Research

Inferring genetic patterns of ongoing recolonization of Central Europe by elusive, large carnivores using novel SNP marker systems for noninvasive samples

Background
The currently observed recolonisation of Central and Western Europe by previously near-extinct carnivores is an important topic for science and society. A detailed, cross-border knowledge about migration routes population growth and gene flow patterns are important for comprehensive wildlife monitoring, and are also the foundation for the scientific analysis of this natural experiment about the coexistence between large carnivores and humans in densely populated areas.

The project
The aim of the SAW project is the establishment of a competence network for the development of novel genetic markers systems for studying endangered European carnivores. To meet this aim we first analyse variable nucleotide positions (single nucleotide polymorphisms; SNPs) in the genome of five large carnivores (brown bear, Eurasian otter, wolf, lynx, wildcat) by next generation sequencing or large scale SNP chips. These markers are tested for their variability across European populations by data base queries. In parallel, we investigate different SNP genotyping technologies for their applicability for non-invasively collected sample material. The final marker systems will be tested on multiple populations of each species and compared with the results of other methods. By means of genotyping of samples from Central and Eastern Europe we will study general genetic diversity, gene flow and the impact of landscape genetic factors, in order to identify the genetic patterns of Europe’s recolonisation. All data will be stored in a genetic data base to facilitate future projects and search queries with regard to the origin of samples of unknown status, and thereby deciphering large-scale gene flow.

Project partners
Marker development and population genetic analyses are carried out cooperatively by the three primary project partners Senckenberg (Dr. Carsten Nowak, Dr. Robert Kraus), Leibniz Institute for Zoo and Wildlife Research (Dr. Jörns Fickel, Dr. Daniel Förster), and TU Munich (Prof. Dr. Ralph Kühn, Dipl.-Biol. Helmut Bayerl). The combination of the aforementioned factors – joint expertise, development of standardised marker systems and installation of a genetic data base – allows the inference of dispersal patterns, isolation factors, and distribution shifts across multiple studies on scales that surpass single, regional studies.

Background
Since 2008, the Location Gelnhausen of the Senckenberg Research Institute has been carrying out molecular genetic DNA studies to differentiate between wildcats and domestic cats. Until now, Senckenberg has examined more than 15,000 wild cat samples for over 200 clients. Previous highlights of the project include the first evidence of wildcats in numerous regions of Germany, e.g. the first evidence of wildcats in the only national park in Hesse, the Kellerwald-Edersee National Park, in 2007, which was celebrated as a “small sensation”. The genetic evidence of wildcats in the Thayatal National Park in Austria is also spectacular. In 2009, a first detailed overview of the German wildcat population was obtained for the “Wildcat Rescue Network” of the BUND (Friends of the Earth Germany) using lure stick samples. Current projects include the “Wildcat Leap Project” of the BUND and the study of the barrier effect of highways and rivers on wildcats.

The wildcat
The European wildcat (Felis s. silvestris) serves as flagship species for the conservation of unfragmented, near-natural broad-leave forests. Historic persection  as well as habitat loss and fragmentation has led to a severe population decline. Since the second half of the 20th century, the wildcat is expanding in Germany. Data on wildcat presence was, however, only sparely distributed, due to the elusiveness of this species and the difficulty to discriminate it from its domestic form –  wildcats show well-distinct black tail-rings, 4-5 black stripes on their neck, and a blurred stripe pattern at the flanks, which is best visible around the shoulders (Krüger et al. 2009). Road kills are analysed by morphometric determination of skull and gut size.

Krüger, M., Hertwig ST, Jetschke G, Fischer MS (2009) Evaluation of anatomical characters and the question of hybridization with domestic cats in the wildcat population of Thuringia, Germany. Journal of Zoological Systematics and Evolutionary Research 47: 268-282.

