Acta Theriol (2011) 56:117–122 product, one primer of each pair was labeled with a fluorescent dye (FAM or JOE) on the 5′-end, which allowed analyses with the use of an ABI 3100 Avant automated sequencer (Applied Biosystems). Statistical analysis The data were statistically analyzed with the application of two different approaches to the genetic structure of the Łęgucki Młyn/Popielno hybrid zone: 1. First, the samples from the nine sites were considered as nine geographical populations; 2. Second, the nine geographical populations were grouped according to their location in the area occupied. Populations P, LK, L, NM, and ZL situated in the area of the Łęgucki Młyn race formed group I; populations KI, KII, Z, and KM from the area of the Popielno race formed group II. For the geographical populations, allelic richness (A), the observed (HO), and expected (HE) heterozygos- ity were computed for all loci using SAS v. 8.0 (User’s Guide 2000) and ARLEQUIN v. 3.1 (Excoffier et al. 2005). Inbreeding coefficients (FIS) were estimated to indicate the within-population heterozygote deficiency due to non- random mating. FIS was calculated per locus, overall loci and per population for the geographical populations. Fixation index (FST) was estimated per locus and for overall micro- satellite loci. Permutations were used for testing the values of FIS and FST for significant departure from zero. These tests were done using 10.000 permutations of alleles within samples (FIS) and 10.000 permutations of genotypes among samples for FST (Arlequin v. 3.1). 119 Micro-Checker program v. 2.2.3 (Van Oosterhout et al. 2004) was used to identify genotyping errors due to alleles. To estimate the gene flow between the races, conventional F-statistics were used (Weir and Cockerham 1984). These statistics are recommended rather than R- statistics for studies with small sample sizes (ten individ- uals or less) and limited numbers of loci (less than 20) (Gaggiotti et al. 1999). The genetic structure of populations was calculated using the analysis of molecular variance (AMOVA) as implemented in Arlequin v. 3.1. A hierarchical analysis of variance divided the total variance into different parts of covariance which were then used to calculate different fixation indices (Excoffier 2000). Significance levels of the different fixation indices were obtained by permuting genotypes for FST, among geographical populations; for FSC, among geographical populations within the races; and for FCT, among chromosomal races (Excoffier et al. 1992). Results Polymorphism and genetic variability Most autosomal loci displayed a high level of polymor- phism. One hundred and thirty six alleles were found at seven autosomal microsatellite loci. The most variable locus (L97) had 30 alleles, whereas the least variable one (L67) had 12 alleles (Table 1). The average allelic richness (A) in the different geo- graphical populations ranged from 2.75 to 10.11, and was smallest in population KM and greatest in population Z (Table 2). Observed heterozygosities (HO) per locus ranged Table 1 Number of alleles (na), observed heterozygosity (HO), expected heterozygosities within (HS) and between (HT) populations, inbreeding coefficient (FIS), and fixation index (FST) estimated for geographical populations and both races Locus L9 L14 L33 L45 L67 L68 L97 All loci *P<0.01 na 27 14 26 13 12 14 30 136 HO 0,86 0.82 0.68 0.57 0.68 0.85 0.79 0.75 HS 0.83 0.79 0.80 0.69 0.68 0.79 0.75 0.76 HT 0.94 0.89 0.93 0.78 0.73 0.87 0.94 0.87 FIS P Geographic populations Race group 0.08 0.07 0.27* 0.28* 0.08 0.00 0.15* 0.13* 0.0200 0.0986 0.0000 0.0000 0.1025 0.5489 0.0009 0.000 FST 0.010 0.023 0.020 0.033 0.011 0.008 0.038* 0.020 Łg FST 0.002 0.040 0.002 0.063 0.013 0.030 0.043 0.027 Po FST 0.034 0.019 0.033 −0.055 −0.019 −0.030 −0.020 0.003