Human land use promotes range expansion of soil protists from temperate to subtropical regions in China

Significance

Land-use changes are altering the distribution pattern of a broad range of species. However, the impacts of land use change on soil microbiota distribution and their consequences for large-scale biodiversity patterns are poorly understood. We present evidence that human land-use practices facilitate the range expansion of generalist taxa of soil protists from temperate to subtropical regions. This cross-regional species flow contributes to biological homogenization at a continental scale. Our findings indicate that land-use changes create potential hotspots of microbial invasions, particularly in subtropical and tropical regions, highlighting that understudied regions are likely to be strongly affected by biological homogenization related to introduction of exotic species.

Abstract

Land-use changes are reshaping the distribution of aboveground species worldwide. However, the impact of land-use changes on the distribution of soil organisms remains poorly understood. In particular, we lack a mechanistic understanding of the environmental factors reshaping the distribution of soil microbiota in response to global biological homogenization. Here, we used metabarcoding to investigate the biogeography of protists and their relationships with prey and hosts in three human-dominated ecosystem types, i.e., farmlands, residential areas, and parks, along with natural forests, in subtropical and temperate climatic regions across China. We found that human land-use systems extended the distribution range of habitat-generalist protists compared to forests. This human-facilitated spread of protists was highly directional and mainly driven by temperate to subtropical range expansion of soil taxa. Put simply, increases in soil pH associated with human land uses mitigate the natural acidity barrier typically found in subtropical ecosystems, facilitating the temperate to subtropical range expansion of protist species. However, in temperate regions, the northward expansion of subtropical species is likely restricted by a more arid climate with even higher soil pH. The cross-region spread of soil protists was more pronounced in phagotrophs than phototrophs and parasites, reflecting codispersal of phagotroph protists and microbial prey (especially bacteria) related to tight predator–prey specialization and/or similar responses to environmental changes. Our findings indicate that land-use changes create hotspots of potential microbial invasions, particularly in subtropical and tropical regions, highlighting that understudied regions are likely to be strongly affected by biological homogenization related to introduction of exotic species.

More than 70% of the global land surface has been transformed to human land-use systems, posing a major threat to biodiversity and its functioning (1, 2). Land-use changes often lead to vegetation simplification, soil erosion, and pollution as well as increased aridity and temperature due to more open canopies compared to natural forest systems (2, 3). These changes not only contribute to local species loss (4) but also promote the spread of species into regions where they previously did not occur (5, 6), with potentially dramatic effects on recipient communities and their functioning (7, 8). Although many studies have addressed the impact of land use on range shifts of plants, birds, insects, and pathogenic microorganisms (9–12), the effects of land-use changes on the spatial distribution of soil-dwelling species remain unclear.

The hyperdiverse soil communities, estimated to account for nearly 60% of species on earth (13), determine ecological processes including decomposition, nutrient cycling, C sequestration, and aboveground community dynamics (14, 15). Protists, i.e., predominantly unicellular eukaryotes, are among the most diverse and abundant members of soil communities (16, 17). Yet, they stay relatively understudied compared with other microorganisms such as fungi and bacteria. Most species of protists are phagotrophs, regulating microbial communities and their functions via preying on bacteria, fungi, other protists (18), and even microfauna such as nematodes (19, 20). Other major protist groups, including phototrophs and parasites of animals and plants, are involved in phototrophic carbon input to soil and regulating animal and plant communities, respectively. A recent global-scale biodiversity survey highlighted the vulnerability of soil protist diversity in urban greenspaces due to the loss of community dissimilarity between sites (i.e., beta diversity) resulting in biological homogenization (21). This loss of community heterogeneity suggests that human land use may facilitate a large-scale range expansion of certain soil-dwelling species, potentially through breaking down ecological barriers (i.e., environmental filtering) and/or enhancing dispersal processes.

Human land uses have the potential to remove ecological barriers and thereby facilitate the introduction of non-native species (22, 23). Notably, environmental factors restricting range expansion of species may differ between climatic regions, depending on the specific factors preventing colonization of species from other regions. For instance, in tropical and subtropical regions dryer conditions in human land-use systems have been suggested to facilitate the colonization by species from temperate regions (4, 10). By contrast, in high-latitude regions warmer climates and management practices such as irrigation may provide climate refuges, which facilitate colonization by species originating from hot and wet climate zones (24, 25). Besides modification of environmental conditions, human activities may facilitate the range expansion of species that are dispersal-limited by transportation and trading networks (22).

