Fig. 1

Sample collection and preparation, data collection, and post-processing are inextricably linked in MME techniques and can all potentially affect estimates of diversity. The effect of researcher choices on S, N (middle row), and SAD (bottom row) during data generation is shown below each step. The true diversity in two samples is shown in red and blue, and the measured diversity is shown as dotted lines. Technical errors during sample collection and storage can increase S (e.g., due to non-specific contamination [19]), resulting in higher estimates for S and steeper SAD (a). In contrast, sample preparation can reduce the detectability of certain molecular entities (e.g., during PCR amplification in metabarcoding [20]) resulting in a lower S and flatter SAD (b). Technical limitations on the number of observations are imposed by some of the data collection instruments used (e.g., sequencer), placing technical limits on N, and potentially resulting in more even communities (i.e., a flatter SAD) (c). During processing, applying a less stringent species definition can result in reduced S and a flatter SAD (d)