Multiple extractions per plant revealed severe undersampling of
FFE
From the six leaves collected from plants in September 2016, we
extracted DNA from all remaining leaf tissue (1240-2460 mg per plant).
Despite increasing the processed plant tissue by two orders of
magnitude, species accumulation curves for individual plants still
failed to approach a plateau (Figure 5). This was true even when
clustering SVs at 98.5% using the LULU method. Post-clustering curation
using the LULU method removed fewer than 10% of SVs and did not change
the overall results. Where extractions from subsamples of 30 mg of leaf
tissue (6 discs from 6 different leaves) had recovered 7-26 total foliar
fungal SVs per plant, extracting from tissue representing our entire
bulk collection of 6 leaves per plant or 10x as much leaf tissue,
resulted in 76-142 total SVs per plant. The average richness recovered
by each extraction was 24 ± 8 SVs and the average number of unique SVs
added by each consecutive extraction was 12 ± 6 SVs. Extractions from
the same plant overlapped as little as 21% of SVs, showing that
extractions from the same homogenized plant tissue can vary
substantially, even when extracting from much larger volumes. Perhaps
not surprisingly, larger leaves contained more FFE SVs than did smaller
leaves, indicating that overall richness increases with plant size.