Population genetic structure of wolves in the northwestern Dinaric-Balkan region.

The Balkan Peninsula and the Dinaric Mountains possess extraordinary biodiversity and support one of the largest and most diverse wolf (Canis lupus) populations in Europe. Results obtained with diverse genetic markers show west-east substructure, also seen in various other species, despite the absence of obvious barriers to movement. However, the spatial extent of the genetic clusters remains unresolved, and our aim was to combine fine-scale sampling with population and spatial genetic analyses to improve resolution of wolf genetic clusters. We analyzed 16 autosomal microsatellites from 255 wolves sampled in Slovenia, Croatia, Bosnia and Herzegovina (BIH), and Serbia and documented three genetic clusters. These comprised (1) Slovenia and the regions of Gorski kotar and Lika in Croatia, (2) the region of Dalmatia in southern Croatia and BIH, and (3) Serbia. When we mapped the clusters geographically, we observed west-east genetic structure across the study area, together with some specific structure in BIH-Dalmatia. We observed that cluster 1 had a smaller effective population size, consistent with earlier reports of population recovery since the 1980s. Our results provide foundation for future genomic studies that would further resolve the observed west-east population structure and its evolutionary history in wolves and other taxa in the region and identify focal areas for habitat conservation. They also have immediate importance for conservation planning for the wolves in one of the most important parts of the species' European range.

Combining phylogenetic and demographic inferences to assess the origin of the genetic diversity in an isolated wolf population.

The survival of isolated small populations is threatened by both demographic and genetic factors. Large carnivores declined for centuries in most of Europe due to habitat changes, overhunting of their natural prey and direct persecution. However, the current rewilding trends are driving many carnivore populations to expand again, possibly reverting the erosion of their genetic diversity. In this study we reassessed the extent and origin of the genetic variation of the Italian wolf population, which is expanding after centuries of decline and isolation. We genotyped wolves from Italy and other nine populations at four mtDNA regions (control-region, ATP6, COIII and ND4) and 39 autosomal microsatellites. Results of phylogenetic analyses and assignment procedures confirmed in the Italian wolves a second private mtDNA haplotype, which belongs to a haplogroup distributed mostly in southern Europe. Coalescent analyses showed that the unique mtDNA haplotypes in the Italian wolves likely originated during the late Pleistocene. ABC simulations concordantly showed that the extant wolf populations in Italy and in south-western Europe started to be isolated and declined right after the last glacial maximum. Thus, the standing genetic variation in the Italian wolves principally results from the historical isolation south of the Alps.

Monitoring the effective population size of a brown bear (Ursus arctos) population using new single-sample approaches.

The effective population size (N(e) ) could be the ideal parameter for monitoring populations of conservation concern as it conveniently summarizes both the evolutionary potential of the population and its sensitivity to genetic stochasticity. However, tracing its change through time is difficult in natural populations. We applied four new methods for estimating N(e) from a single sample of genotypes to trace temporal change in N(e) for bears in the Northern Dinaric Mountains. We genotyped 510 bears using 20 microsatellite loci and determined their age. The samples were organized into cohorts with regard to the year when the animals were born and yearly samples with age categories for every year when they were alive. We used the Estimator by Parentage Assignment (EPA) to directly estimate both N(e) and generation interval for each yearly sample. For cohorts, we estimated the effective number of breeders (N(b) ) using linkage disequilibrium, sibship assignment and approximate Bayesian computation methods and extrapolated these estimates to N(e) using the generation interval. The N(e) estimate by EPA is 276 (183-350 95% CI), meeting the inbreeding-avoidance criterion of N(e) > 50 but short of the long-term minimum viable population goal of N(e) > 500. The results obtained by the other methods are highly consistent with this result, and all indicate a rapid increase in N(e) probably in the late 1990s and early 2000s. The new single-sample approaches to the estimation of N(e) provide efficient means for including N(e) in monitoring frameworks and will be of great importance for future management and conservation.

Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples.

Historical samples, like tanned hides and trophy skulls, can be extremely important for genetic studies of endangered or elusive species. Selection of a sampling protocol that is likely to provide sufficient amount and quality of DNA with a minimum damage to the original specimen is often critical for a success of the study. We investigated microsatellite genotyping success of DNA isolated from three different types of Eurasian lynx historical samples. We analysed a total of 20 microsatellite loci in 106 historical samples from the endangered Dinaric lynx population, established from re-introduction of three pairs of lynx in 1973 from Slovakian Carpathians. Of the three tested sample types, turbinal bone and septum from the nasal cavity of the trophy skulls had the lowest percentage of samples successfully genotyped for all 20 microsatellite loci. Footpad samples, collected using a cork drill, exhibited better results in polymerase chain reaction amplification and genotyping than samples of footpad epidermis cut with a scalpel. We report simple and efficient sampling protocols, which could be widely applied for future studies utilizing historical samples.

Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification.

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.