Alexander Kopatz

A forensic DNA profiling system for Northern European brown bears (Ursus arctos)

A set of 13 dinucleotide STR loci (G1A, G10B, G1D, G10L, MU05, MU09, MU10, MU15, MU23, MU26, MU50, MU51, MU59) were selected as candidate markers for a DNA forensic profiling system for Northern European brown bear (Ursus arctos). We present results from validation of the markers with respect to their sensitivity, species specificity and performance (precision, heterozygote balance and stutter ratios). All STRs were amplified with 0.6ng template input, and there were no false bear genotypes in the cross-species amplification tests. The validation experiments showed that stutter ratios and heterozygote balance was more pronounced than in the tetranucleotide loci used in human forensics. The elevated ratios of stutter and heterozygote balance at the loci validated indicate that these dinucleotide STRs are not well suited for interpretation of individual genotypes in mixtures. Based on the results from the experimental validations we discuss the challenges related to genotyping dinucleotide STRs in single source samples. Sequence studies of common alleles showed that, in general, the size variation of alleles corresponded with the variation in number of repeats. The samples characterized by sequence analysis may serve as standard DNA samples for inter laboratory calibration. A total of 479 individuals from eight Northern European brown bear populations were analyzed in the 13 candidate STRs. Locus MU26 was excluded as a putative forensic marker after revealing large deviations from expected heterozygosity likely to be caused by null-alleles at this locus. The remaining STRs did not reveal significant deviations from Hardy-Weinberg equilibrium expectations except for loci G10B and MU10 that showed significant deviations in one population each, respectively. There were 9 pairwise locus comparisons that showed significant deviation from linkage equilibrium in one or two out of the eight populations. Substantial genetic differentiation was detected in some of the pairwise population comparisons and the average estimate of population substructure (F(ST)) was 0.09. The average estimate of inbreeding (F(IS)) was 0.005. Accounting for population substructure and inbreeding the total average probability of identity in each of the eight populations was lower than 1.1×10(-9) and the total average probability of sibling identity was lower than 1.3×10(-4). The magnitude of these measurements indicates that if applying these twelve STRs in a DNA profiling system this would provide individual specific evidence.

Limited gene flow among brown bear populations in far Northern Europe? Genetic analysis of the east–west border population in the Pasvik Valley

Noninvasively collected genetic data can be used to analyse large‐scale connectivity patterns among populations of large predators without disturbing them, which may contribute to unravel the species’ roles in natural ecosystems and their requirements for long‐term survival. The demographic history of brown bears (Ursus arctos) in Northern Europe indicates several extinction and recolonization events, but little is known about present gene flow between populations of the east and west. We used 12 validated microsatellite markers to analyse 1580 hair and faecal samples collected during six consecutive years (2005–2010) in the Pasvik Valley at 70°N on the border of Norway, Finland and Russia. Our results showed an overall high correlation between the annual estimates of population size (Nc), density (D), effective size (Ne) and Ne/Nc ratio. Furthermore, we observed a genetic heterogeneity of ∼0.8 and high Ne/Nc ratios of ∼0.6, which suggests gene flow from the east. Thus, we expanded the population genetic study to include Karelia (Russia, Finland), Västerbotten (Sweden) and Troms (Norway) (477 individuals in total) and detected four distinct genetic clusters with low migration rates among the regions. More specifically, we found that differentiation was relatively low from the Pasvik Valley towards the south and east, whereas, in contrast, moderately high pairwise FST values (0.91–0.12) were detected between the east and the west. Our results indicate ongoing limits to gene flow towards the west, and the existence of barriers to migration between eastern and western brown bear populations in Northern Europe.

