and mean annual temperature. Importantly, microbial interactions had a more significant influence on Anisodus tanguticus physiological and ecological functions compared to individual microorganisms. Network analyses revealed that bacteria occupy a central position of the co-occurrence network and play a crucial role of connector within this community. The addition of protists increased the stability of bacterial, fungal, and archaeal networks. Overall, our findings indicate that trophic relationships play an important role in assembly and for functioning of the rhizosphere soil microbiota. Bacterial communities serve as a crucial link between different kingdoms of microorganisms in the rhizosphere community. These findings help us to fully harness the beneficial functions of rhizosphere microorganisms for plants and achieve sustainable use of biological resources.

Environmental factors influencing the diversity and distribution of dictyostelid cellular slime molds in forest and farmland soils of western China.

IMPORTANCE: Soil protists are an essential yet seriously understudied component of the soil microbiome. In this study, 11 new records of dictyostelids belonging to 2 orders, 3 families, and 4 genera were identified from 99 soil samples collected from different elevations and habitats in central Gansu and the southeastern and southcentral portions of Guizhou Province, China. We found that dictyostelid communities were significantly different between Gansu and Guizhou Provinces, apparently in response to different environmental factors. Moreover, dictyostelids were found to have the highest species diversity in mixed forests. Soil pH, temperature, and elevation were determined to be the primary factors that affect the distribution and occurrence of dictyostelids in Guizhou and Gansu Provinces. This work supplements the survey data available for dictyostelids elsewhere in China. These new findings have significant implications for our understanding of the diversity of soil microorganisms.

An elevation transect study of testate amoeba communities up to 4000 m a.s.l. on Mount Kinabalu, Borneo.

Testate amoebae are a polyphyletic assemblage of unicellular eukaryotes with a specific shell. They occur frequently in soil and freshwater habitats all over the world, important for ecosystem functioning and useful for bio-indication. Terrestrial amoeba data from Borneo or Malaysia are rarely available. In this study, data on species composition and relative abundance were presented along a transect from 1730 m up to 4000 m above sea level from 10 elevational levels on Mt. Kinabalu, Malaysian Borneo. In total 78 morphotypes of testate amoebae were recorded. Most were small (under 100 µm) and supposedly cosmopolitan. However, Certesella certesi with limited geographical distribution and some rare taxa (e.g., Padaungiella lageniformis cordiformis, Placocista jurassica, Trinema chardezi) and two undescribed morphospecies were also observed. Presence/absence and relative abundance data of testate amoebae were associated to elevation with a pronounced change at the tree line, as revealed by non-metric multidimensional scaling ordination analysis. Our data from Southeast Asia contribute to the actual discussion on distributional patterns considering elevation gradients and biogeography of eukaryotic microbes.

Winogradsky columns as a strategy to study typically rare microbial eukaryotes.

Winogradsky columns have been widely used to study soil microbial communities, but the vast majority of those investigations have focused on the ecology and diversity of bacteria. In contrast, microbial eukaryotes (ME) have been regularly overlooked in studies based on experimental soil columns. Despite the recognized ecological relevance of ME in soil communities, investigations focused on ME diversity and the abundance of certain groups of interest are still scarce. In the present study, we used DNA metabarcoding (high-throughput sequencing of the V4 region of the 18S rRNA locus) to survey the ME diversity and abundance in an experimental Winogradsky soil column. Consistent with previous surveys in natural soils, our survey identified members of Cercozoa (Rhizaria; 31.2%), Apicomplexa and Ciliophora (Alveolata; 12.5%) as the predominant ME groups, but at particular depths we also detected the abundant presence of ME lineages that are typically rare in natural environments, such as members of the Vampyrellida (Rhizaria) and Breviatea (Amorphea). Our survey demonstrates that experimental soil columns are an efficient enrichment-culture approach that can enhance investigations about the diversity and ecology of ME in soils.

Contrasting Responses of Protistan Plant Parasites and Phagotrophs to Ecosystems, Land Management and Soil Properties.

Functional traits are increasingly used in ecology to link the structure of microbial communities to ecosystem processes. We investigated two important protistan lineages, Cercozoa and Endomyxa (Rhizaria) in soil using Illumina sequencing and analyzed their diversity and functional traits along with their responses to environmental factors in grassland and forest across Germany. From 600 soil samples, we obtained 2,101 Operational Taxonomic Units representing ∼18 million Illumina reads (region V4, 18S rRNA gene). All major taxonomic and functional groups were present, dominated by small bacterivorous flagellates (Glissomonadida). Endomyxan plant parasites were absent from forests. In grassland, Cercozoa and Endomyxa were promoted by more intensive land use management. Grassland and forest strikingly differed in community composition. Relative abundances of bacterivores and eukaryvores were inversely influenced by environmental factors. These patterns provide new insights into the functional organization of soil biota and indications for a more sustainable land-use management.

Protist communities are more sensitive to nitrogen fertilization than other microorganisms in diverse agricultural soils.

