Correlation between empirical and estimated allele frequencies calculated from 20 (left) and 50 (right) individuals. Each point represents the mean allele frequency calculated from nine replicates within each of the four pools (= four points per locus). Empirical allele frequencies were determined from the full dataset (N = 514). R2 is the adjusted R2 value from a linear regression (red line). NB. Note that some points are far removed from the regression line; these are cases where clusters in duplicated regions of the genome have been placed incorrectly due to the small number of reference individuals.