Admixture and gene flow from Russia in the recovering Northern European brown bear (Ursus arctos)

Large carnivores were persecuted to near extinction during the last centuries, but have now recovered in some countries. It has been proposed earlier that the recovery of the Northern European brown bear is supported by migration from Russia. We tested this hypothesis by obtaining for the first time continuous sampling of the whole Finnish bear population, which is located centrally between the Russian and Scandinavian bear populations. The Finnish population is assumed to experience high gene flow from Russian Karelia. If so, no or a low degree of genetic differentiation between Finnish and Russian bears could be expected. We have genotyped bears extensively from all over Finland using 12 validated microsatellite markers and compared their genetic composition to bears from Russian Karelia, Sweden, and Norway. Our fine masked investigation identified two overlapping genetic clusters structured by isolation-by-distance in Finland (pairwise FST = 0.025). One cluster included Russian bears, and migration analyses showed a high number of migrants from Russia into Finland, providing evidence of eastern gene flow as an important driver during recovery. In comparison, both clusters excluded bears from Sweden and Norway, and we found no migrants from Finland in either country, indicating that eastern gene flow was probably not important for the population recovery in Scandinavia. Our analyses on different spatial scales suggest a continuous bear population in Finland and Russian Karelia, separated from Scandinavia.

Evidence of rapid change in genetic structure and diversity during range expansion in a recovering large terrestrial carnivore

Recovery of natural populations occurs often with simultaneous or subsequent range expansions. According to population genetic theory, genetic structuring emerges at the expansion front together with decreasing genetic diversity, owing to multiple founder events. Thereupon, as the expansion proceeds and connectivity among populations is established, homogenization and a resurgence of genetic diversity are to be expected. Few studies have used a fine temporal scale combined with genetic sampling to track range expansions as they proceed in wild animal populations. As a natural experiment, the historical eradication of large terrestrial carnivores followed by their recovery and recolonization may facilitate empirical tests of these ideas. Here, using brown bear (Ursus arctos) as model species, we tested predictions from genetic theory of range expansion. Individuals from all over Finland were genotyped for every year between 1996 and 2010 using 12 validated autosomal microsatellite markers. A latitudinal shift of about 110 km was observed in the distribution and delineation of genetic clusters during this period. As the range expansion proceeded, we found, as theory predicts, that the degree of genetic structure decreased, and that both genetic variation and admixture increased. The genetic consequences of range expansions may first be detected after multiple generations, but we found major changes in genetic composition after just 1.5 generations, accompanied by population growth and increased migration. These rapid genetic changes suggest an ongoing concerted action of geographical and demographic expansion combined with substantial immigration of bears from Russia during the recovery of brown bears within the large ecosystem of northern Europe.

Identification and evaluation of novel di- and tetranucleotide microsatellite markers from the brown bear (Ursus arctos)

We here describe 34 novel microsatellite loci from the brown bear (Ursus arctos) identified using a next-generation sequencing approach. While we found allelic variation in 30 of these, we selected 16 polymorphic microsatellites (11 di- and 5 tetranucleotide repeats) for four multiplex-PCR assays based on genetic variability, PCR performance and interpretability. Applying these assays for the 16 novel microsatellites, we screened 31 brown bears from a northern European population. The number of alleles encountered at single loci was between 4 and 10, and expected and observed heterozygosities ranged between 0.66–0.85 and 0.60–0.97, respectively. Seven of 120 pairwise locus combinations showed linkage disequilibrium and one locus deviated from Hardy–Weinberg equilibrium, while no null-alleles were detected. Our novel multiplex-PCR assays may be useful in forensics, parentage and population genetic studies.

Sex-specific genetic analysis indicates low correlation between demographic and genetic connectivity in the Scandinavian brown bear ( Ursus arctos )

The degree of gene flow within and among populations, i.e. genetic population connectivity, may closely track demographic population connectivity. Alternatively, the rate of gene flow may change relative to the rate of dispersal. In this study, we explored the relationship between genetic and demographic population connectivity using the Scandinavian brown bear as model species, due to its pronounced male dispersal and female philopatry. Thus, we expected that females would shape genetic structure locally, whereas males would act as genetic mediators among regions. To test this, we used eight validated microsatellite markers on 1531 individuals sampled noninvasively during country-wide genetic population monitoring in Sweden and Norway from 2006 to 2013. First, we determined sex-specific genetic structure and substructure across the study area. Second, we compared genetic differentiation, migration/gene flow patterns, and spatial autocorrelation results between the sexes both within and among genetic clusters and geographic regions. Our results indicated that demographic connectivity was not a reliable indicator of genetic connectivity. Among regions, we found no consistent difference in long-term gene flow and estimated current migration rates between males and females. Within regions/genetic clusters, only females consistently displayed significant positive spatial autocorrelation, indicating male-biased small-scale dispersal. In one cluster, however, males showed a dispersal pattern similar to females. The Scandinavian brown bear population has experienced substantial recovery over the last decades; however, our results did not show any changes in its large-scale population structure compared to previous studies, suggesting that an increase in population size and dispersal of individuals does not necessary lead to increased genetic connectivity. Thus, we conclude that both genetic and demographic connectivity should be estimated, so as not to make false assumptions about the reality of wildlife populations.

