Mature queens of single-queen colonies such as Pogonomyrmex and L. niger are almost impossible to collect due to the depth and size of the colonies. Fortunately, the genotype of a queen can be determined by genotyping male allates from the colony. As males are the product of unfertilized eggs, a sample size of 6 will cover 99% of the queen's genome. The genotype of the father(s) of the colony, as well as the queen's, can also be reconstructed by sampling workers. In both cases, such sampling will not negatively impact large colonies and represents a benign way to monitor the genetic structure of extant, wild long-lived populations.
Social insects should be sampled at the level of colony or group of colonies for molecular studies. When the queen has been fertilized by only one male, all of her daughters are full sisters and share the same haploid father and diploid mother. Thus, workers from a mono-gynous colony headed by a singly mated queen are identical for 75% of their genome. This can lead to pseudo-replication, as colonies represent closely related families with related genetic backgrounds. Measurements of a group of 25 workers consisting of 5 workers from 5 different colonies cannot be considered 25 independent samples because of within-colony relatedness. Instead, this example contains measurements of 5 independent colonies, each consisting of the average from 5 workers. Thus, in most cases, worker colony samples should be averaged and one value taken per colony for statistical tests.
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