2. Materials and methods
(a) Field data
The wolf population has been monitored since 1978, based on snow tracking and, from 1998, also on radio telemetry. Territorial pairs were distinguished and the number of animals in packs counted (Wabakken et al. 2001). A 'pair' is two breeding adults producing offspring together, while a 'pack' is the total number of individuals in a family, for example, the pair and its dependent offspring. The 'territory' is the geographical area where the pair is living. As a fitness measure, we used the number of pups per litter surviving until the first winter after birth ('winter litter size'). We used data for first-born litters of each breeding pair only, because for subsequent litters, tracks from pups of the year could not be separated from those of yearlings and older philopatric siblings (Mech 1970). In darted wolves, ageing was based on the growth zone in the tibia for pups and tooth wear for adults, and in retrieved dead wolves annual tooth cementum layers (C1) were counted.
(b) Genetic analyses
Samples were derived from the blood of captured wolves, the muscle of dead wolves ('tissue'), from oestrus blood on snow and from scats. Genomic DNA from tissue was isolated using standard phenol/chloroform-isoamylalcohol extraction protocols. Two isolates were extracted from faecal samples with a Qiamp DNA stool mini kit (Qiagen, Valencia, CA, USA). Faecal and oestrus blood samples were extracted in a separate workspace treated with ultraviolet light to avoid contamination (Sarkar & Sommer 1990). Negative extraction controls were used throughout.
We scored tissue samples for allelic variation at 32 autosomal microsatellite loci, and faecal samples on a subset of 16 (for details see Electronic Appendix). To minimize scoring errors associated with low quality DNA (Taberlet et al. 1999), faecal samples were amplified a minimum of four times (twice per isolate). Heterozygotes were accepted if both alleles were present in two amplifications and homozygotes if four positive amplifications showed only one allele. If neither condition was met, samples were re-amplified. Problematic samples were amplified up to 10 times. In the few samples, where an ambiguous result still occurred, we recorded a half-locus (Miller et al. 2002).
The pedigree was determined by parentage analysis. We used material from 163 wolf individuals; 113 of these were based on muscle from dead wolves or blood from anaesthetized wolves, the rest from faeces and/or from oestrus blood found in snow. A missing genotype of one parent was reconstructed from genotypes of the known parent and pups of that pair. Of the 48 breeding wolves in the pedigree used in the analysis, genotypes of 16 were reconstructed, 25 were based on tissue (muscle or blood drawn directly from the animal) and seven were based on faeces/oestrus blood. The three incestuous pairs in the period 1987-1990 were completely reconstructed from genotypes of 10 wolves born during this period. Here several alternatives were possible. We chose the most parsimonious alternative, but tested all possible alternatives, and none changed the results of this study other than marginally. Inbreeding coefficients were calculated with the software PEDIGREE VIEWER 5.0 (© Brian and Sandy Kinghorn).
(c) Statistical analyses
We used parametric statistics (ANCOVA) in the analyses of inbreeding effects, including the interaction terms between the independent variables in the initial model. Ages of breeding females were treated as a two state variable: young (2-3 years) and old (4 years or older). Genetic load is expressed in terms of lethal equivalents, based on viability data (Kalinowski & Hedrick 1998). We calculated an analogous parameter, litter-reducing equivalents, by regressing litter size (Wi) against the inbreeding coefficient (fi) using the relationship lnWi=ln(W0-Bfi), where W0 is the litter size for outbred litters (f0). Inbreeding effects on population growth rate () were tested using a Leslie matrix with five age classes. We used data from our study population for survival and reproduction, adjusted to give a baseline growth rate similar to the one observed in the period 1991-2000 of =1.29 (Wabakken et al. 2001).
For further details on material and methods, see the Electronic Appendix.