A Modification of the Hair-Trapping Method for Surveillance of Problematic Bear Activity Close to a Farm — a Case Study from the Pasvik Valley in Norway

Human—bear conflicts occur frequently in the Pasvik Valley, Norway. We used a variant of the hair-trapping method with higher densities of traps (2.5 × 2.5 km grid) to detect brown bears moving near human settlements and livestock. We distributed 20 hair traps for one month close to a farm with frequent observations of grazing bears. The study area consisted of one area close to the farm, and one adjacent area without settlements. We collected 85 hair samples and identified 13 different individuals by STR analysis. In the farm area, we detected 4 different males once, and a female that was detected in both areas. In comparison, nine bears (2 males and 7 females) were detected for more than one week in the area without settlements, suggesting lower roaming activity. Conclusively, hair trapping has the potential to survey bears at specific locations of importance to the wildlife management.

Genetic substructure and admixture as important factors in linkage disequilibrium-based estimation of effective number of breeders in recovering wildlife populations

Abstract The number of effective breeders (N b) and effective population size (N e) are population parameters reflective of evolutionary potential, susceptibility to stochasticity, and viability. We have estimated these parameters using the linkage disequilibrium‐based approach with LDNE through the latest phase of population recovery of the brown bears (Ursus arctos) in Finland (1993–2010; N = 621). This phase of the recovery was recently documented to be associated with major changes in genetic composition. In particular, differentiation between the northern and the southern genetic cluster declined rapidly within 1.5 generations. Based on this, we have studied effects of the changing genetic structure on N b and N e, by comparing estimates for whole Finland with the estimates for the two genetic clusters. We expected a potentially strong relationship between estimate sizes and genetic differentiation, which should disappear as the population recovers and clusters merge. Consistent with this, our estimates for whole Finland were lower than the sum of the estimates of the two genetic clusters and both approaches produced similar estimates in the end. Notably, we also found that admixed genotypes strongly increased the estimates. In all analyses, our estimates for N e were larger than N b and likely reflective for brown bears of the larger region of Finland and northwestern Russia. Conclusively, we find that neglecting genetic substructure may lead to a massive underestimation of N b and N e. Our results also suggest the need for further empirical analysis focusing on individuals with admixed genotypes and their potential high influence on N b and N e.

Genetic evidence of female kin clusters in a continuous population of a solitary carnivore, the Eurasian lynx

Abstract Large terrestrial carnivores can sometimes display strong family bonds affecting the spatial distribution of related individuals. We studied the spatial genetic relatedness and family structure of female Eurasian lynx, continuously distributed in southern Finland. We hypothesized that closely related females form matrilineal assemblages, clustering together with relatives living in the neighboring areas. We evaluated this hypothesis using tissue samples of 133 legally harvested female lynx (from year 2007 to 2015), genotyped with 23 microsatellite markers, and tested for possible spatial genetic family structure using a combination of Bayesian clustering, spatial autocorrelation, and forensic genetic parentage analysis. The study population had three potential family genetic clusters, with a high degree of admixture and geographic overlap, and showed a weak but significant negative relationship between pairwise genetic and geographic distance. Moreover, parentage analysis indicated that 64% of the females had one or more close relatives (sister, mother, or daughter) within the study population. Individuals identified as close kin consistently assigned to the same putative family genetic cluster. They also were sampled closer geographically than females on average, although variation was large. Our results support the possibility that Eurasian lynx forms matrilineal assemblages, and comparisons with males are now required to further assess this hypothesis.