## 2 Materials and Methods  
This study was conducted in two study areas in Sweden (Sweden North and Sweden South), and two study areas in Norway (Hedmark North and Hedmark South, Figure 1). Sweden North consisted of red fox individuals from Dalarne County, while Sweden South consisted of red fox individuals from the Counties of Sodermanland and Ostergotland. Hedmark North consisted of red fox individuals from Amot, Trysil, and Engerdal municipalities; and Hedmark South consisted of red fox individuals from Hamar, Elverum, and Stange municipalities (Figure 1). Our Swedish sampling area contained three of the five areas where _Echinococcus multilocularis_ had been detected [27]. A total of 150 individual red fox samples were used in this study, with 101 sampled from Hedmark County in Norway and 49 sampled from Dalarna, Sodermanland, and Ostergotland Counties in Sweden (Table 1, Figure 1). Individual red fox samples were grouped into the nearest geographical area to ensure a large enough sample size. Samples from Hedmark South consisted of 52 individuals and samples from Hedmark North consisted of 49 individuals. For Sweden, 24 samples were collected for Sweden North and 25 samples were collected for Sweden South. Red fox samples were either collected as hair from live trapped individuals (\(n=33\)) or collected as tissue samples from shot foxes (\(n=117\)). The sampling was opportunistic in that we collected material from ongoing studies. Sampling was performed from 2012 to 2013. DNA was extracted from selected hair root or dissected muscle samples according to standard protocols [28] involving lysis with proteinase K, sodium dodecyl sulfate (SDS), phenol/chloroform extraction, and ethanol precipitation. Other dissected muscle samples were processed for DNA extraction according to the manufacturer's instructions using the MasterPure Complete DNA and RNA Purification Kit (Epicentre, Madison, WI, USA). Figure 1: Geographical localization of the four study areas in Norway and Sweden. Sites where red fox samples were collected within the study areas is marked with a star. Fifteen established microsatellite markers and one polymorphic sex-linked locus previously employed in multiplex [29; 30; 31; 32; 33] and 11 relatively new markers tested before only in uniplex [33] were processed groupwise using the MPPrimer software package [34] to design primers for increased degrees of multiplexing. Newly designed primers for all 27 loci were tested and optimized for use in three multiplex panels consisting of 11, 10, and 6 primer sets. Marker and primer information is provided in Supplementary Table S1. All multiplex PCR reactions using the final primer concentrations listed in Table S1 were composed as follows: 1 \(\times\) Buffer B1, 1.5 mM MgCl2, 0.1 mM deoxyribonucleoside triphosphates (dNTPs), 0.1 mg/mL bovine serum albumin (BSA), 10-100 ng genomic DNA, and 1 U of Hot FIREpol DNA polymerase (Solis Biodyne, Tartu, Estonia); the thermocycler profile consisted of one cycle of 10 min at 95 \({}^{\circ}\)C followed by 35-40 cycles of 95 \({}^{\circ}\)C for 15 s, 60 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 1 min; and a final extension step of 72 \({}^{\circ}\)C for 60 min concluded the profile. Reactions were analyzed using capillary electrophoresis with an ABI 3100 \(\times\)1 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using five-dye spectral calibration. Allele binning and genotype scoring were performed using GeneMapper 4.0 (Applied Biosystems). All samples were genotyped for all markers in the three panels in at least two independent multiplex reactions; uniplex reactions were also performed for some few recalcitrant markers and/or samples. Microsatellites were analyzed using MICRO-CHECKER 2.2.3 [35] to detect signs of stutter and null alleles when using the total material. In addition, to search for specific loci being candidates under directional or balancing selection, compared to neutral expectations, all microsatellites were analyzed in Arlequin 3.5.2.2 [36].