Notably, the range expansion of taxa may depend on the distribution pattern of other biota with which they are associated. For instance, the range expansion of protists is likely to depend on the range expansion of their prey and host species. This is especially evident for phagotrophs and parasites of metazoans, whose biogeographic distribution depends on that of their prey and host species, respectively (26–28). Phagotrophs may be more successful invaders than animal parasites given that microorganisms, such as bacteria and fungi, are less limited by dispersal than larger-sized metazoan hosts like earthworms and arthropods (23). Phototrophic protists are likely to be more vigorous invaders than animal and plant parasites due to low nutritional specialization and dependence on other biota (27–29).

Here, we aimed at obtaining a better understanding of the impacts of human land use on cross-region range expansion of species, its determining factors, and its link to biological homogenization in soil systems. For that, metabarcoding approaches were used to investigate large-scale biodiversity patterns of protist communities and their potential prey (bacteria and fungi) and hosts (Metazoa) in human land-use systems including farmlands, residential areas, and parks as well as more natural forest systems across subtropical and temperate regions in China. We further cross-validated our results using a global database available from the literature (21). We hypothesized that: H1: Human land-use systems extend the distribution range of protist taxa by promoting their spread across subtropical and temperate regions. H2: The range expansion of protist taxa in human land-use systems results in reduced beta-diversity compared to forests, with a more significant impact at the cross-region scale than at the region and city scale. H3: The determinants of cross-region range expansion of protist taxa differ between climatic regions, with dryer conditions facilitating temperate-to-subtropical expansion, while increases in temperature, precipitation, and soil moisture promoting subtropical-to-temperate expansion. H4: The range expansion of phagotrophic and animal-parasitic protists is determined by the range expansion of their microbial prey and host biota, respectively.

1. Results

1.1. Impact of Human Land-Use Systems on Range Expansion of Soil Protists.

Levins’ niche breadth of shared operational taxonomic units (habitat-generalist OTUs) between forests and human land-use systems was higher than that of OTUs (habitat-specialist OTUs) exclusively detected in either forests or human land-use systems (Fig. 1A). Except for parasitic protists, the niche breadth of habitat-generalist OTUs was higher in human land-use systems than in forests for all protist groups. This observation aligned with the lower minimum latitude of habitat-generalist OTUs in human land-use systems, suggesting more southern distribution boundary compared to the forest counterparts (Fig. 1 A and B).

Fig. 1.

Levins’ niche breadth, minimum latitude, maximum latitude (A), and radar plot of average distribution boundary of OTUs of total protists as well as protist functional groups (phagotrophs, phototrophs, and animal parasites) (B). Boxes sharing the same letter are not significantly different in a specific functional group of protists (P < 0.05; Tukey’s HSD test). In the radar plot, northern (N), southern (S), eastern (E), and western (W) boundaries are averages of standardized maximum latitudes, minimum latitudes, maximum longitudes, and minimum longitudes, respectively. nat.gen, habitat-generalist OTUs in forests; human.gen, habitat-generalist OTUs in human land-use systems; nat.spe, habitat-specialist OTUs of forests; human.spe, habitat-specialist OTUs of human land-use systems.

For different land-use types in different climatic regions, about 50% of protist OTUs were tracked back to subtropical and temperate forest sources (Fig. 2A). The exotic-to-native ratio differed between climatic regions, while the variation between land-use types was minor. Specifically, the exotic-to-native ratio was higher in the subtropical than in the temperate region for total, phagotrophic, and phototrophic protists (Fig. 2B). In trend this also applied for animal parasitic protists but the magnitude was relatively minor.

Fig. 2.

Proportion of OTUs of subtropical or temperate origin in total (protists) and different functional groups of protists (phagotrophs, phototrophs, and animal parasites) in different human land-use systems from the subtropical and temperate regions (A). Ratio between the proportion of exotic and native OTUs (ratio exotic:native) of total (protists) and different functional groups of protists in different land-use systems from the subtropical and temperate regions (B). Boxes sharing the same letter are not significantly different in a specific functional group of protists (P < 0.05; Tukey’s HSD test). Native and exotic OTUs are defined as OTUs originating from the same and different climatic regions, respectively.

Following the higher expected exotic-to-native ratio in the subtropical human land-use systems, community composition of protists in subtropical land-use systems differed from that in subtropical forests and was more similar to those in temperate forests and human land-use systems (SI Appendix, Fig. S1A). As a result, the community dissimilarity between natural and human land-use systems within cities was more pronounced in the subtropical than in the temperate region (F_1,628 = 344.97, P < 0.001; SI Appendix, Fig. S1B). This pattern was mainly driven by the increase in the turnover component of community dissimilarity in the subtropical region (turnover, F_1,628 = 298.1, P < 0.001; nestedness, P > 0.05).

