Population genetic studies reveal biodiversity patterns and inform about drivers of evolutionary differentiation and adaptation, including gene flow, drift and selection. This can advance our understanding and aid decision making regarding management and conservation efforts. Microsatellites have long been used in population genetic studies.Thanks to the development of newer techniques, sequencing approaches such as restriction site associated DNA sequencing (RADseq) are on their way to replace microsatellites for some applications. However, the performance of these two marker types in population genetics have rarely been systematically compared. We utilized three neutrally and adaptively differentiated populations of anadromous pike (Esox lucius) to assess the relative performance of microsatellites and RADseq with respect to resolution and conclusiveness of estimates of population differentiation and genetic structure. To this end, the same set of individuals (N = 64) were genotyped with both RADseq and microsatellite markers. To assess effects of sample size, the same subset of 10 randomly chosen individuals from each population (N = 30 in total) were also genotyped with both methods. Comparisons of estimated genetic diversity and structure showed that both markers were able to uncover genetic structuring. The full RADseq dataset provided the clearest detection of the finer scaled genetic structuring, and the other three datasets (full and subset microsatellite, and subset RADseq) provided comparable results. A search for outlier loci performed on the full SNP dataset poninted to signs of selection potentially associated with salinity and temperature, exemplifying the utility of RADseq to inform about the importance of different environmental factors. To evaluate whether performance differences between the markers are general or context specific, the results of previous studies that have investigated population structure using both marker types were synthesized. The synthesis revealed that RADseq performed as well as, or better than microsatellites in detecting genetic structuring in the included studies. The differences in the ability to detect population structure, both in the present and the previous studies, are likely explained by the higher number of loci typically utilized in RADseq compared to microsatellite analysis, as increasing the number of markers will (regardless of the marker type) increase power and allow for clearer detection and higher resolution of genetic structure.

In the present study, DNA-samples from a total of 64 individuals from three subpopulations of anadromous pike (Harfjärden, Lerviksbäcken and Oknebäck) that spawn in the Southeastern part of Sweden were used. The pike were captured using fyke nets placed in the inlet of the streams leading up to the spawning locations during the spawning migration of 2016. DNA sampling (fin clip) were conducted for each individual, and the individuals were then released back into the water. The DNA samples were immediately placed in separate 1.5 mL Eppendorf tubes with 70% ethanol and kept on ice until transported to the Kalmar Sound Laboratory of Linnaeus University in Kalmar, Sweden, where they were subsequently stored in a freezer (-20 °C) until the molecular work was conducted.

Tissue from each individual was ground using stainless steel beads (Next Advance, USA) and a Bullet Blender, and DNA was extracted using DNeasy blood and tissue kit (Qiagen, USA) according to the manufacturer’s instructions. All 64 samples were genotyped for 10 microsatellite loci (Elu 2, Elu 6, Elu 19, Elu 37, Elu 51, Elu 64, Elu 76, Elu 78, Elu 87 and Elu 276; Hansen, Taggart, & Meldrup, 1999; Miller & Kapuscinski, 1996; Miller & Kapuscinski, 1997), using Extract-N-Amp Blood PCR Kit for PCR amplification. Fragment analysis was performed by the Uppsala Genome Center (Uppsala, Sweden) using the size standard GeneScan™ 500 ROX™ (Thermo Fisher Scientific), and alleles were scored in the Microsatellite Plugin 1.4.4 in Geneious® Pro 11.0.3 (Biomatters Ltd., New Zealand).

All individuals were also genotyped for SNPs derived from Restriction-site Associated DNA sequencing (RADseq). For this, the extracted DNA was digested with HF EcoRI (New England Biolabs, USA) at 37°C for 16 hours. Size-selection of the fragments, paired-end library construction and sequencing was conducted by Science for Life Laboratory (SciLifeLab; Stockholm, Sweden). The 64 samples used in this study were sequenced along with additional samples (166 samples in total) on two lanes of Illumina HiSeq2500 with a 2 x 126 bp setup. The sequence data (raw reads) are publically available in the NCBI Sequence Read Archive (SRA)(Accession number: PRJNA586770).