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 2006 mitochondrial DNA sequencing of hair samples was used to delineate wildcat from domestic cat in the Senckenberg Conservation Genetics Lab. Early 2009 we added microsatellite analysis in order to allow for individual assignment, hybrid detection and fine-scale analysis of population divergence, reproductive isolation and the identification of landscape barriers. So far we have analysed several thousands of hair samples collected from valerian-treated lure sticks in Germany, Luxemburg, Austria and further regions. These data have reshaped the known distribution range of wildcats in central Europe and have led to the discovery of wildcat presence in numerous regions, such as the Kellerwald-Edersee National Park and UNESCO world heritage site, the Rhön mountains, the Steiermark region in Austria or in Saxony.  Moreover, we estimate population size of regional populations and have estimated the effects of major landscape elements, such as the Rhine river valley or the Autobahn A3 in Germany (Hartmann et al., 2013). Our analyses are done in close cooperation with the BUND (Friends of the Earth Germany). 

Kathrin and Annika conduct the wildcat research in the frame of their PhD projects, and Susanne, Dino, and Mascha conduct lab analyses, sample logistics, and help in methodological improvements.
 
The wildcat
The European wildcat 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
The lure stick method (Simon and Hupe, 2007; Steyer et al., 2013) allows for simple and effective wildcat detection and monitoring. Since its routine implementation in wildcat monitoring activities in Germany it has rapidly expanded the knowledge on wildcat distribution across the country. The method is straightforward: A simple wooden stick (60 cm) is roughened with a steel brush and sharpened on one side. Drive the stick into the ground, so that it stands solid and treat it with valerian extract (from the pharmacy) as scent lure. Cats will be attracted, rub, and leave hairs that can be sampled every few days up to a week.  Check this clip for Kathrin’s charming YouTube instructions. Enjoy! 

DNA analysis of hair samples
Hairs contain low amounts of DNA, mostly in the hair follicle. This DNA has to be preserved and treated with caution until it arrives in the laboratory where we will take care of it. Please consider the following golden rules:

• Keep hairs dry, humidity leads to rapid DNA degradation, please use our envelopes with silica gel.
• Sample several hairs with follicles from the lure stick. The more hairs we have the higher is the chance of successful analysis.
• Do not freeze or cool the hairs in the fridge. Place them at constant room temperature or slightly below, keep samples dark, send them to us asap.
• Keep samples in a closed bag, to avoid contamination with cat hairs.
• Transfer hairs with forceps, do not use tape. 

Analysis of mitochondrial DNA haplotypes
Mitochondrial sequence analysis is widely used for species discrimination and identification. DNA-barcoding, for instance, relies on a small stretch of mitochondrial DNA (mtDMA). MtDNA is more frequent in the cell than nuclear (chromosomal) DNA and is therefore easier to extract from hair samples compared to nuclear DNA. MtDNA analysis is often successful with only a single hair, it allows for a reasonable distinction between wild- and domestic cats, but it will not account for the effects of hybridisation nor is it 100% safe. It only reveals the maternal ancestral lineage of a sample, as mtDNA is inherited via the mother’s egg only. A hybrid between a domestic male cat and a wildcat female will show a mitochondrial wildcat haplotype. Moreover, wildcats and domestic cats share some common haplotypes. Thus, we use mtDNA mostly as a fast prescreening to identify potential wildcat samples that can be used for microsatellite analysis.

A genetic fingerprint for cats: microsatellites

A nuclear microsatellite analysis is feasible if a hair sample contains several hairs with bulbs. For this we run 14 variable microsatellite markers and a sex marker with three PCR replicates per hair sample. Fragment lengths of the replicates are compared to exclude genotyping errors and generate a consensus genotype. The resulting genotype information is used to assign individuals, calculate relatedness and assess population structure. Our reference dataset of >500 morphologically checked wild- and domestic cats of various origins ensures safe discrimination of both forms and the identification of hybrids.

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.

Since the1st of July 2015 the sample form (Excel) cannot be used any more. Please use our online tool: www.wildtiergenetik.de.

• Handling and shipping of sample material
• Costs and contact details

Important Notes

Please read the information sheets above thoroughly and particularly note the following:

Use of data: We reserve the use of the data that we obtained from your samples for ourselves in the form of presentations and publications. Exceptions from this rule must be discussed in advance of sample processing. The wolf monitoring data remain in the possession of the federal states of Germany.
Online tool: You must use our online tool to place an order.
Options for scientific cooperation

In the framework of scientific cooperation, the Conservation Genetics Section provides the following services regarding the identification and analysis of mammal species:

• DNA extraction
• Analysis of mitochondrial DNA
• Analysis using Microsatellites

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 is 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 is to conserve the remaining garden dormouse populations in Germany and to implement long-term conservation measures.

We conduct 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 conduct 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 runs from 2018 – 2024 and is 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”, options to join in, and an online reporting tool for garden dormouse observations 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. Since 2018 we conduct 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 are

• 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).