1.2. The Association Between Range Expansion and Loss in Beta-Diversity of Protists in Human Land-Use Systems.

Beta-diversity of total protists in human land-use systems was lower than in forests, with the magnitude of reduction being the highest at the cross-region scale, followed by the region scale (subtropical > temperate) and being lowest at the city scale (Fig. 3). The patterns of phagotrophs and phototrophs were similar to that of total protists, except for a more pronounced decline in beta-diversity of phototrophs in farmlands of the subtropical region than that at the cross-region scale. In contrast to phagotrophs and phototrophs, the reduction in beta-diversity of animal parasites in human land-use systems was relatively minor (Fig. 3).

Fig. 3.

Effect size of human land-use systems on community dissimilarity of total (protists) and different functional groups of protists (phagotrophs, phototrophs, and animal parasites) compared to forests at the city-, region- (temperate, subtropical), and cross-region (All) scale.

The distance to community centroid (community uniqueness) of protists, in particular phagotrophs, was negatively linked to their exotic-to-native ratio in the subtropical region, and this correlation strengthened with the increase in spatial scale (Fig. 4). In contrast to the pattern in the subtropical region, the correlation between the exotic-to-native ratio and community uniqueness was generally not significantly negative in the temperate region, except for phototrophs at the cross-region scale. For more details on comparisons of alpha-, beta-, and gamma-diversity between natural and human land-use systems for different taxonomic and functional groups of protists please see (30).

Fig. 4.

Relationships between distance to community centroid and ratio between proportion of exotic and native OTUs (ratio exotic:native; log scale) of total and different functional groups of protists (phagotrophs, phototrophs, and animal parasites) at the city, region (temperate, subtropical), and cross-region (All) scale. r, Spearman’s correlation coefficients.

1.3. Determinants of Range Expansion of Protists in Human Land-Use Systems.

The correlation between the exotic-to-native ratio and environmental factors differed between the subtropical and temperate regions (Fig. 5A). Specifically, mean annual precipitation (MAP) and soil moisture were strong and positive determinants for the exotic-to-native ratio of total, phagotrophic, and phototrophic protists in the temperate region, while negative and/or neutral correlations were detected in the subtropical region. In addition, the exotic-to-native ratio of total, phagotrophic, and phototrophic protists correlated positively with soil pH in the subtropical region, but negatively in the temperate region (Fig. 5 A and B). The correlations between the exotic-to-native ratio of animal parasites and environmental factors were generally weak compared to other protist groups. Random forest models based on projected climatic conditions in 2,100 deciphered the possibility of a substantial increase in the exotic-to-native ratio in the temperate region while the change in the subtropical region was minor (SI Appendix, Fig. S2).

Fig. 5.

Relationship between different environmental variables (Materials and Methods) and the ratio between proportion of exotic and native OTUs (ratio exotic:native) of total (protists) and different functional groups of protists (phagotrophs, phototrophs, and animal parasites) in different land-use systems from the subtropical and temperate regions, respectively; Upper panel Spearman’s correlation coefficients (A), Lower panel regressions of significant relationships (B). Native and exotic OTUs are defined as OTUs originating from the same and different climatic regions, respectively. Determinants in (B) refer to the respective environmental variables given at the Top of the figure and were presented at log10 scale for the exotic:native ratio of bacteria, fungi, phototrophs, and Metazoa. MAT, mean annual temperature; MAP, mean annual precipitation; TC, soil total carbon; TP, soil total phosphorus; C.N, soil carbon-to-nitrogen ratio; r, Spearman’s correlation coefficients.

The exotic-to-native ratio of bacteria positively correlated with that of protists, with the strongest correlation in phagotrophic protists (Spearman’s correlation coefficients of 0.75 and 0.71 for the subtropical and temperate region, respectively) followed by phototrophic protists (0.40 in the temperate region) (Fig. 5A). The exotic-to-native ratio of fungi was also positively correlated with that of total, phagotrophic, and phototrophic protists (0.29 to 0.54), but the strength of the correlations was lower compared to that with bacteria. The exotic-to-native ratio of Metazoa did not correlate significantly with total and functional groups of protists (P > 0.05).

