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.