The threat of isolation to red deer (Cervus elaphus) has been described in numerous European studies. The consequences range from reduced genetic diversity and increased inbreeding to inbreeding depression. It has been shown that the underlying factors cannot be generalised, but vary greatly in their effects depending on local conditions. The aim of this study was to analyse in detail the genetics of red deer in a large German federal state with a population density of 532 inhabitants per km² and 23.8% settlement and traffic area, in order to generate data for future management of the region. 1199 individual samples of red deer were collected in all 21 Administrative Management Units (AMUs) and compared with existing results from the neighbouring state of Hesse (19 AMUs). All 2400 individuals from both states were clustered using Bayesian methods and connectivity between neighbouring AMUs was quantified. Overall, 30% of the AMUs were found to be highly isolated, mostly with effective population sizes (Ne) < 100. In contrast, 47.5% of the AMUs still had clear connectivity, allowing them to be merged into 4 larger red deer regions. For the small isolated areas, low genetic diversity was found in unit with high homozygosity and low Ne. With high sampling density and identical methodology, detailed information on AMUs can be obtained and the degree of vulnerability of individual AMUs as part of the overall population can be validated. These data will be available for future red deer management in the study area and beyond.

Study area, red deer populations and sampling

The study area covers the entire region of the federal states of Hesse and North Rhine-Westphalia (NRW) in central and western Germany with a north-south extension of 350 km, a west-east extension of 290 km and a total area of approximately 55,213 km².

Compared to the German average of 237 persons per km², the population density in 2023 was about 531.7 persons per km² in North Rhine-Westphalia and 302.6 persons per km² in Hesse (Statistisches Bundesamt 2024). Hesse consists of different types of land use, mainly forests (42.5%), pastures (13.4%) and agriculture (22.6%). Hesse is the German state with the largest forest area. The corresponding land use proportions for forest, pasture and agriculture in NRW were 26.9%, 12.1% and 31.1, respectively.

The AMUs are scattered across the two states and vary considerably in size from 41.3 (RK) to 787.2 km² (TAU) (Table 1). Forty AMUs were surveyed. The distances between AMUs ranged from 7.64 (DB-SIO) to 321.41 km (OD-RK) (centre to centre). They were separated by residential areas, fenced motorways, country roads or by larger contiguous agricultural areas. In the ‘red deer–free’ areas between AMUs, establishment of red deer populations should be prevented to protect vegetation from damage. Hunting in these areas could impede the migration of red deer between AMUs (Herzog et al. 2020). According to the local authorities and the red deer management associations legally responsible for the management of the respective AMUs, the estimated population size of the AMUs in spring ranged from 59 animals in Wattenberg-Weidelsburg to 3480 animals in Eifel-Zitterwald-Mürrel (Table 1). However, these figures are based on estimates, as exact numbers are not available. The estimates are based on calculations of hunting bag numbers according to the Oberste Jagdbehörde Hessen/HMUKLV (2023) in Hesse and according to Müller et al. (2020) in NRW (Forschungsstelle für Jagdkunde/LANUV 2022a, b).

Samples were labelled with information on the animal (sex and age class) and the hunting area, and frozen until processed in the laboratory. A total of 1199 samples were collected in NRW during the 2020-2022 hunting seasons. No animals were killed specifically for the study. No live animals were sampled and no shed antlers were sought or collected for the study. Due to the impact of sample size on the accuracy of population genetic results (Reiner et al. 2019), the goal was to collect 60 samples per area. In the end, an average of 63.8 samples were collected per area (0.194 individuals per km²). The minimum sample size was 12 samples in Nutscheid (see Tables 1A and B for details). AMUs with close geographical/political links were grouped together to form ‘larger regions’, e.g. the Eifel (Point 1) with the individual AMUs 1.1 (EHU), 1.2 (ENP), 1.3 (EZM) and 1.4 (EFW) (see Table 1A).

No new samples were collected for Hesse. Rather, data from the already genotyped samples from 1291 individuals collected during the 2018/2019 hunting season (Table 1B) (Reiner et al. 2021) were used and analysed together with the newly collected and genotyped samples from NRW. In total, samples from 2490 red deer individuals were analysed.

 DNA extraction and genotyping

DNA was extracted using a commercially available kit (Instant Virus RNA Kit, Analytik Jena, Germany). For this purpose, 30 to 50 mg of tissue was processed according to the manufacturer's instructions. DNA concentration was determined photometrically and adjusted to 5 ng/μl with RNAse-free water. The presence of high molecular weight DNA was confirmed by agarose gel electrophoresis. Sixteen microsatellites were used to genotype red deer, as described in detail by Willems et al. (2016). Primers were combined in two multiplex PCRs (Reiner et al. 2021). PCR was performed in a volume of 10 μl consisting of 5 μl 2× multiplex mastermix (Qiagen, Germany), 4 μl primer mix and 1 μl (5 ng) extracted DNA. After an initial denaturation step of 15 min, the DNA was amplified in 26 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C (multiplex PCR 4 at 50 °C) for 90 s and extension at 72 °C for 30 s. After a final step at 60 °C for 30 min, the PCR reactions were cooled to 4 °C.

Capillary electrophoresis

One microliter of fluorescently labelled PCR product and 0.375 µl DNA Size Standard 500 Orange (Nimagen, The Netherlands) were added to 12 µl Hi-Di-formamide (Thermofisher Scientific, Germany) and electrophoresed on an ABI PRISM 310 automated sequencer. Allele sizes were determined using Peakscanner 2.0 software (Thermofisher Scientific, Germany).