2. Discussion

2.1. The Impact of Human Land-Use Systems on the Range Expansion of Soil Protists.

In line with our first hypothesis, Levins’ ecological niche of protist taxa was higher in human land-use systems than in natural systems, which was driven by the broader distribution of habitat generalists in human land-use systems than in forests. This finding is in line with previous studies on plants, birds, and insects, showing that human disturbance facilitates generalist species and their spread across large spatial scales (31–33). Importantly, this land-use assisted range expansion was mainly related to the lower latitudinal boundary of habitat-generalist taxa. Together with the species flow analysis, this indicates that the range expansion of soil protists induced by land-use change is highly directional, and largely driven by the introduction of exotic taxa from the temperate into the subtropical region.

Notably, the lower number of protist sequences in subtropical forests paralleled by the decline in the generalist-to-specialist sequence ratio compared to human land-use and temperate forest systems may bias estimates of the distribution range of protist taxa (SI Appendix, Figs. S3 and S4). However, further analyses controlling for the differences in sequence number of protist generalists across samples showed highly consistent results (SI Appendix, Figs. S5 and S6), indicating that the reported broader and more southern distribution of protist generalists in human land-use systems is robust. Importantly, the coupled reduction in the sequence number of protists and in the dominance of generalists suggests that ecological barriers limit the range expansion of generalist species into subtropical forests, and these barriers are likely to be diminished in human land-use systems.

Previous studies found that community composition was more sensitive to land-use changes in tropical than in high-latitude regions, which was attributed to a higher proportion of specialists in the tropics and higher tolerance of temperate species to human disturbances (4). In addition to this explanation, our results indicate that land use induced range expansion of species of temperate origin into tropical regions also contributes to the more pronounced differences in community composition between natural and managed systems in the tropics. Notably, these increased community differences were driven by enhanced species turnover (rather than nestedness), suggesting that in tropical and subtropical regions land-use impacts on community structure and functioning of protists, especially phagotrophs, are tightly correlated with the addition of exotic species but less with species loss.

2.2. Association Between Cross-Region Range Expansion and Loss in Beta-Diversity of Protists in Human Land-Use Systems.

Beta-diversity of protists was lower in human land-use systems than in forests, which is in line with previous studies, suggesting that biological homogenization is among the most prominent impacts of land-use changes (34, 35). Importantly, our results identified increased reduction in beta-diversity with the increase in spatial scale, highlighting the important role of processes operating at larger scales. Supporting this conclusion, community uniqueness estimated at the cross-region scale negatively correlated with the exotic-to-native ratio of soil protists in subtropical land-use systems. This indicates that the range expansion of protists of temperate origin into subtropical land-use systems reduces community heterogeneity between climatic regions. Consequently, this contributed to the decline in overall beta-diversity in human land-use systems compared to natural systems. In the temperate region, the exotic-to-native ratio was not negatively correlated with community uniqueness, rather, in some cases, it increased with community uniqueness. This corresponds to the relatively low exotic-to-native ratio in the temperate region, in line with previous findings that invasion events occurring at an early stage and/or in limited locations may increase rather than reduce beta-diversity within regions (36).

Notably, range expansion across climatic regions is not the only mechanism underlying biological homogenization. Other mechanisms such as range expansion of species with broad habitat preferences and high tolerance to environmental changes in their native region are also important (37). In our study, beta-diversity of phototrophic protists was more strongly reduced at the regional compared to the cross-region scale. In addition, at a small spatial scale (city scale), biological homogenization of phototrophs exceeded that of phagotrophs and parasites. Therefore, range expansion of native species appears to be a stronger driver of biological homogenization of phototrophs compared to other protist groups.

2.3. Determinants of Range Expansion of Protists in Human Land-Use Systems.

In contrast to our third hypothesis that the introduction of exotic species in subtropical regions is driven by drier conditions in human land-use systems, we found that the increase in soil pH played a predominant role in determining the colonization of subtropical regions by exotic species. Soil pH in subtropical forests was generally lower than in forests of the temperate region. High heterogeneity in pH has been identified as a major environmental filter determining the large-scale distribution of species in soils in natural systems (38, 39). Soil pH in subtropical land-use systems exceeded that in subtropical forests and was similar to that in temperate ecosystems (SI Appendix, Fig. S7). This breakdown of ecological barrier apparently facilitates the colonization of subtropical soils by protist species from higher latitude regions. In addition, the contrasting environmental conditions between human land-use and forest systems in the subtropical region may also hinder the colonization of human land-use systems by native taxa, and thus facilitate the establishment of exotic taxa by reducing competition.

