National Reference Center for Genetic Analysis of Wolf and Lynx

All commissioned samples are processed according to strict scientific standards, which include the use of separate laboratory rooms and the use of analytical replicates in all investigations. The basic method for the nationwide genetic monitoring of wolf and lynx is microsatellite analysis (also called STR or SSR) based on nuclear DNA, which yields an individually unique genetic fingerprint and allows inferences on individual numbers, sex, relationships, and the occurrence of hybrids.

Furthermore, a section of the mitochondrial control region is sequenced for all commissioned samples, which allows identification of the maternal lineage. This procedure allows species identification even for samples with very low DNA content and provides clues to population assignment (haplotype determination).

Since the derivation of hybridization levels in wolves via microsatellites usually only allows the reliable detection of F1 hybrids (direct offspring from the mating of a wolf with a domestic dog), we additionally use a SNP chip optimized for non-invasively collected samples to reliably detect hybridization events that occurred longer ago (Harmoinen et al., 2021; Kraus et al., 2015). This is based on numerous point mutations (SNPs) distributed across the entire genome,  which can be used to reliably distinguish wolves from domestic dogs regardless of their geographic origin (Galaverni et al., 2017; von Holdt et al., 2012). The method can be used to reliably detect hybridization events beyond the third hybrid generation (= second backcross generation).

The results of the commissioned genetic studies within the framework of the official monitoring of wolf and lynx are immediately reported to the responsible offices of the federal states so that they can be promptly incorporated into the monitoring and wildlife management. The results can be viewed on the corresponding information pages of the responsible federal state offices. In the case of wolves, a great deal of detailed information on population patterns and pack numbers can be found on the DBBW homepage, which incorporates the genetic data produced in our lab. We regularly publish scientific findings from the genetic monitoring of wolf and lynx in scientific journals.

Summary of previous findings from the genetic monitoring of large carnivores in Germany

(i) Lynx

Wildliving lynx in Germany originate from three reintroduction projects in the Bavarian Forest (Bayerischer Wald), Harz Mountains and Palatinate Forest (Pfälzerwald). The lynx in the Palatinate Forest and in the Bavarian/Czech border area originate from the Slovakian Carpathians. They carry the mitochondrial haplotype 4, which is characteristic for this region, and also group in genome-wide comparison to lynx samples from the Carpathian region. As in most other European lynx reintroductions, the genetic diversity of lynx in the Bavarian Forest is significantly reduced compared to free-ranging populations (Mueller et al., in review). For the long-term conservation of the species, European conservation strategies are needed that enable the connection of the source population to largely isolated subpopulations. The lynx population in the Harz Mountains does not originate from lynx captured in the Carpathian Mountains, like most other European reintroductions, but from enclosures with a variety of origins. As origins, Baltic/Scandinavian, Carpathian and Asian origins can be determined by genome-wide comparisons. The mixing of these lineages has resulted in a comparatively high genetic diversity in the Harz Mountains, which, is slowly decreasing despite the expansion of the lynx population (Mueller et al. 2020). In this case, connectivity to adjacent populations will be important for the long term preservation of genetic diversity. Occasional migrations of mostly male Harz lynx over distances of several hundred kilometers, detected via genetic matching, show that genetic exchange between local lynx populations is possible in the long term even in densely populated Central Europe (Gajdárová et al., in review).

(ii) Wolf

Genetic comparisons with surrounding wolf populations show that wolves in Germany derive from northeastern Poland via long-distance dispersal since the late 1990s. Starting in the Lausitz region of Saxony, wolves have since spread mainly across the northern German lowlands, while suitable low mountain regions are currently being colonized more slowly. Through the ongoing genetic wolf monitoring, more than 5000 samples with suspected wolves are processed annually, sent in by the state authorities and commissioned for analysis. From these samples, wolf relationships are continuously updated to determine pack structures and migration patterns. Examples include several wolves detected in Denmark that originated in Lusatia, as well as the wolf known as “Billy” (GW1554m), whose migration route was traced in 2020 based on DNA analysis of livestock kills. From its original territory in Lower Saxony, it migrated through the Netherlands, Belgium, Rhineland-Palatinate and France. The reconstruction of the migration route of more than 1000 km Euclidean distance is a collaboration of the research institutes of the CEwolf Consortium and Antagène in France.

Wolves in Germany appear to be genetically quite homogeneous; however, inbreeding occurs rather rarely. Most wolves carry the mitochondrial haplotypes HW01 and HW02, which are also typical for most regions of Northeastern Europe. More rarely, however, the haplotype HW22, which is characteristic in Italy and the Alpine region, is detected. Matching of nuclear DNA shows that wolves in Germany, western and central Poland and some surrounding regions form a genetically largely uniform population that is genetically separated from surrounding populations in the Baltic region (Szewczyk et al., 2019, 2021). In the future, a stronger mixing of the genetically separated European wolf populations is to be expected. In the Bavarian Forest, for example, there was a successful mating of wolves from the Central European and Alpine populations in 2017. Kinship analyses show that wolf packs in Germany mostly consist of the parents and their offspring from the last one to two years. While two cases of wolves escaping from enclosures have been documented since 2010 (both made public), there is no genetic indications of illegally released wolves or wolf-dog hybrids so far, despite of occasional media reports hinting in this direction.

