We are nearing the 20-year anniversary of a groundbreaking paper which details how microsatellite marker development in Lepidoptera is “extremely difficult for no apparent reason.” How far have we come in these past 20 years? Microsatellites are still the marker of choice in many population genetics studies for their ease of use, high degrees of polymorphism, species-specificity, and low cost. The rise of next-generation sequencing technologies (e.g. 454, Illumina, PacBio, etc.) has greatly advanced our abilities to generate many microsatellite markers per species. In this paper, we summarize the improvements in marker development using next-generation technology. Using case studies, we review the use and implementation of microsatellite markers in different conservation programs. Lastly, we provide a guide to data interpretation of microsatellite data generated for butterflies, with the goal of supporting student researchers and conservation practitioners in evaluating the meaning in their data.

We investigated the genetic structure and diversity between populations of a rare butterfly, the Florida duskywing (Ephyriades brunnea floridensis E. Bell and W. Comstock, 1948) (Lepidoptera: Hesperiidae) across a network of South Florida pine rockland habitat fragments. Based on 81 individuals from seven populations and using multiple polymorphic microsatellite loci, our analyses support the presence of mainland Florida (peninsular) and Florida Keys (island) population groupings, with a moderate, asymmetrical gene flow connecting them, and the presence of private alleles providing unique identities to each. We additionally found that despite a prevalence in many Lepidoptera, the presence of Wolbachia was not identified in any of the samples screened. Our findings can be used to inform conservation and recovery decisions, including population monitoring, organism translocation, and priority areas for management, restoration or stepping-stone creation to help maintain the complex genetic structure of separate populations.