In contrast to the subtropical region, lower soil pH and higher precipitation and soil moisture promoted the colonization of temperate land-use systems by subtropical taxa. Along with the markedly lower exotic-to-native ratio in the temperate region, this suggests that dry and alkaline soils hamper northward migration of subtropical taxa. Additionally, the exotic-to-native ratio in the temperate region did not increase with annual mean temperature. These results are in line with previous findings that precipitation is a more important determinant than temperature for the large-scale biogeography of protists (39, 40). Notably, given the projected increase in precipitation in China especially in the northern region (41, 42), the northward range expansion of protist species and other soil biota may increase substantially in the future and reach levels comparable to the southward expansion, but this needs further investigation.

Partially in line with our fourth hypothesis that the cross-region range expansion pattern of protists is determined by their prey or host biota, the exotic-to-native ratio of phagotrophic protists strongly and positively correlated with that of microorganisms, particularly bacteria. Tight association between phagotrophic protists and their microbial prey has been widely reported (23, 27, 28). Notably, the cross-region range expansion of bacteria was closely and positively correlated with soil pH in the subtropical region, suggesting that the impact of pH on the cross-region range expansion of phagotrophic protists is likely to be mediated by the expansion of their microbial prey. In contrast to phagotrophic protists, the cross-region range expansion of animal-parasitic protists was low and decoupled from that of soil Metazoa. This supports the hypothesis that free-living microbes might spread faster than obligate symbionts and parasites because their spread is not limited by the availability of hosts (23, 43). In addition, the association between the diversity of parasites and soil animals has been found to be strongly disrupted by land-use changes (26) resulting in mismatch between animal hosts and parasites.

Although our study covered a wide range of climatic regions, it primarily represented regions with natural backgrounds of forests in China. To test the generality of our conclusion, we estimated the distribution range of protist taxa in the global dataset published in ref. 21 (SI Appendix, Fig. S8). Supporting our conclusion, the distribution range of habitat-generalists was larger in urban than in natural systems across continents, including Asia, Europe, and Australia, while differences in habitat specialists were not observed. Further supporting our findings for China, in Asia, South, and North America, the distance to the equator for habitat generalist taxa decreased in urban compared to natural systems. Notably, however, also contrasting patterns were detected, e.g. poleward expansion of generalists in European urban systems. This biogeographic disparity indicates that land-use impacts on large-scale soil biodiversity patterns are affected by continent-specific factors, such as the size of continent and its range across latitude, which determine the climatic pattern in a continent. Our study emphasizes that geographic regions characterized by tropical and subtropical moist climates are especially susceptible to land-use changes, which drive biological homogenization by facilitating introduction of temperate species.

Our study was primarily based on OTU data, which may limit extrapolation of our conclusions to finer taxonomic resolution, e.g. species level. However, analyses based on OTUs and ASVs showed highly consistent patterns, suggesting that our conclusions are robust. Notably, the magnitude of southward range expansion of protist taxa was lower at the ASV than the OTU level, potentially due to more variable response of taxa of high taxonomic resolution to land-use changes. Future studies using higher-resolution approaches, such as long-read or metagenomic sequencing, are needed to further test these patterns.

3. Conclusions

Our study demonstrates that human land-use systems are important corridors facilitating the spread of habitat-generalist protists beyond their native range. Importantly, exchange of species between regions is highly biased in direction and predominated by southward range expansion of protist species originating from the temperate zone into subtropical regions. Consequently, this results in a decline in beta-diversity in human land-use systems at the large spatial scale. The directional range expansion of protists is due to two major mechanisms: 1) elevation in soil pH in human land-use systems removing the ecological barrier preventing the southward migration of soil protists, with this being most pronounced in phagotrophs and their microbial prey; 2) dry conditions and high soil pH restricting northward range expansion of subtropical taxa. Among functional groups of protists, cross-region range expansion was most pronounced in phagotrophs, with this being closely related to the expansion of microbial prey, in particularly bacteria, implying invasion meltdown mediated by prey taxa. Weak cross-region expansion of animal parasites likely reflects that long-distance dispersal of symbionts is more restricted than in free-living organisms. Our results provide important insights into how human land-use systems reshape the distribution pattern of soil biodiversity. In the future, the potential risk of land-use systems as bridgehead for invasion of natural systems in subtropical and tropical regions needs to be considered. In addition, human land-use systems in subtropical regions expose species from temperate regions to new climatic conditions, such as high precipitation and temperature, with potentially important consequences for the structure and functioning of land-use systems in these regions.

4. Materials and Methods

4.1. Soil Sampling and Site Characteristics.

From May to September 2021, soil samples were taken in thirteen cities across subtropical and temperate regions in China (SI Appendix, Fig. S9). Subtropical cities (24.6 to 32.1°N; 106.6 to 121.7°E), including Xiamen, Fuzhou, Guiyang, Ningbo, and Nanjing, were characterized by high mean annual temperature (MAT; > 15°) and precipitation (MAP; > 1,000 mm). Temperate cities (34.2 to 45.6°N; 102.5 to 126.8°E) had low MAT (4 to 13°) and MAP (200 to 700 mm) and included Xi’an, Wuwei, Kaifeng, Taiyuan, Liaocheng, Shenyang, Changchun, and Harbin.

