Advancements in molecular technology have reduced the constraints that the grain of observation, or the spatial resolution and volume of the sampling unit, has on the characterization of plant-associated microbiomes. With discrete ecological sampling and massive parallel sequencing, we can more precisely portray microbiome community assembly and microbial recruitment to host tissue over space and time. Here, we differentiate rarefied community richness and relative abundance in bacterial microbiomes of Salvia lyrata dependent on three spatial depths, which are discrete physical distances from the soil surface within the rhizosphere microhabitat as a proxy for the root system zones. To assess the impact of sampling grain on rarefied community richness and relative abundance, we evaluated the variation of these metrics between samples pooled prior to DNA extraction and samples pooled after sequencing. A distance-based redundancy analysis with the quantitative Jaccard distance revealed that rhizosphere microbiomes vary in richness between rhizosphere soil depths. At all orders of diversity, rarefied microbial richness was consistently lowest at the deepest samples taken (approximately 4 cm from soil surface) in comparison with other rhizosphere soil depths. We additionally show that finer grain sampling (i.e., three samples of equal volume pooled after sequencing) recovers greater microbial richness when using 16S rRNA gene sequencing to describe microbial communities found within the rhizosphere system. In summary, to further elucidate the extent host-specific microbiomes assemble within the rhizosphere, the grain at which bacterial communities are sampled should reflect and encompass fine-scale heterogeneity of the system.
Keywords: 16S rRNA amplicon sequencing; Alpha diversity; Hill numbers; Indicator species analysis; Rhizosphere microbiomes; Salvia.