Delimitation and identification of microeukaryotic OTUs
The three different OTU generation methods infer somewhat different
community compositions from sequenced long read rDNA environmental DNA
amplicons. For instance, the methods resulted in very different
estimates of total non-singleton OTU richness, ranging from 1,336 OTU_A
detected based on inference of ASVs, compared to 2,046 OTU_S and 2,488
OTU_C for sequence similarity-based clustering using single-linkage or
centroid-based clusters respectively (Table 1). The OTU_A dataset
represents only 28% of the raw reads while the two other methods were
comparable, capturing 81–83% of the reads into OTUs (Table 1). After
pooling all reads, OTU accumulation curves for the three methods
indicate that sequencing depth was sufficient to reach comparable
asymptotic OTU richness estimates in both mesic-dry and wet soil
conditions (Fig. S4a). For individual samples, however, increased
sequencing depth would be expected to increase OTU detection for all
three methods (Fig. S5). Further, the estimated asymptotic OTU richness
increased for all methods when analyzing the pooled reads based on
number of samples (Fig. S4b), indicating that taking additional samples
would be expected to increase the number of detected taxa for all
methods. Across the three methods, 38–42% of the detected OTUs were
taxonomically assigned to kingdom Fungi. Based on read abundance, the
estimated proportion of fungi ranged from 34% for OTU_As compared to
just over 40% for the two other methods (Table 1). Protists thus
dominated the sequenced microeukaryotic soil community at this site.