Lure stick method
Previous genetic studies on wildcats in Germany, such as hybridization rates and population structure calculations, have exclusively used dead specimens and museum specimens, and only very few wildcat populations have been genetically characterized to date. The non-invasive lure stick method can be used to collect sufficient genetic sample material from wildcats in the field with the aid of hair traps, without the wildcats being directly affected. The lure stick method has proven to be very effective in numerous studies as a supplier of hair samples for subsequent genetic studies.  Lure sticks are simple wooden stakes that are sprayed with valerian as an attractant. The valerian causes the animals to rub against the stakes, leaving hairs on the stakes. The hairs are genetically analyzed after collection, making it possible to differentiate between domestic cats and wildcats and to individualize them.

DNA analysis of hair samples
It is important to note that moisture, temperature changes and sunlight lead to DNA degradation in hair. Therefore, the number of dry stored hairs with hair roots is decisive for the success of the genetic analysis. The amount of DNA molecules in the hair roots is a thousand times higher than in the rest of the hair. Against the light, hair roots appear as a transparent thickening. For this reason, hair should never be cut from a piece of fur with scissors. Hair should also never be fixed to adhesive surfaces, as this greatly impairs subsequent analyses. Fixing hair on adhesive strips etc. is only useful in exceptional cases (e.g. when searching surfaces for hair and tissue residues, for example in forensic examinations). As the root of the hair contains most of the DNA, it is not possible to create a genetic fingerprint if there are fewer than 5 hairs with roots. Nevertheless, a distinction between domestic and wild cats can be made using a mitochondrial marker. In the case of >5 hairs with roots, the number of DNA molecules is usually sufficient and 14 microsatellite genes can be examined. This examination is used to create a genetic fingerprint, which can be used to identify species and differentiate between individuals.  

Genetics – the analysis of mitochondrial DNA
The analysis of mitochondrial DNA (mtDNA) offers the advantage that due to the high copy number in each cell, even small sample quantities contain enough DNA for analysis. However, mtDNA is inherited purely maternally, so when interpreting the data later, only the individual’s affiliation to the maternal line of inheritance can be determined.

Genetics – Genotyping via genome-wide SNPs
Using genome-wide SNPs (single nucleotide polymorphisms = single point mutations), individual genetic profiles (genotypes) of wildcats are created using a 96 SNP chip (Fluidigm microfluid platform) in accordance with Thaden et al. 2020 and compared with the genotype databases at the Center for Wildlife Genetics. If a hybrid is suspected, samples are examined according to the method of Nussberger et al. 2014 and Tiesmeyer et al. 2020 in order to reliably detect first-generation hybrids as well as backcross hybrids and more distant hybridization events.

Projects
In the framework of the following projects, we cooperate with several project partners:

• Wildcat monitoring in Germany and adjacent regions (several project partners)
• Wildcat Leap (BUND Friends of the Earth Germany)
• Monitoring of the wildcat in the Kellerwald-Edersee National Park (Kellerwald-Edersee National Park, Institut für Tierökologie und Naturbildung)
• Analysis of the population structure and barrier effects on the wildcat in the Rheingau-Taunus Area (Hessen Forst FENA)
• Regional population structure and reconstruction of  the recolonisation of wildcat habitats in the Rhön (RhönNatur e.V., Rhön Biosphere Reserve)

Options for scientific cooperation
We conduct genetic analyses of hair samples in order to identify and individualise wildcats. Click here for further information.

Further information can be found on the website of the Center for Wildlife Genetics.

(Funded by DAAD (stipend grant for Sarah A. Mueller))

Background
The Eurasian lynx is a large carnivore that was extirpated from central Europe until the late 1800s due to habitat fragmentation and hunting pressure. Beginning in the early 1970s, several reintroduction attempts have been made across central Europe to return this species to its native range. Despite the numerous attempts, small founder population sizes, high levels of inbreeding, illegal hunting, and a number of diseases led to the failure of many projects. Those that were considered successful are now seeing considerably reduced levels of genetic diversity and a marked increase in inbreeding. As more reintroductions are planned in the future, it is important to understand the degree of genetic diversity loss and the degree of gene flow needed to for a viable European lynx metapopulation.

