Metagenomic insights into the effect of mixed plantations with N-fixing species on microbial functional diversity, gene assembly processes and soil multi-nutrient cycling

Abstract

Nitrogen-fixing legumes such as Robinia pseudoacacia are widely used in vegetation restoration due to their rapid growth and N-fixation capacity. However, large-scale single-species forests of N-fixing species often lead to long-term unsustainability, such as functional simplification and soil desiccation. Mixed-species forests that integrate N-fixing trees with native drought-tolerant species may help overcome these limitations, but their effects on soil functional microbial communities and nutrient cycling remain poorly understood. This study examined the 24-year effects of mixed plantations combining Robinia pseudoacacia with native species (Prunus sibirica, Platycladus orientalis, and Pinus tabuliformis) on soil microbial functional diversity and gene assembly processes based on metagenomic functional profiles, and explored their relationships with soil multi-nutrient cycling. Compared to monocultures, mixed-species forests exhibited significantly higher microbial functional α-diversity (gene richness and Shannon index) and soil multi-nutrient cycling index, with the most pronounced effects in mixed-species forests combining Robinia pseudoacacia with Pinus tabuliformis. Mixed plantations also exhibited more diverse functional gene compositions and higher abundances of genes associated with nutrient cycling processes, including carbon decomposition, aerobic respiration, fermentation, carbon and nitrogen fixation, nitrification, organic phosphorus mineralization and inorganic phosphorus solubilization. Spatial and environmental distance-decay relationships of functional gene composition were more pronounced in monocultures than in mixed-species forests. In contrast, gene assembly in mixed-species forests was more strongly influenced by stochastic processes, reflecting greater variability in functional composition and reduced environmental constraint. Furthermore, the richness of core genes in the functional network was a strong determinant of soil multi-nutrient cycling in mixed-species forests, highlighting the pivotal role of these keystone functions in sustaining biogeochemical processes. These findings emphasize that microbial functional communities should be prioritized in evaluating the ecological consequences of afforestation and provide guidance for optimizing mixed-plantation design to enhance soil function and ecosystem sustainability.

Graphical abstract

Keywords

multispecies forests / soil functional genes / nutrient cycling / afforestation

Highlight

	● Mixed plantations enhance microbial functional diversity and soil nutrient cycling.
	● Stochastic processes dominate gene assembly in mixed forests, boosting resilience.
	● Core genes in complex networks strongly drive nutrient cycling in mixed plantations.
	● Microbial functional traits key for evaluating afforestation ecological consequences.

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