Sample size shapes metabarcoding-driven biodiversity assessments across body sizes in soil

Lu Wang , April Lyn Leonar, Simone Cesarz, Nico Eisenhauer, Antonis Chatzinotas, Stephanie Jurburg

Understanding how sample size influences biodiversity detection across taxonomic
groups differing in body size is critical for designing robust and cost-efficient
metabarcoding studies of soil eukaryotes. Using a soil mass gradient (0.25-32 g)
combined with a universal 18S rRNA metabarcoding approach, we quantified how
sample mass shapes diversity estimates across eukaryotic taxa. Diversity metrics
(richness and inverse Simpson diversity; q = 0, 2) and community dispersion exhibited
clear body size-dependent responses. Larger-bodied taxa (e.g., Nematoda, Collembola, Insecta) showed pronounced increases in detected diversity and reduced
community dispersion with increasing soil mass, indicating that small soil samples fail
to capture their full diversity. Conversely, microeukaryotic groups such as fungi and
protists displayed weak or even negative relationships with increasing soil mass,
implying limited improvement in detection with increased sampling effort. These findings demonstrate the interactive effect organismal body size and sampling
approach on the detection of soil biodiversity. We suggest optimal soil sample sizes for different groups of soil biota, and propose that a taxon-specific analytical framework can enhance both the ecological representativeness and cost efficiency in
metabarcoding-based soil biodiversity assessments and monitoring.