The project
We gathered samples from 18 lynx populations to assess genome-wide genetic diversity and structure using Restriction-site Associated DNA (RAD) sequencing. These populations consist of reintroduced (Swiss Alps, Jura Mountain, Luno Mountains, Harz Mountains, Bohemian-Bavarian, Dinaric) and natural populations, including the Western Carpathian source region for most central European reintroductions. Genomic DNA from 190 lynx samples have been sequenced at a minimum of 15,000 loci of on average 150bp on an Illumina HiSeq NGS machine using the NextRad approach (Fu et al. 2017). The obtained data is used to inform applied conservation and forms a genetic baseline for a harmonized European lynx strategy as envisioned by leading lynx researchers.

Cooperation Partners
Ole Anders, Nationalpark Harz, Germany
Christine & Urs Breitenmoser, KORA, Switzerland
Peter Klinga, Technical University in Zvolen, Slovakia
Jarmila Krojerová-Prokešová, Institute of Vertebrate Biology, Czech Republic
Krzysztof Schmidt, Mammal Research Institute, Poland
Tomaž Skrbinšek, University of Ljubljana, Slovenia

Combining citizen science and conservation genomics to reveal the causes of rapid population decline in the garden dormouse

Background
The garden dormouse (Eliomys quercinus) is a medium-sized rodent that is related to the edible dormouse. Historically, garden dormice have been distributed across large parts of central and Eastern Europe. During the last decades, however, the species has disappeared from 50 percent of its former distribution and is considered extinct in some countries. The reasons for the drastic declines remain unknown. Large parts of the current garden dormouse populations live in Germany. This is why Germany bears special responsibility for conserving and protecting the species, in accordance with the National Strategy on Biological Diversity.

The project
The project “In search of the garden dormouse” was started in order to investigate the underlying causes for the drastic declines and to develop a suitable conservation strategy. The project was conducted as a joint effort of three research institutions and conservation NGOs, namely Senckenberg, the research group for wildlife biology at Justus-Liebig University of Giessen and The Friends of the Earth Germany (BUND). The main aim of the project was to conserve the remaining garden dormouse populations in Germany and to implement long-term conservation measures.

We conducted accompanying genomic research to reveal possible genetic causes for the population declines. While the species has deserted large sections of its eastern habitats, an opposing trend is observed in parts of its western distribution, where garden dormice are regionally abundant and occupy new areas. The underlying reasons for these divergent range dynamics as well as the genetic differentiation between the populations remain unclear. To elucidate this issue, we conducted genomic sequencing and develop a SNP-based marker system in order to be able to assign individuals to genetic lineages based on noninvasive samples.

The project ran from 2018 – 2024 and was funded by the Federal Agency for Nature Conservation with resources from the Federal Ministry for the Environment, Nature Conservation and Nuclear Safety. In May 2020 the project was awarded as “project of the UN-decade for biodiversity”.

Further information
Detailed information about the project “In search of the garden dormouse” can be found on www.gartenschlaefer.de.

Background 
Since 2012 we investigate the genetic structure and diversity within populations of the critically endangered Common hamster (Cricetus cricetus) in Central Europe. Another focus is on the genetic assessment of captive hamster breeding programmes and the monitoring of reintroduction success. Our work is closely linked to applied hamster conservation.

Between 2018 and 2023 we conducted the scientific supervision of the project Feldhamsterland funded by the german Federal Agency for Nature Conservation. The Feldhamsterland project aims at halting the dramatic decline of the species in five German model regions by implementing stategies of coexistence between agricultural farming and hamster conservation. All our common hamster projects are led by Tobias E. Reiners.