To date, three cases of wolf-dog mating have been detected in Germany (2003 in Saxony and 2017 and 2019 in Thuringia). Genome-wide comparisons with wolves from across Eurasia show that wolves in Germany do not carry high proportions of domestic dog DNA in their genome. Currently, international research projects are underway to search for small traces of historic admixture of canine DNA in European – including German – wolves.

Species determinations based on swab samples from suspected wolf or lynx kills take an average of 4-5 business days between sample arrival and delivery of results. If high-priority samples are ordered, individual and pack membership are also determined during this period. Only samples with a special necessity can be accepted as urgent samples.

In case of an unclear result, the analysis of a B-sample (reserve sample) is often ordered, which extends the analysis time accordingly. When a result becomes known to the public is at the discretion of the client. The determination of the causative agent of livestock depredations is a complex process in which genetic analysis is only a partial step. Therefore, the elapsed time between a kill incident and the announcement of the result cannot be used to infer the duration of the genetic analysis.

The genetic tests are financed by the responsible state offices. The payment is by sample basis. The costs per sample depend on the type and methodology of the commissioned examination and usually amount to 100 to 200 € per analysis plus VAT. The examination of non-invasively collected sample material such as faeces, urine or kill samples is time-consuming and therefore more expensive than is usual for standard applications in the clinical-diagnostic field.

Senckenberg does not make any profit from the sample analyses. All income generated by the genetic analysis service is used to finance the staff required for this purpose as well as consumables and laboratory maintenance. Any surplus income is invested in optimizing methods and performing further research in wildlife genetics.

de Groot GA, Nowak C, Skrbinšek T, Andersen L, Aspi J, Fumagalli L, Godinho R, Harms, V, Jansman HAH, Liberg O, Marucco F, Mysłajek RW, Nowak S, Pilot M, Randi E, Reinhardt I, Śmietana W, Szewczyk M, Taberlet P, Vilà C, Muñoz-Fuentes V (2016) Decades of population genetic research call for harmonization of molecular markers: the grey wolf, Canis lupus, as a case study. Mammal Review 46, 44-59.

Galaverni M, Caniglia R, Pagani L, Fabbri E, Boattini A, Randi E (2017) Disentangling timing of admixture, patterns of introgression, and phenotypic indicators in a hybridizing wolf population. Molecular Biology & Evolution 34, 2324-2339.

Harmoinen J, von Thaden A, Aspi J, Kvist L, Cocchiararo B, Jarausch A et al. (under review) Reliable wolf-dog hybrid detection in Europe using a reduced SNP panel developed for non-invasively collected samples, 01 December 2020, PREPRINT (Version 1) available at Research Square.

Kraus RHS, von Holdt B, Cocchiararo B, Harms V, Bayerl H, Kühn R, Förster DW, Fickel J, Roos C, Nowak C (2015) A single-nucleotide polymorphism-based approach for rapid and cost-effective genetic wolf monitoring in Europe based on non-invasively collected samples. Molecular Ecology Resources 15, 295-305.

Mueller SA, Reiners TE, Middelhoff L, Anders O, Nowak C (2020) The rise of a large carnivore population in Central Europe: Genetic evaluation of lynx reintroduction in the Harz Mountains. Conservation Genetics 21, 577-587.

Szewczyk M, Nowak S, Niedźwiecka N, Hulva P, Špinkytė-Bačkaitienė R, Demjanovičová K, Černá Bolfíková B, Antal V, Fenchuk V, Figura M, Tomczak P, Przemysław Stachyra, Stępniak K M, Zwijacz-Kozica T & Mysłajek R W (2019) Dynamic range expansion leads to establishment of a new, genetically distinct wolf population in Central Europe. Scientific Reports 9, 1-16.

Szewczyk M, Nowak C, Hulva P, Mergeay J, Stronen A, Bolfíková B, Czarnomska S, Diserens T, Fenchuk V, Figura M, de Groot A, Haidt A, Hansen M, Jansman H, Kluth G, Kwiatkowska I, Lubinska K, Michaux J, Niedzwiecka N, Nowak S, Olsen K, Reinhardt I, Romanski M, Schley L, Smith S, Spinkyte-Backaitiene R, Stachyra, P, Stepniak K, Sunde P, Thomsen P, Zwijacz-Kozica T & Myslajek R (2021) Genetic support for the current discrete conservation unit of the Central European wolf population. Wildlife Biology, doi: 10.2981/wlb.00809.

von Holdt BM, Pollinger JP, Earl DA, Parker HG, Ostrander EA, Wayne RK (2012) Identification of recent hybridization between gray wolves and domesticated dogs by SNP genotyping. Mammalian Genome 24, 80-88.