Initial Cell Number Regulates Microscale Community Assembly and Structure

<title>Abstract</title> <p>Background Soil is the most complex biological material on Earth, with highly diverse microbial communities showing pronounced spatial heterogeneity at the microscale as small aggregates of tens to hundreds of cells within biofilms. We hypothesized that the initial population size (initial cell number) in these micro-aggregates profoundly affects community assembly and function. To test this hypothesis, we used flow cytometry to construct thousands of random communities with precisely controlled initial cell numbers (10, 50, 100, or 250 cells per well) across four different carbon sources that mimic natural soil microhabitat heterogeneity. We systematically evaluated the effects of initial cell number on community phenotypes, structure, diversity, and association networks. Results Initial cell number exerted carbon-source-dependent effects on growth but consistently showed a strong positive correlation with biofilm formation. After incubation, communities underwent sharp declines in diversity and converged toward structures dominated by Pseudomonas and Klebsiella. Pseudomonas species were strongly enriched at low initial cell numbers, whereas Klebsiella species increased at high initial cell numbers. Evenness increased with initial cell number; low-cell-number communities harbored more unique amplicon sequence variants and negative-correlation networks, while high-cell-number communities promoted shared variants and positive connections. This cell-number-dependent switching of dominant taxa extended to intra-genus niche differentiation at the amplicon sequence variant level. Conclusions Our findings identify initial cell number as a critical driver of microscale soil microbial community assembly. Initial cell number regulates competition–coexistence dynamics of dominant taxa, intra-genus ASV-level niche differentiation, and microbial association networks. This study highlights initial cell number as a key factor shaping community structure, diversity, and function in soil microbiomes.</p>