3. Results and discussion
(a) Pedigree, inbreeding coefficients and litter sizes
We traced the complete ancestry for both male and female in 24 of the 28 breeding wolf pairs registered during the period 1983-2002, constructing the first complete pedigree back to its founders that has been published for a wild mammal population (Keller & Waller 2002), and calculated inbreeding coefficients (F; figure 1). The first founding wolf pair reproduced for 3 years (1983-1985) until the female was shot in 1985, but offspring from this pair continued to breed within the same territory until 1994 through incestuous matings (figure 1). In 1991, an immigrant male mated with a daughter of the first breeding pair and contributed to the large variation in the inbreeding coefficient F in the population (0.00-0.41). Apart from the early incestuous matings, we recorded only two later cases of full sibling pairings (pairs O and U in figure 1). Nevertheless, most animals born after 1997 have inbreeding coefficients close to or higher than 0.25, a level corresponding to full sibling mating (figure 1).
The sizes of winter litters for first breeding pairs were strongly affected by the inbreeding coefficient of the pups (n=24, R2=0.39, pfigure 2), while the inbreeding coefficients of the mother (partial R2=0.04, p=0.23), of the father (partial R2=0.07, p=0.11), the age of the mother (partial R2=0.09, p=0.076) and time (partial R2=0.10, p=0.058) did not contribute significantly to the same model. After removing offspring inbreeding coefficients, there was indeed an effect of the mother's inbreeding coefficient (n=24, R2=0.27, p=0.01), but not from the father's (partial R2=0.02, p=0.41), nor from age of the mother (partial R2=0.08, p=0.13) or time (partial R2=0.10, p=0.075). Inbreeding of the father (R2=0.06, p=0.25), or time (R2=0.001, p=0.88) had no effect alone. The inbreeding coefficient increased over the years for pups and mothers (r=0.49, p=0.016 and r=0.58, p=0.003).
We are confident that the demonstrated inbreeding effect was not a by-product of association with coincidental trends in the environment, for example weather or food, as time itself had no effect on litter size. Change in prey availability can also be discarded considering that the number of moose (Alces alces), the most important prey for wolves in Scandinavia (Sand et al. in press), stayed high (greater than 1 moose per km2 in all wolf territories) during the study period (Hörnberg 2001). It was well above the threshold (0.5 moose per square kilometre) under which wolf populations are reported to be affected (Messier, 1994).
(b) Effects of inbreeding on demography
The quantitative inbreeding effect was a reduction of 1.15 winter pups per litter for each increase of 0.1 in the F for pups (winter litter size=6.54-11.51F; figure 2). In our population model, an increase of offspring inbreeding coefficient F of 0.1 reduced the growth rate from 1.29 to 1.21, assuming all litters were affected equally by inbreeding. Zero population growth (=1) would be reached at an average F of 0.48. Our chosen fitness measure, winter litter size, actually represents a combination of fecundity and early survival. It is possible that more fitness components, for example, yearling or adult survival, could be affected, which would make the demographic consequences even more severe. The Scandinavian wolf population thus may have a gloomy future unless it can be purged of its genetic load through natural selection, or receives new genetic variation from outside. However, the effectiveness of purging in small populations has been questioned (Hedrick & Kalinowski 2000), and the probability of natural immigration also seems low, as no new immigrants have appeared in the last 13 years. In an earlier report concerning this population, it was claimed that the male immigrating in 1991 'rescued' the population (Vila et al. 2003). Our interpretation is that before this male arrived there was no population but just a strongly inbred family. The arrival of this newcomer allowed young wolves to find partners outside of their own family, and this sparked off a rapid initial increase, but has not prevented the succeeding inbreeding.
(c) Conservation implications
This study has general implications for the 'small population paradigm' (Caughley 1994), and is especially relevant for the conservation of large carnivores. These are charismatic species with large public support, but as powerful predators also highly controversial, they are often forced into small fragmented populations. The wolf could be useful as a model species for this dilemma, in part because there are several studies of inbreeding in captive populations of this species. A captive Swedish wolf population, partly founded from the same source as our study population, also expressed severe inbreeding effects (Laikre 1999), while in two American captive populations of red and Mexican wolf, no effects were noted on demographic parameters (Kalinowski et al. 1999), although effects on body size were noted in the Mexican wolves (Fredrickson & Hedrick 2002). The genetic load of our wild population (6.043.44), 95% CI) was substantially heavier than that for the red and Mexican wolves (0.63 and 0.71, respectively), and also clearly higher than the average estimate of 3.14 in a study of 40 captive mammal populations (Ralls et al. 1988). This indicates that impact of inbreeding can vary substantially, even within the same species, depending on the random subset of genes from the source population drawn by the founders, and succeeding random drift. For the famous wild wolf population on Isle Royale in Minnesota, USA, 50 years after founding by only two individuals there still is only some indirect evidence of demographic effects of inbreeding (Wayne 1991; Peterson et al. 1998), but a detailed analysis of inbreeding, of the type demonstrated in this paper, has not been used.
The conservation implication for our study population is that genetic exchange with the source population should be strongly promoted. In the meanwhile, the close demographic and genetic monitoring of the population should be continued. The potential for further exploration of inbreeding effects on more demographic parameters should be pursued.
Funding was provided to O.L., H.A. and H.S. by the Swedish Environmental Protection Agency, the World Wide Fund for Nature (Sweden), the Swedish Association for Hunting and Wildlife Management, the private foundation 'Olle och Signhild Engkvists Stiftelser', to H-C.P. and P.W. by the Norwegian Research Council, the Norwegian Directorate for Nature Management, the Norwegian Institute for Nature Research, Norwegian Ministry of Environment, and the Hedmark University College and to S.B. by the Swedish Research Council. We thank Philip Hedrick, John Vucetich and Josephine Pemberton and two anonymous referees for insightful comments and suggestions on an earlier version of the manuscript.