Sampling constrains our characterization of plant microbiomes: Are we
losing site of the trees in the forest of microbial diversity?
Abstract
Plants host diverse microbial communities, but there is little consensus
on how we sample and characterize these communities, and this has
unknown consequences. Using root and leaf tissue from 20 showy milkweed
(Asclepias speciosa) plants in the field, we compared two common
sampling strategies by: 1) homogenizing after subsampling a small
proportion of tissue (30 mg), and 2) homogenizing bulk tissue before
subsampling 30 mg. Due to potential differences in richness and spatial
distributions among microorganisms, we targeted bacteria, arbuscular
mycorrhizal (AM) fungi and non-AM fungi in roots, as well as foliar
fungal endophytes (FFE) in leaves. We also sampled FFE twice across the
season using sampling strategy 1 to assess temporal dynamics, and we
extracted DNA from all remaining homogenized bulk leaf tissues to
determine the extent of potential undersampling. Bacterial richness was
higher under sampling strategy 2, and all microbial groups except AM
fungi differed in composition. Community overlap between the two
sampling strategies increased when rare taxa were removed, but FFE and
bacterial communities remained more different than alike and showed
largely non-overlapping communities within individual plants. Increasing
the extraction mass 10x also increased FFE richness
~10x, confirming the severe undersampling indicated in
the sampling strategy comparisons. Even so, seasonal patterns in FFE
communities were apparent, suggesting that strong drivers may be
identified despite severe undersampling. Our findings highlight that
current sampling practices poorly characterize many microbial groups and
that increased sampling intensity is necessary to identify subtler
patterns and to increase the reproducibility of studies.