High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.

Community measures collected at a single instance or over a short temporal period rarely provide a complete accounting of biological diversity. The gap between such "snapshot" measures of diversity and actual diversity can be especially large in systems that undergo great temporal variation in environmental conditions. To adequately quantify diversity in these temporally varying ecosystems, individual measures of diversity collected throughout the range of environmental variation, i.e., temporal alpha-diversity measures, must be combined to obtain temporal gamma-diversity. Such a time-integrated gamma-diversity measure will be a much closer approximation of a site's true alpha-diversity and provide a measure better comparable to spatial alpha-diversity measures of sites with lower temporal variation for which a single or a few "snapshot" measures may suffice. We used aquatic-macroinvertebrate community-composition data collected over a 24-year period from a complex of 16 prairie-pothole wetlands to explore the rate that taxa accumulate over time at sites with differing degrees of temporal variation. Our results show that the rate of taxa accumulation over time, i.e., the slope of the species-time relationship, is steeper for wetlands with ponds that frequently dry compared to those with more-permanent ponds. Additionally, we found that a logarithmic function better fit species accumulation data for seasonally ponded wetlands whereas a power function better fit accumulations for permanently and semi-permanently ponded wetlands. Thus, interpretations of ecological diversity measures, and conservation decisions that rely on these interpretations, can be biased if temporal variations in community composition are not adequately represented.