Microsatellite analysis
A total of 408 samples belonging to 14 different populations were genotyped at twenty microsatellite markers (BT10, B11, B96, B100, B, B118, B119, B124, B126, BT26, BT28, B132, BT06, BT09, BT20, BTMS0033, BTMS0119, BTMS0131, BTMS0082, BTMS0124 and BTMS0045) previous developed for B. terrestris (Estoup, Scholl, Pouvreau, & Solignac, 1995; Estoup, Solignac, Cournet, Goudet, & Scholl, 1996; Reber-Funk, Schmid-Hempel, & Schmid-Hempel, 2006; Stolle, Rohde, Vautrin, Solignac, Schmid-Hempel, Schmid-Hempel,& Moritz, 2009). PCR amplification was performed in 20 µl volumes containing 2-2.5 µl of template DNA, 2 µl, 10X colorless buffer (Geneall), 1.2 µl HQ buffer (Geneall), 2 µl dNTPs (2.5mM), 0.3 µl (10 pmol) of each primer, and 2.5 U of Taq polymerase (Geneall). The PCR conditions consisted of an initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing temperature at 48-60 °C for 1 min, extension at 72 °C for 30 s and a final extension at 72 °C for 10 min. Allele sizing was performed using 96-part automatic capillary electrophoresis system (Fragment Analyzer-Advanced Analytical Technologies-AATI, Ames, Iowa, USA) by comparing alleles with an internal size standard (dsDNA 900 Reagent Kit, 35bp/500 bp).