BACKGROUND: Agricultural food production is at the base of food and fodder, with fertilization having fundamentally and continuously increased crop yield over the last decades. The performance of crops is intimately tied to their microbiome as they together form holobionts. The importance of the microbiome for plant performance is, however, notoriously ignored in agricultural systems as fertilization disconnects the dependency of plants for often plant-beneficial microbial processes. Moreover, we lack a holistic understanding of how fertilization regimes affect the soil microbiome. Here, we examined the effect of a 2-year fertilization regime (no nitrogen fertilization control, nitrogen fertilization, and nitrogen fertilization plus straw amendment) on entire soil microbiomes (bacteria, fungi, and protist) in three common agricultural soil types cropped with maize in two seasons. RESULTS: We found that the application of nitrogen fertilizers more strongly affected protist than bacterial and fungal communities. Nitrogen fertilization indirectly reduced protist diversity through changing abiotic properties and bacterial and fungal communities which differed between soil types and sampling seasons. Nitrogen fertilizer plus straw amendment had greater effects on soil physicochemical properties and microbiome diversity than nitrogen addition alone. Moreover, nitrogen fertilization, even more together with straw, increased soil microbiome network complexity, suggesting that the application of nitrogen fertilizers tightened soil microbiomes interactions. CONCLUSIONS: Together, our results suggest that protists are the most susceptible microbiome component to the application of nitrogen fertilizers. As protist communities also exhibit the strongest seasonal dynamics, they serve as the most sensitive bioindicators of soil changes. Changes in protist communities might have long-term effects if some of the key protist hubs that govern microbiome complexities as top microbiome predators are altered. This study serves as the stepping stone to promote protists as promising agents in targeted microbiome engineering to help in reducing the dependency on exogenous unsustainably high fertilization and pesticide applications.

Functional Traits and Spatio-Temporal Structure of a Major Group of Soil Protists (Rhizaria: Cercozoa) in a Temperate Grassland.

Soil protists are increasingly appreciated as essential components of soil foodwebs; however, there is a dearth of information on the factors structuring their communities. Here we investigate the importance of different biotic and abiotic factors as key drivers of spatial and seasonal distribution of protistan communities. We conducted an intensive survey of a 10 m2 grassland plot in Germany, focusing on a major group of protists, the Cercozoa. From 177 soil samples, collected from April to November, we obtained 694 Operational Taxonomy Units representing >6 million Illumina reads. All major cercozoan taxonomic and functional groups were present, dominated by the small flagellates of the Glissomonadida. We found evidence of environmental selection structuring the cercozoan communities both spatially and seasonally. Spatial analyses indicated that communities were correlated within a range of 3.5 m. Seasonal variations in the abundance of bacterivores and bacteria, followed by that of omnivores suggested a dynamic prey-predator succession. The most influential edaphic properties were moisture and clay content, which differentially affected each functional group. Our study is based on an intense sampling of protists at a small scale, thus providing a detailed description of the biodiversity of different taxa/functional groups and the ecological processes involved in shaping their distribution.

Erratum: Functional Traits and Spatio-Temporal Structure of a Major Group of Soil Protists (Rhizaria: Cercozoa) in a Temperate Grassland.

[This corrects the article DOI: 10.3389/fmicb.2019.01332.].

Toxicity of cadmium and zinc to small soil protists.

Small heterotrophic protists (flagellates and naked amoebae) are very abundant in soil and play a key role in maintaining soil services. Hence, knowledge on how xenobiotics affect these organisms is essential in ecosystem management. Cadmium (Cd) is an increasing environmental issue as both industrial deposition and recycling of heavy metal rich waste products have led to Cd enrichment of soils. Evaluation of toxicity of Cd to micro-organisms is often performed using a solution of pure Cd (e.g. CdCl) in liquid culture. This approach may be highly misleading as interactions between Cd and other substances, e.g. various ions or inherent soil components often strongly modify Cd toxicity. Hence, we compared the toxic effect of Cd to small heterotrophic protists in soil microcosms and liquid culture. We also evaluated how zinc (Zn) affects Cd toxicity, as Zn usually accompanies Cd in a ratio of c. 100:1, and is known to impede Cd toxicity. In the soil microcosms, we also monitored the primary food source of the protists, i.e. culturable bacteria, and used soil respiration as a proxy of soil functioning. Finally, we examined to what extent Cd actually sorbs to soil. We found 1) that c. 103 times more Cd was required to obtain the same effect in the soil microcosms compared to the liquid culture, 2) that soil sorption explains why Cd, even though highly toxic in aqueous solutions, has very limited effect when applied to soil, and 3) (very surprisingly) that in our experimental systems Zn was as toxic as Cd. Our study suggests that Cd toxicity to soil protists will be small because most Cd in soil will be sorbed to the soil matrix and because the Zn:Cd ratio of 100:1 in most substances, incl. pollutants, will mean that lethal Zn effects will occur before Cd reaches toxic levels.

Molecular Identification of Soil Eukaryotes and Focused Approaches Targeting Protist and Faunal Groups Using High-Throughput Metabarcoding.

While until recently the application of high-throughput sequencing approaches has mostly been restricted to bacteria and fungi, these methods have now also become available to less often studied (eukaryotic) groups, such as fauna and protists. Such approaches allow routine diversity screening for large numbers of samples via DNA metabarcoding. Given the enormous taxonomic diversity within the eukaryote tree of life, metabarcoding approaches targeting a single specific DNA region do not allow to discriminate members of all eukaryote clades at high taxonomic resolution. Here, we report on protocols that enable studying the diversity of soil eukaryotes and, at high taxonomic resolution, of individual faunal and protist groups therein using a tiered approach: first, the use of a general eukaryotic primer set targeting a wide range of eukaryotes provides a rough impression on the entire diversity of protists and faunal groups. Second, more focused approaches enable deciphering subsets of soil eukaryotes in higher taxonomic detail. We provide primers and protocols for two examples: soil microarthropods and cercozoan protists.

Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea.

Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.