Different sampling strategies yield different microbial communities
The sampling strategies compared in this study (homogenizing tissue before subsampling and homogenizing tissue after subsampling ) are common methods found in the literature for characterizing plant-associated microbial communities (Jumpponen and Jones 2009; Zimmerman and Vitousek 2011; Busby et al ., 2016; Barge et al ., 2019). Procrustes analyses and community overlap between sampling strategies demonstrated that different strategies can capture disparate microbial communities within plants, with the extent of these differences depending on the community targeted and plant tissue type sampled. In FFE as well as bacterial and non-AM fungal communities in roots, subsamples from the same plant resulted in completely different sets of species recovered, illustrating the severe undersampling that is inherent to each of these strategies. With these sampling strategies, we are undoubtedly sacrificing power and accuracy to characterize the subtler aspects of plant microbiome interactions, despite often seeing community differences across landscapes, treatments or seasons.
Richness was higher when homogenizing before subsampling for bacteria only, despite differences observed in composition for all groups. It is perhaps surprising that homogenizing plant tissues before subsampling did not recover more species than homogenizing after subsampling for fungi as well, because with the former approach, more plant tissue is initially represented. Indeed, sample pooling or homogenizing before subsampling has resulted in a higher richness of soil fungi compared to equally sized individual samples (Song et al ., 2015). In the Songet al . study they also found that multiple individual subsamples, rather than the single homogenized subsample, resulted in higher richness. This may suggest that the scale at which we are physically able to break down the particle size of plant tissues, as opposed to soil, is not always fine enough to sufficiently homogenize the fungi within. Because of this, plant-associated microbial communities may require a greater sampling effort than soil microbes. Additionally, the removal of low-abundant SVs did not result in differences in richness between the two sampling strategies for any microbial group (data not shown), suggesting that neither strategy is better at capturing rare species. Although this study was performed only on milkweed plants, we believe that these results are applicable to other plant species as well. The richness reported here is similar to other studies of plant-associated microbes (e.g. Furtado et al. , 2019; Kuźniaret al ., 2020), indicating that differences in subsamples were not due to extreme richness of milkweed-associated microbes.