Space Beats Seasons: Soil Protist Communities Shaped More by Place Than Time on European Mountains

Beneath the forest floors and alpine meadows of Europe’s mountains lives an enormous, largely unmapped cast of microscopic eukaryotes. Protists — every eukaryotic organism that is not a plant, animal, or fungus — drive nutrient cycling, sustain the soil food web, and underpin ecosystem functioning, yet most of their species remain formally undescribed. A new study published in Frontiers of Biogeography delivers one of the most rigorous tests to date of how these tiny organisms are distributed in space and time, and its conclusion is strikingly simple: where you sample matters far more than when you sample.

Researchers from the University of Neuchâtel and partner institutions collected 104 soil samples along five elevation gradients in two distant mountain ranges, the Spanish Sierra Nevada and the Swiss Alps, spanning two distinct biogeographic regions. Sampling took place in 2019 at 52 sites in forests and grasslands below the treeline, with every site visited twice — once in spring and once in summer. This design allowed the team to directly compare the magnitude of spatial turnover in protist communities against the magnitude of seasonal change, a question that has rarely been addressed for soil microeukaryotes across multiple gradients.

The methodological backbone of the study was environmental DNA metabarcoding. The team extracted DNA from roughly a gram of sieved soil, amplified the hypervariable V4 region of the 18S rRNA gene with universal eukaryotic primers, and sequenced the products on an Illumina MiSeq platform. After stringent quality filtering, chimera removal, error-model-based inference of amplicon sequence variants (ASVs), and a dedicated procedure to detect and correct tag-jumps — sequencing artifacts in which reads are assigned to the wrong sample — the final dataset contained more than 6.5 million cleaned reads representing nearly 20,000 eukaryotic ASVs. Of these, 11,706 ASVs were assigned to protists, with rarefaction curves indicating that sequencing depth had reached saturation for every sample regardless of region, habitat, or season.

The taxonomic picture was dominated by Cercozoa, a phylum of predominantly predatory and bacterivorous amoebae and flagellates, which contributed 4,445 ASVs and the largest share of reads. Ciliophora (1,725 ASVs), Apicomplexa (1,176 ASVs), the amoeboid group traditionally labeled Lobosa (1,131 ASVs), Conosa (968 ASVs), and Chlorophyta (488 ASVs) followed. The strong showing of Apicomplexa, a group famous for animal parasites such as the malaria agent Plasmodium, most likely reflects the pervasive parasitism of soil animals, a signal that has been increasingly recognized in environmental sequencing surveys. Green algae of the phylum Chlorophyta were more abundant in open grasslands than under forest canopies, consistent with their phototrophic lifestyle.

To quantify the relative roles of space and time, the authors decomposed total (gamma) diversity into three additive components: local alpha diversity, temporal beta diversity (the turnover of ASVs between spring and summer at the same plot), and spatial beta diversity (the turnover among plots within a habitat and region). The result was unambiguous. In the Swiss Alps, spatial turnover accounted for an average of 85.06 percent of total diversity, compared with just 3.84 percent for temporal turnover. In the Spanish Sierra Nevada the corresponding figures were 70.40 percent versus 14.17 percent. Local alpha diversity contributed the remainder and, except in Sierra Nevada grasslands, always exceeded the temporal component.

Ordination and statistical testing reinforced this hierarchy. Non-metric multidimensional scaling of Bray-Curtis dissimilarities revealed four distinct sample clusters separated along two axes: one splitting forests from grasslands and the other separating the Sierra Nevada from the Alps. Season, by contrast, left no visible imprint. A permutational analysis of variance confirmed that community composition differed significantly between regions and habitats and, to a lesser degree, across elevations, while the temporal variable and all its interactions were statistically non-significant. Variance partitioning showed that space, soil properties, and climate together explained 29 percent of community variation, whereas season alone explained none, contributing only about 3 percent in combination with the other factors.