Our project partners:

• Dr. Ulrich Weinhold – Institut für Faunistik & LUBW (Landesanstalt für Umwelt Baden-Württemberg), Germany
• Julien Eidenschenk & Charlotte Kourkgy, ONCFS – (Office National de la Chasse et de la Faune Sauvage) Elsass, France
• Maurice La Haye & Gerard Müskens – Radboud Universität Nijmegen & Alterra Wageningen, The Netherlands
• Johanna Ziomek- Adam Mickiewicz University in Poznań, Poland
• Agata Banaszek – University of Białystok, Poland
• Dr. Carina Siutz, Universität Wien, Austria

Distribution in Germany and Europe

The Common hamster (Cricetus cricetus L., 1758) is a critically endangered species throughout Western Europe and listed in Annex IV of the European Habitats Directive. Population decline has been particularly dramatic in its most western distribution edge within The Netherlands, Belgium, France, and Northwestern Germany. New data show that currently a similar decline is ongoing in several parts of Central and Eastern Europe. Together with international partners we contributed to the change of the IUCN Red List status of the Common hamster to ‟critically endangered”.

Banaszek, A., Bogomolov, P., Feoktistova, N., La Haye, M., Monecke, S., Reiners, T. E., Rusin, M., Surov, A., Weinhold, U. & Ziomek, J. (2020). Cricetus cricetus. The IUCN Red List of Threatened Species 2020: e.T5529A111875852. doi.org/10.2305/IUCN.UK.2020-2.RLTS.T5529A111875852.en.

Common Hamster
The Common hamster is the only species within the genus Cricetus. A characteristic is the invert colouring of his fur, which is rare among native mammals. Adults may reach a body size of 200-300 mm and weight 200-650g. Females may reproduce up to three times per season. The size of the litter ranges from 2-8 youngs. The offspring is already weaned after 25 days. Juveniles reach sexual maturity with a few weeks only and females may reproduce within the first season.

Projects

• Genetic evaluation of the Dutch captive breeding programmes (Alterra Wageningen & Radboug University Nijmegen)
• Genetic study on wild and captive-bred common hamsters in Alsace (ONCFS – Office National de la Chasse et de la Faune Sauvage)
• Genetic monitoring of common hamsters in the Rhein-Neckar County (IFF – Institut für Faunistik, LUBW – Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg)
• Genetic monitoring of Common Hamsters in Bavaria, Germany (2018)
   

eDNA – background and analysis
Organisms continually release genetic traces in the environment. These DNA traces are known as ‘environmental DNA’ (eDNA). eDNA can be obtained from environmental samples such as soil, sediment, water and air. Primary eDNA sources are excretion and secretion products as well as degrading tissue. eDNA can be detected using different highly sensitive molecular genetic methods. eDNA barcoding allows for the (semi) quantitative assessment of individual species using real-time PCR methodology. eDNA metabarcoding via Next Generation Sequencing enables to assess entire species communities and ecosystems. Both methods provide important information on the occurrence and abundance of species and community assemblages.

eDNA research focus
The main focus of our work is the eDNA-based monitoring of aquatic organisms. The genetic monitoring includes endangered species (amphibians: great crested newt Triturus cristatus, common spadefoot toad Pelobates fuscus; fish: spirlin Alburnoides bipunctatus, Atlantic salmon Salmo salar), invasive species (e.g. pumpkinseed Lepomis gibbosus, topmouth gudgeon Pseudorasbora parva) and waterborne pathogens (the crayfish plague agent Aphanomyces astaci). We primarily use eDNA barcoding of individual species due to very reliable (semi-) quantitative results.

Within the scope of our research, basic and applied research go hand in hand. New sampling and analysis methods for accurate eDNA analytics are continuously examined and optimized. Another focus lies on the characterization of the behavior of eDNA in the environment (e.g. distribution mechanisms, accumulation/degradation rates). Our aim is to standardize eDNA-based detection methods for routine use in applied biomonitoring and species protection.

We are currently working on the development and establishment of an eDNA detection system based on microfluid technology for fish (FishChip) and amphibians (FrogChip) as part of a LOEWE-TBG project. These eDNA chips are intended to (semi-) quantitatively detect a large number of aquatic species quickly, precisely and in parallel at numerous sample sites. These microfluidic chips will combine the advantages of eDNA-based barcoding (robustness, semi-quantitative data) and metabarcoding (parallel detection of numerous species).