In each city, we selected four different types of land use: forest, park, residential area, and farmland. Forests consisted of natural or seminatural stands of typically more than 20,000 m². Farmland sites were mostly cultivated with maize and located at the outskirts of the cities. Urban parks and residential sites were located within cities. In each city and for each land-use type four sampling sites were established spaced by at least 200 m. At each sampling site, we randomly established a 20 m × 20 m plot located 20 to 50 m away from the edge for soil sampling. Within each plot, we randomly took nine soil cores using an auger of a diameter of 5.5 cm to a depth of 10 cm which were mixed as composite sample. In total, we sampled 208 composite samples (13 cities × 4 land-use types × 4 sampling sites). The samples were frozen at −20 °C until further analyses. Notably, our sampled forests were mostly located in the outskirt of cities and therefore likely also were exposed to some human activities, although certainly much less than urban ecosystems. For more details on the sampling sites see the supplementary data in ref. 44.

Air-dried soil subsamples were ground and sieved through 0.2 mm mesh before soil physical and chemical analyses. Soil pH was measured using a pH meter (PHS-3C, Shanghai Leici). Soil total carbon (TC) and total nitrogen (TN) were measured using an elemental analyzer (Elemental Analyzer System Vario Macro Cube, Langenselbold, Germany), and the carbon-to-nitrogen ratio (C/N) was calculated. Total phosphorus (TP) was measured by inductively coupled plasma-atomic emission spectrometry (ICPS-7500) using the triacid digestion-ICP-AES method (Agilent Technologies, USA). Soil moisture was measured using 10 g of fresh soil dried at 105 °C for 48 h. The soil texture was determined using a laser particle size analyzer (Bettersize 2000, Dandong Better Instrument Co. Ltd., CN). Information on MAP and MAT was retrieved for each plot from the Worldclim database (www.worldclim.org). To investigate the impacts of urban development pressure, urban area, population density, and urbanization rate of residents were collected from https://www.mohurd.gov.cn/.

4.2. Analysis of Soil Biota.

Each composite soil sample (fresh, not sieved) was thoroughly mixed and 10 g was homogenized. Genomic DNA was extracted from 0.5 g of soil using the FastDNA^® Spin Kit for soil (MP Biomedicals, Santa Ana, CA), according to the manufacturer’s protocol. The quality and quantity of the extracted DNA were assessed with 1% agarose gel electrophoresis and spectrophotometric analyses using NanoDrop ND-2000 (Thermo Fisher Scientific, Wilmington, USA), respectively. Eukaryotes, bacteria, and fungi were analyzed using high-throughput sequencing by targeting the V4 region of the eukaryotic 18S rRNA gene, the V4-V5 region of the 16S rRNA gene, and the fungal internal transcribed spacer 1 (ITS1) region amplified with the primer sets F-TAReuk454FWD1 (5’-CCAGCASCYGCGGTAATTCC-3’) and R-TAReukREV3 (5′-ACTTTCGTTCTTGATYRA-3′) (45); 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) (46); and ITS1F (5′- CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) (47), respectively.

PCR conditions of the 18S rRNA gene were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s and single extension at 72 °C for 10 min, and ended by holding at 4 °C. PCR conditions of the 16S rRNA gene were as follows: an initial 5 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. PCR conditions of the ITS1 region were as follows: an initial denaturation at 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C, annealing for 30 s at 55 °C and elongation for 45 s at 72 °C, the last step being extension at 72 °C for 10 min. The 18S rRNA and 16S rRNA genes were amplified in triplicate in 20 μL mixtures consisting of 0.4 μL TransStart^® FastPfu DNA Polymerase (Transgen Biotech Co., Ltd., Beijing, China), 4 μL of 5 × TransStart^® FastPfu Buffer, 2 μL dNTPs (2.5 mM), 0.8 μL of each primer (5 μM), 0.2 μL BSA (2 mg·mL⁻¹), 2 μL template DNA (10 ng·μL⁻¹), and 9.8 μL of sterilized ddH₂O. The ITS1 region was amplified in a total volume of 20 μL in triplicate, the PCR mix contained 0.2 μL Premix Taq™ DNA Polymerase (Takara Biotechnology, Dalian, China), 4 μL of 10 × Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.2 μL BSA (2 mg·mL⁻¹), 2 μL template DNA (10 ng·μL⁻¹), and 11.6 μL of sterilized ddH₂O. Negative control samples were included throughout the PCR assay to ensure reaction systems were not contaminated. The PCR products were recovered from a 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA) according to the manufacturer’s protocols, quantified using Quantus™ Fluorometer (Promega, USA) (48). Purified amplicons were pooled and sequenced on the Illumina MiSeq^® PE 300 platform (Illumina, San Diego, CA) by Majorbio (Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China).

