Assessment of genetic diversity, population structure and wolf-dog hybridisation in the Eastern Romanian Carpathian wolf population.

The Carpathian Mountains have been constantly inhabited by grey wolves and present one of the largest distribution areas in Europe, comprising between 2300 and 2700 individuals in Romania. To date, however, relatively little is known about the Romanian wolf population. We aimed to provide a first assessment of genetic diversity, population structure and wolf-dog hybridisation based on 444 mostly non-invasively collected samples in the Eastern Romanian Carpathians. Pack reconstruction and analysis of population genetic parameters were performed with mitochondrial DNA control-region sequencing and microsatellite genotyping. We found relatively high levels of genetic diversity, which is similar to values found in previous studies on Carpathian wolves from Poland and Slovakia, as well as to the long-lasting Dinaric-Balkan wolf population. We found no significant population structure in our study region, suggesting effective dispersal and admixture. Analysis of wolf-dog hybridisation using a Single Nucleotide Polymorphism panel optimised for hybrid detection revealed low rates of admixture between wolves and domestic dogs. Our results provide evidence for the existence of a genetically viable wolf population in the Romanian Carpathians. The genetic data obtained in this study may serve as valuable baseline information for the elaboration of monitoring standards and management plans for wolves in Romania.

Reliable wolf-dog hybrid detection in Europe using a reduced SNP panel developed for non-invasively collected samples.

BACKGROUND: Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples. RESULTS: Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations. CONCLUSIONS: We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the third-generation backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales.

How the west was won: genetic reconstruction of rapid wolf recolonization into Germany's anthropogenic landscapes.

Following massive persecution and eradication, strict legal protection facilitated a successful reestablishment of wolf packs in Germany, which has been ongoing since 2000. Here, we describe this recolonization process by mitochondrial DNA control-region sequencing, microsatellite genotyping and sex identification based on 1341 mostly non-invasively collected samples. We reconstructed the genealogy of German wolf packs between 2005 and 2015 to provide information on trends in genetic diversity, dispersal patterns and pack dynamics during the early expansion process. Our results indicate signs of a founder effect at the start of the recolonization. Genetic diversity in German wolves is moderate compared to other European wolf populations. Although dispersal among packs is male-biased in the sense that females are more philopatric, dispersal distances are similar between males and females once only dispersers are accounted for. Breeding with close relatives is regular and none of the six male wolves originating from the Italian/Alpine population reproduced. However, moderate genetic diversity and inbreeding levels of the recolonizing population are preserved by high sociality, dispersal among packs and several immigration events. Our results demonstrate an ongoing, rapid and natural wolf population expansion in an intensively used cultural landscape in Central Europe.

Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays.

Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.

Population expansion and individual age affect endoparasite richness and diversity in a recolonising large carnivore population.

The recent recolonisation of the Central European lowland (CEL) by the grey wolf (Canis lupus) provides an excellent opportunity to study the effect of founder events on endoparasite diversity. Which role do prey and predator populations play in the re-establishment of endoparasite life cycles? Which intrinsic and extrinsic factors control individual endoparasite diversity in an expanding host population? In 53 individually known CEL wolves sampled in Germany, we revealed a community of four cestode, eight nematode, one trematode and 12 potential Sarcocystis species through molecular genetic techniques. Infections with zoonotic Echinococcus multilocularis, Trichinella britovi and T. spiralis occurred as single cases. Per capita endoparasite species richness and diversity significantly increased with population size and changed with age, whereas sex, microsatellite heterozygosity, and geographic origin had no effect. Tapeworm abundance (Taenia spp.) was significantly higher in immigrants than natives. Metacestode prevalence was slightly higher in ungulates from wolf territories than from control areas elsewhere. Even though alternative canid definitive hosts might also play a role within the investigated parasite life cycles, our findings indicate that (1) immigrated wolves increase parasite diversity in German packs, and (2) prevalence of wolf-associated parasites had declined during wolf absence and has now risen during recolonisation.