Identification of microbial keystones in complex soil ecosystems and determination of their activity and niche preferences

Determining the functional role of individual types of microorganisms and their specific relevance in the environment remains very challenging, especially in highly complex environments such as soils. One promising approach is to try to detect and characterize so-called keystone taxa, which are critical to the stability and functioning of the entire communities. We used co-occurrence networks from high-throughput sequencing data to elucidate significant associations, and to thereby infer microbial sequence types that might be of particular importance to the overall microbial communities, hence representing keystones. This approach was applied to bacterial and fungal communities in 300 different soils of the German Biodiversity Exploratories. The sites encompass grassland and forest ecosystems under different land use regimes, which were sampled over a period of 11 years. 16S rRNA and the internal transcribed spacer (ITS2) were used as markers for bacteria and fungi, respectively. Keystones were identified by a ranking procedure based on the susceptible-infected-recovered model after perturbation of the original networks. Keystone taxa were specific to distinct soil environments within different geographic regions and remained rather stable in their habitat over time. We hypothesized that keystone taxa must be more active than co-occurring microorganisms to exert their ecological influence over longer time periods. Accordingly, active bacterial taxa were identified using both 16S rRNA/ 16S rDNA ratios and also by determining bacterial growth rate from the peak-to-trough ratio of genomic sequence coverage. Notably, we found a significant overlap between keystone and active species, corroborating the network analysis approach. Using the molecular signatures of keystone taxa, their distinct ecological niche preferences were analysed by modelling abundances against environmental parameters, and metagenome assembled genomes (MAGs) from bacterial keystones provided further insights into their functional potential. Our results thus pave the way towards dissecting the ecological roles of individual microbial taxa in highly complex environments.