Pair-end reads were matched using FLASH (v1.2.11) (49) and high-quality reads were merged on FASTP (v0.19) (50). The Quantitative Insights Into Microbial Ecology (QIIME v1.90) pipeline was used to generate high-quality processed and analyzed sequences (51). After removal of chimera, sequences were assigned to operational taxonomic units OTUs at 97% identity using UPARSE (v7.0.1090) (52). The Protist Ribosomal Reference (PR², v4.14, https://github.com/vaulot/pr2_database) (53), SILVA rRNA database (v138, http://www.arb-silva.de) (54), and UNITE fungal ITS database (v8.0, http://unite.ut.ee/index.php) (55) were used for alignments and classification of eukaryotes, bacteria, and fungi, respectively.

4.3. Assignment of the Protist Functional Group.

Here, Rhodophyta, Streptophyta, Fungi, Metazoa, and unclassified taxa in Eukaryotes were excluded to obtain protist OTUs. We further assigned protist OTUs (at the genus level) to three major functional groups (17, 40), including phagotrophs, phototrophs, and parasites. Phagotroph protists, primarily Cercozoa, Lobosa, Conosa, and Ciliophora, are mainly consumers of microbes preying on bacteria, fungi, and other protists. Phototrophs are protists that obtain energy via photosynthesis and mainly comprised Ochrophyta and Chlorophyta. Parasites are a group of protists (mainly Apicomplexa) parasitizing metazoan animals. Parasites of plants or other organisms were not included in our analysis as their relative abundance was low and data on their hosts were lacking. Our study mainly focused on variations between functional groups of protists as the degree of biological homogenization has been shown to be closely related to differences in trophic groups (30). For more information on the OTU number of different protist taxonomic groups and their assignment to different functional groups see SI Appendix, Table S1 and Dataset S1.

4.4. Statistical Analysis.

All statistical analyses were conducted in R (version 3.6.2, http://www.r-project.org/). Overall, we obtained 17,375 eukaryotic OTUs. Protists were the predominant group, accounting for 45% of eukaryotic OTUs, while fungi and Metazoa only accounted for 13 and 9% of eukaryotic OTUs, respectively. The OTU table of eukaryotic communities (including protists, fungi, Metazoa, and other eukaryotic taxa) was rarefied to a sequence depth of 28,222 using the rarefy function in the “vegan” package (56). In rarefaction curves of individual samples, richness of different taxonomic and functional groups of protists generally reached saturation levels (SI Appendix, Figs. S10 and S11). In addition, there were strong positive correlations between rarefied and nonrarefied datasets for richness (Pearson’s correlation coefficient > 0.96, P < 0.001) and community dissimilarity (> 0.93, P < 0.001) (SI Appendix, Fig. S12). These results indicate that our rarefied datasets well represent the biodiversity of protist communities. For analyses using protist ASVs based on the same dataset, see SI Appendix, Fig. S13. Overall, the patterns of ASVs were highly similar to those of OTUs.

4.4.1. Land-use impacts on range expansion and cross-climatic-region range expansion.

The distribution range of each OTU was estimated by Levins’ niche breadth according to the following equation:

$$B_j=\frac{1}{{\sum }_{i=1}^N{P_{\mathit{ij}}}^2},$$

where B_j represents the niche breadth of OTU j, N the total number of samples, and P_ij the proportion of OTU j in sample i. A high B-value for a given OTU indicates its wide distribution range across sites. The potential northern, southern, eastern, and western boundaries of distribution of individual OTU were represented by the maximum latitude, minimum latitude, maximum longitude, and minimum longitude of the samples in which they were detected. Protist OTUs were identified as habitat-generalists if they occurred in both natural (forests) and human land-use systems. Protist OTUs occurred in either natural or human land-use systems were assigned to habitat-specialists of their respective systems. To examine whether the distribution range of protists differed between natural and human land-use systems, the differences in Levins’ niche breadth, maximum latitude, minimum latitude, maximum longitude, and minimum longitude of habitat-generalist and -specialist protist taxa were tested using general linear models. The average of standardized distribution boundaries across OTUs was visualized by radar charts (R package “ggradar”) (57).

