Molecular Ecology Resources (2008) 8, 1285–1287 doi: 10.1111/j.1755-0998.2008.02356.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Isolation and characterization of 10 highly polymorphic di- and trinucleotide microsatellite markers in the mayfly Ameletus inopinatus (Ephemeroptera: Siphlonuridae) KATHRIN THEISSINGER,*‡ KEVIN A. FELDHEIM,† JULIA TAUBMANN,‡ ALFRED SEITZ‡ and STEFFEN U. PAULS*† *Research Institute Senckenberg, Department of Limnology and Conservation, Clamecystrasse 12, 63571 Gelnhausen, Germany, †Pritzker Laboratory for Molecular Systematics and Evolution, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605-2496, USA, ‡Institute of Zoology, Department of Molecular Ecology, Johannes Gutenberg University, J.J. Becherweg 13, 55128 Mainz, Germany Abstract We describe the isolation of ten polymorphic microsatellite loci from the mayfly Ameletus inopinatus. Loci had di- or trinucleotide repeat motifs and were highly variable with three to 17 alleles (mean = 7.15). Observed heterozygosity ranged from 0.143 to 0.905. One locus (Ami_202) showed significant deviation from Hardy–Weinberg equilibrium in one population, but no evidence for null alleles. One locus (Ami_73) was significantly linked with three other loci. The remaining nine loci should prove highly informative for population genetic studies. Keywords: mayfly, microsatellite enrichment, PCR Received 17 July 2008; revision accepted 30 July 2008 The mayfly Ameletus inopinatus Eaton 1887 inhabits mountain ranges of high elevation on the British Isles and in Central Europe. In northern Eurasia, it is known from Scandinavia to Western Siberia, where it also occurs at lower altitudes. It is considered a representative of the Eurasic, boreo-montane biome type (Haybach 2003). Its absence in the Alps and the Pyrenees indicates that A. inopinatus was not distributed in Europe during the last ice age (Haybach 2003), and is a recent colonizer of Central Europe with postglacial retreat to higher elevations (Malicky 1988; Haybach 2003). Ameletus inopinatus can be found in slow running water systems as well as little puddles or big lakes, exhibiting a high plasticity in niche occupancy. Here we present the development of polymorphic microsatellite markers which will be used to examine the fine-scale population genetic structure of A. inopinatus. Microsatellite markers were developed using an enrich- ment protocol developed by Glenn & Schable (2005). We extracted genomic DNA (gDNA) from one individual of A. inopinatus from the Andertal Moor, St. Lorenzen, Austria, using the DNEasy tissue kit (QIAGEN). Approximately 4 μg gDNA was digested with BstUI and XmnI (New England Biolabs), and SuperSNX24 linkers were ligated onto the gDNA fragments. Linkers serve as priming sites for subsequent polymerase chain reactions (PCR) throughout the protocol. Different combinations of biotinylated tri- nucleotide and tetranucleotide probes were hybridized to gDNA. The biotinylated probe-gDNA complex was added to streptavidin-coated magnetic beads (Dynabeads M-280, Invitrogen). This mixture was washed twice with 2× SSC, 0.1% SDS and four times with 1× SSC, 0.1% SDS at 53 °C. Between washes, a magnetic particle-collecting unit was used to capture the magnetic beads. After the last wash, enriched fragments were removed from the biotinylated probe by denaturing at 95 °C and precipitated with 95% ethanol and 3 m sodium acetate. To increase the amount of enriched fragments, a ‘recovery’ PCR was performed in a 25-μL reaction containing 1× PCR buffer (Roche), 1.5 mm MgCl 2 , 1× BSA, 0.16 mm of each dNTP, 0.52 μm SuperSNX24 forward primer, 1 U Taq DNA polymerase, and approxi- mately 25 ng enriched gDNA fragments. Thermal cycling was performed in an MJ Research DYAD as follows: 95 °C for 2 min followed by 25 cycles of 95 °C for 20 s, 60 °C for Correspondence: Steffen U. Pauls, Department of Entomology, University of Minnesota, 219 Hodson Hall, 1980 Folwell Ave, St Paul, MN 55108, USA. Fax: +1 612 625 5299; E-mail: pauls497@umn.edu.