Page overview
Background to the genetic analyses
Methods used
Utilization of the genetic findings
Summary of previous findings from genetic wolf monitoring in Germany
Duration and funding of the studies
Information on wolf haplotypes in Germany
Literature
Background to the genetic analyses
Since 2010, all samples collected as part of the nationwide wolf monitoring program have been centrally examined in the laboratory of the Senckenberg Center for Wildlife Genetics. The results are used to gain insights into the occurrence of wolves in Germany, distinguish between individuals, determine relationships and continuously check the wolf population for possible mixing with domestic dogs (hybridization).
DNA-based investigations of livestock kills to detect wolves and other predators play an important role in monitoring and management. These are carried out on behalf of the federal states. Accordingly, all results obtained are forwarded to the federal states, which are responsible for publishing the results.
The Senckenberg Centre for Wildlife Genetics was recommended to the federal states as a national reference center for genetic studies on wolves and lynx following a selection process supervised by the Federal Agency for Nature Conservation (BfN). In October 2009, the Working Group of the Federal States for Nature Conservation (LANA) decided to follow the BfN's recommendation. The use of a central analysis laboratory ensures the comparability of all data collected nationwide. Due to the lack of standards for wildlife genetic analysis methods (de Groot et al. 2016), this centralized processing within the framework of official wildlife monitoring is internationally common and well established.
Methods used
All commissioned samples are processed according to strict scientific standards, which include the use of separate laboratory rooms and the use of analytical replicates for all tests. The basic method for nationwide genetic monitoring of wolves is microsatellite testing (also known as STR or SSR) based on nuclear DNA, which produces an individually unique genetic fingerprint and allows conclusions to be drawn about the number of individuals, sex, relationships and the presence of hybrids.
In addition, a section of the mitochondrial control region is sequenced in all samples ordered, which allows the maternal lineage to be identified. This procedure enables the species to be determined even for samples with a very low DNA content and provides information on the 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 also use a SNP chip optimized for non-invasively collected samples for the reliable detection of hybridization events that occurred longer ago (Harmoinen et al., 2021). 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 geographical 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).
Utilization of the genetic findings
The results of the genetic studies commissioned as part of the official monitoring are immediately forwarded to the responsible authorities in the federal states so that they can be promptly incorporated into monitoring and wildlife management. The results can be viewed on the relevant information pages of the responsible federal state authorities. In the case of wolves, the DBBW website contains a wealth of detailed information on population patterns and pack numbers, which includes genetic data. Senckenberg regularly publishes scientific findings from genetic monitoring and the research work based on it in international journals (e.g. Jarausch et al. 2021, Szewczyk et al. 2021).
Summary of previous findings from genetic wolf monitoring in Germany
At the Center for Wildlife Genetics, each genetically identified wolf is given an individual label consisting of GW (“Genetik Wolf”), a serial number and the sex code (m - male, f - female). Genetic comparisons with surrounding wolf populations show that the wolves in Germany migrated from north-eastern Poland in the late 1990s. Starting from Lusatia in Saxony, the wolves have since spread mainly across the northern German lowlands, while suitable low mountain regions are only slowly being colonized. The ongoing genetic wolf monitoring program processes more than 6,000 samples of suspected wolves every year, which are sent in by the state authorities and commissioned for analysis. These samples are used to continuously update the wolves' relationships and thus determine pack structures and migration movements. Examples of this include several wolves detected in Denmark that originate from Lusatia, as well as the wolf known as “Billy” (GW1554m), whose migration route was traced in 2020 based on DNA analyses 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 over 1000 km as the crow flies is a collaboration between the research institutes of the CEwolf consortium and the Antagène company in France.
Wolves in Germany are genetically quite homogeneous; however, inbreeding is rather rare. Most wolves carry the mitochondrial haplotypes HW01 and HW02, which are also typical for most regions of north-eastern Europe. In contrast, the haplotype HW22, which is characteristic of Italy and the Alpine region, is found less frequently. Comparisons of nuclear DNA show 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 future, however, greater mixing of the genetically largely separate European wolf populations is to be expected. In the Bavarian Forest, for example, wolves from the Central European and Alpine populations successfully mated 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. There have been two cases of wolves escaping from enclosures since 2010 that have been reported in the media; there is no genetic evidence of illegally released wolves or wolf-dog hybrids to date.
So far, five cases of wolf-dog hybridization events have been documented in Germany (2003 in Saxony, 2017 and 2019 in Thuringia and 2022 in the Rhön in the border region of Thuringia/Hesse/Bavaria). In another case in 2022 in Brandenburg, a hybrid that had migrated from central Poland (Szubin region) mated with a German wolf female. Genome-wide comparisons with Eurasian wolves show that, apart from minor traces of historical hybridization events, wolves in Germany do not carry an increased proportion of DNA from domestic dogs in their genome (Stenøien et al., 2021).
Duration and financing of the investigations
Deadlines for genetic test results were agreed with the responsible federal state authorities. Genetic pack reconstruction based on the DNA samples collected in a wolf monitoring year (May 1 to April 30) is carried out once a year. An average of 4-5 working days is required for species determinations based on swab samples from suspected wolf kills. In the case of commissioned urgent samples, individual and pack affiliation are also determined during this period. Only samples with a particular urgency can be accepted as urgent samples.