To test whether range expansion of protists induces species flow across the subtropical and temperate regions, FEAST analysis was conducted to identify the contribution of subtropical and temperate taxa sources to local communities in each sample of human land-use systems in different climatic regions (58). Given the assumption that distribution patterns of taxa in forests largely reflected their original distribution pattern, we linked local communities in human land-use systems to the species pool in forests in different climatic regions to estimate the intensity of exotic species flow for each sampling plots. Specifically, we quantified the proportion of taxa of subtropical and temperate origin in each sample in human land-use systems by using function “FEAST” (package FEAST) (58). In this analysis, each sample in human land-use samples was taken as taxa sink, while samples in subtropical or temperate forests were pooled respectively as subtropical or temperate taxa sources. The magnitude of introduction of exotic taxa in a given climatic region was represented using exotic-to-native ratio. For instance, the magnitude of introduction of exotic taxa in the subtropical region was estimated by the ratio between the proportion of taxa of temperate origin to the proportion of taxa of subtropical origin.

To investigate how range expansion links to changes in community composition of protists, principal coordinate analysis (PCoA) was used to visualize the community variation of soil protists across different land-use types and climatic regions (function “cmdscale”, R package “vegan”) (56). In addition, we examined community dissimilarity (beta diversity) between human land-use and natural systems in the subtropical and temperate region. As different components of beta diversity are associated with different ecological processes, beta diversity was partitioned into turnover (Sim) and nestedness (Sne) using the function “betapart” (package betapart) (59). The turnover component is related to the replacement of species, that is, the degree to which different species colonize the different local communities. The nestedness component is related to environmental filtering and local species extinction, reflecting the extent to which species at a specific site are a subset of sites richer in species. In these analyses, site-to-site community dissimilarity was calculated using Sørensen index, a binary version of the Bray–Curtis dissimilarity index.

4.4.2. The association between range expansion and biological homogenization.

The site-to-site dissimilarity in community composition (beta-diversity) of total and individual protist groups was calculated using the Sørensen index. The distance to the community centroid in different human land-use systems was calculated using the function betadisper in the vegan package (56). To inspect the influence of spatial scales, the distance to community centroid was calculated based on centroids across all samples (cross-region scale), across samples in different climatic regions (region scale), and across samples in different cities (city scale).

Linear mixed effects models were used to inspect whether community distances of total and different functional groups of protists in human land-use systems were lower than those in forests (biological homogenization impacts) with “city identity” included as random term (“lmer” in package “lme4”) (60). To facilitate comparison of the magnitude of biological homogenization between spatial scales and land-use types, effect sizes of human land-use systems on community distance were estimated based on linear mixed effects models and given as Cohen’s D (“eff_size” in the “emmeans” package) (61). Cohen’s D reflects the differences in means of community distance between human land-use systems and forests divided by the pooled SD.

To test whether the introduction of exotic taxa from other climatic region reduces the beta-diversity at different spatial scales, Spearman’s correlation analysis was used to inspect the relationship between community distance and exotic-to-native ratio for total and different functional groups of protists at different spatial scales.

4.4.3. Determinants of introduction of exotic taxa in different climatic regions.

To identify environmental factors driving the introduction of exotic protists in human land-use systems, Spearman’s correlation analysis was conducted to inspect the relationship between soil, climatic, city factors, and exotic-to-native ratio in the subtropical and temperate region, respectively. The exotic-to-native ratio for other soil biota, including bacteria, fungi, and Metazoa, was also estimated. For Metazoa, microfauna such as nematodes were not included in the analysis because they are not the major hosts of protist parasites (26). Spearman’s correlation coefficients between different functional groups of protists and different nonprotist biota were used to test the role of prey and host range expansion for cross-region range expansion of phagotrophic and parasitic protists, respectively.

To inspect the potential influence of climatic changes on the cross-region range expansion of soil protists in human land-use systems, we used random forests to construct models linking climatic conditions and exotic-to-native ratio in different climatic regions, separately. Then, we used projected climatic conditions based on the high-emission scenario (CMIP6 E2.1 sp585) as input to predict the exotic-to-native ratio in each sampling plot in different climatic regions.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Data, Materials, and Software Availability

The data that support the findings of this study are available in the NCBI Sequence Read Archive (SRA, Bioproject ID PRJNA1114439) (62) and FigShare at https://doi.org/10.6084/m9.figshare.26122408.v1 (63). R codes used in this article are available at https://doi.org/10.6084/m9.figshare.29107655 (64).