Microbial community composition and functional potential changes along a century-scale geothermally warmed soil temperature gradient

Understanding changes in soil organic matter (SOM) dynamics in response to long-term warming is central to predicting carbon stocks under future climate-change scenarios. This study investigates how century-scale soil warming influences microbial community composition and functional potential using a subarctic deciduous forest located on a geothermal hotspring (Takhini Hot Springs, Yukon Territory, Canada) as a model system. The soils affected by this natural geothermal gradient, which has been documented as being active for at least 100 years, range between 0 and 5°C above ambient surface temperature, with 40 - 60 cm subsoils reaching up to +11°C. Previous analyses of SOM from the study site show a decline in C:N ratios with increasing soil temperature, while nitrogen stocks remain largely unchanged, suggesting long-term alterations in organic matter inputs and decomposition processes. This system provides a unique opportunity to study long-term warming effects under field conditions while avoiding artefacts associated with short-term manipulations.

Topsoil and subsoil microbial community population size, taxonomy and functional gene composition at topsoil mean annual temperatures of 3.5, 4.2 and 5.3 and 8.3°C were assessed using qPCR and whole-genome shotgun sequencing. As soil texture and humic acid content varied along the gradient (e.g., 8.5 % to 25.9 % clay), an adapted extraction protocol optimised for humic-rich soils was used for DNA extraction, together with the use of an internal whole-cell spike-in standard of Gram-positive and negative halophilic extremophiles in all qPCR assays to correct for differential extraction efficiency and PCR inhibition. Metagenomic data is used to characterise microbial community shifts and to identify functional genes related to carbon, nitrogen and phosphorus cycling, as well as traits linked to microbial metabolic strategies. Metagenomic analyses indicate that long-term warming restructures microbial communities in a depth-dependent manner, characterised by increased Actinobacteriota in warmer deep soils, reduced Planctomycetota and Chloroflexi with warming, and higher surface-layer abundance of Amorphea in cooler plots. PCA of phylum-level communities revealed clear depth stratification (p<0.001) and warming effects (p=0.003), with surface and subsoil samples clustering separately and warmer plots diverging along PC1.

By integrating microbial community data with soil physicochemical properties, this study aims to clarify how sustained warming alters microbial functional potential and SOM processing in subarctic soils.  Decreases in the relative abundance of eukaryotes (Amorphea) with increasing temperature, and a concomitant increase in Gram-positive Actinobacteriota associated with plant biomass cycling and secondary metabolite production in soils, suggests that temperature-dependent shifts in organisms responsible for SOM cycling may occur under soil warming of > 5 °C. The findings will contribute to improving predictions of climate-driven changes in soil biogeochemistry and the long-term stability of SOM under warming.