If the result is unclear, the analysis of a B sample (reserve sample) is often commissioned, which extends the analysis time accordingly. When a result is made public is at the discretion of the client. Determining the cause of livestock kills is a complex process in which genetic analysis is only a partial step. It is therefore not possible to draw conclusions about the duration of the genetic analysis from the time elapsed between an incident and the announcement of the result.
The genetic tests are financed by the responsible state authorities. Remuneration is based on samples. The costs per sample depend on the type and methodology of the test commissioned and usually amount to around €100 to €200 per analysis plus VAT. The analysis of non-invasively collected sample material such as feces, urine or crack samples is time-consuming and therefore more expensive than standard applications in the clinical-diagnostic field. Senckenberg does not generate any profits from the sample analyses. All income generated by the genetic analysis service is used to finance the necessary staff, consumables and laboratory maintenance.
Information on wolf haplotypes in Germany
A mitochondrial haplotype describes a variant of a contiguous DNA section and is passed on exclusively from the mother's side, allowing the so-called maternal line to be traced.
There is currently no globally standardized nomenclature for mitochondrial wolf haplotypes. In addition, different regions of the mitochondrial DNA sequence are sometimes used to determine the haplotype. Many studies use internal designations, which are often numbered “H” or “W”. However, published DNA sequences of the individual haplotypes can be viewed in freely available online databases and used for comparison (e.g. NCBI genbank).
In Germany and most of the surrounding countries that participate in the CEP, we refer to the haplotypes in accordance with the specialist publication Pilot et al. (2010), which covers a large number of wolf haplotypes. In the publication, the haplotypes are labeled “W”; we prefix them with “HW” (= wolf haplotype) while retaining the numbering from Pilot et al.
With the appearance of long-distance migrants from the Dinaric population, haplotypes are now also represented in Germany which are not covered in Pilot et al. 2010, but are mentioned in other specialist publications. For these haplotypes, the nomenclature used in Germany follows the study by (Montana et al. 2017). Mitochondrial haplotypes are passed on exclusively from the maternal side and thus allow maternal lines to be traced.
The haplotypes found in wolves in Europe are often characteristic of certain geographical areas of occurrence. To date, the following haplotypes have been found in descending frequency in wild wolves in Germany: HW01, HW02, HW22, HW03, HW06, HW10, W3 and W17 with only the first two haplotypes showing a significant frequency in the population so far.
In Germany, >90% of wolves carry the haplotype HW01, which occurs frequently in Central and Eastern Europe and Scandinavia. In recent years, haplotype HW02, which also occurs with a certain frequency in the Polish population of origin, has established itself with a slight upward trend. This is followed by HW22, which occurs in almost all wolves of the Apennine and Alpine populations; it is characteristic of the genetically clearly defined lineage of the “Italian wolf”. To date, four cases are known in which an animal from the Alpine population has mated with an animal from the Central European population. As all cases involve males, the HW22 haplotype is not passed on even in Germany.
Haplotype HW03 is mainly found in south-eastern Poland. Due to an immigrant female from this region, which reproduced in the Barnimer Heide in BB, the haplotype is now also being passed on in Germany and is becoming increasingly common (0.1%)
Nevertheless, a population affiliation cannot be determined with absolute certainty on the basis of the haplotype alone, as haplotypes that are otherwise rather atypical for a population can also occur in low frequencies. This can be explained, for example, by historical migration movements between genetically separate populations. For example, HW06 is characteristic of the Carpathian region. The individual GW1724m detected in Germany (spring 2020 in Saxony-Anhalt and North Rhine-Westphalia), which carries the haplotype HW06, is such a case. Its genetic profile (microsatellites of the nuclear DNA; genotype) is more similar to reference profiles of the Central European Plain Population (CEP) or the Baltic population than to wolves from the Carpathians. A comparison with colleagues from Poland revealed that the animal probably originates from the region around the Koszalin Forest in north-western Poland, where at least one pack with this haplotype lives.
According to Pilot et al. 2010, the HW10 haplotype is found in Poland, Russia, Croatia, Bulgaria, Greece, Turkey, Spain and Portugal. Our own research has shown that the haplotype is also present in the Dinaric wolf population. According to the international NCBI database, the haplotype may also occur in dogs. For this reason, the creation of a genetic profile using microsatellite analysis is necessary for reliable species identification and population assignment.
The haplotypes W3 and W17 are typical for the Dinaric population and have so far only been recorded in Germany in animals originating from there. Through the international exchange of samples and data, it was even possible to determine the exact herd of origin in Slovenia for one of the animals.
Literature
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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.
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Jarausch A, Harms V, Kluth G, Reinhardt I, Nowak C (2021) How the west was won: genetic reconstruction of rapid wolf recolonization into Germany’s anthropogenic landscapes. Heredity 127, 92-106.
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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.
Stenøien HK, Sun X, Martin MD, Scharff-Olsen CH, Alonso GH, Martins NFG, Lanigan L, Ciucani MM, Sinding M-HS, Gopalakrishnan S, Gilbert MTP (2021) Genetisk opphav til den norsk- svenske ulvestammen (Canis lupus lupus) – NTNU Vitenskapsmuseet naturhistorisk rapport 2021-11, 1-53.
Szewczyk M, Nowak C, Hulva P, Mergeay J, Stronen, AV et al. (2021) Genetic support for the current discrete conservation unit of the Central European wolf population. Wildlife Biology, wlb.00809.