Microbial Functional Potentials Differ Among Monospecific and Mixed Moss Biocrusts in an Alpine Sandy Ecosystem

Moss-dominated biological soil crusts are associated with carbon and nitrogen cycling in dryland and alpine sandy ecosystems, but functional differentiation among closely related moss species and their mixed assemblages remains unclear. We used shotgun metagenomic sequencing to compare below-crust soil associated with Didymodon constrictus (Mitt.) K. Saito (D. constrictus) crusts (mossC), ferrugineus (Schimp. ex Besch.) M.O. Hill (D. ferrugineus) crusts (mossF), and visually co-dominated mixed crusts (mossM) in the Gonghe Basin on the northeastern Qinghai-Tibet Plateau. Fifteen spatially separated quadrats per category were pooled into three composite biological replicates (effective n = 3). KEGG, CAZy, and targeted carbon- and nitrogen-cycling annotations showed category-associated differences in relative gene representation. MossC had greater mean representation of glycoside hydrolases and several complex-carbon-processing functions, mossF had greater representation of nitrogen-assimilation and acetate-related functions, and mossM had greater representation of selected carbon-degradation, nitrogen-mineralization, and dissimilatory-nitrate-reduction functions. The full RDA model explained 58.4% of functional variation (adjusted R² = 0.334; exact permutation p = 0.028340), with single-variable associations retained for total carbon and soil water content. Genus-level taxonomic dissimilarity correlated with KEGG, CAZy, carbon-cycling, and nitrogen-cycling dissimilarities after FDR correction. MossM showed both positive and negative descriptive deviations from the approximate unweighted midpoint of mossC and mossF, but no inferential test was applied to these deviations. The small number of composite replicates, visually estimated moss proportions, edaphic confounding, and absence of activity measurements limit causal and confirmatory interpretation.