The team then zoomed in on four of the most species-rich protist groups — Cercozoa, Ciliophora, Apicomplexa, and Lobosa — each exceeding 1,000 ASVs, to test whether the whole-community pattern held at finer taxonomic resolution. It did. None of the four groups showed significant seasonal shifts in alpha diversity indices, ordinations separated samples by habitat and region but not season, and diversity partitioning for every group showed spatial beta diversity dwarfing temporal turnover. The sole exception was Ciliophora, for which season explained a minor portion of community composition, yet even here the overarching pattern of spatial dominance remained intact.

The environmental drivers of community composition also proved temporally stable. Redundancy analyses with forward selection identified the same set of edaphic predictors in spring and summer within each region, even though the explanatory power of individual variables varied between seasons. In the Spanish Sierra Nevada, elevation and a composite variable combining soil organic matter, organic carbon, organic nitrogen, and residual humidity were the significant predictors, the latter likely reflecting the capacity of organic particles to retain water. In the Swiss Alps, soil pH, the carbon-to-nitrogen ratio, the nitrogen-to-phosphorus ratio, and bioavailable phosphorus emerged as key drivers — variables tied to organic matter turnover and nutrient cycling. That different sets of predictors operated in the two regions underscores how bedrock, climate, and vegetation jointly shape belowground communities, while their consistency across seasons within each region strengthens confidence in single-visit sampling.

Why does this matter beyond the world of protist ecology? Microorganisms divide far faster than plants and animals, so ecologists have long worried that microbial communities might fluctuate seasonally enough to confound comparisons among studies conducted at different times of year. If temporal turnover matched or exceeded spatial turnover, a survey of one mountain range sampled in May could not legitimately be compared with another sampled in July. The new findings lift that constraint for temperate mountain ecosystems: sampling once at any point between late spring and late summer should not bias beta diversity comparisons across elevation gradients, regions, or habitats. Given that logistical and financial realities frequently restrict large-scale and global sampling campaigns to a single visit per site, this is genuinely good news for the design of macroecological studies of soil life.

The authors also note important caveats. Their conclusions apply to temperate climates and to the spring–summer window; harsher seasons were excluded because snow cover, road access, and safety make winter and autumn sampling impractical at high elevations, even though snow insulates soils and allows microbial activity to continue beneath it. Hot, dry summer spells can sharply reduce protist abundance and activity, and drought sensitivity varies among protist groups, so climate change could rewrite the seasonal rules the study establishes. The findings may also not transfer to other climates or latitudes, and the eDNA-based approach has yet to be systematically validated against traditional microscopy for seasonal dynamics of soil protists. Moreover, the analyses did not cover phototrophic protists, which play important roles in grasslands. Still, as a baseline for a component of biodiversity long relegated to the margins of biogeography, the study’s message is clear and actionable: in Europe’s temperate mountains, the identity of a soil protist community is written primarily by its place on the landscape — its elevation, habitat, and soil chemistry — not by the calendar. Researchers planning large-scale soil biodiversity surveys can now schedule their fieldwork with one less worry, and conservation assessments of mountain biodiversity gain a firmer foundation for comparing sites across regions and seasons.

Subject of Research: Spatial versus seasonal beta diversity of soil protist communities along elevation gradients in the Swiss Alps and Spanish Sierra Nevada

Article Title: Higher spatial than seasonal beta diversity of soil protists along elevation gradients

Article References: Bruni, E. P., Lorite, J., Peñas, J., Mulot, M., Fournier, B., Vittoz, P., Mitchell, E. A. D., & Lentendu, G. (2024). Higher spatial than seasonal beta diversity of soil protists along elevation gradients. Frontiers of Biogeography, 17, Article e132637. https://doi.org/10.21425/fob.17.132637

Image Credits: AI Generated

DOI: 10.21425/fob.17.132637

Keywords: soil protists, beta diversity, elevation gradients, environmental DNA, metabarcoding, Swiss Alps, Sierra Nevada, seasonality, biogeography, Cercozoa, soil microbiology, mountain ecology