Isolated adders

In 2021, Judith Grünewald and I commenced the fieldwork of our master theses in cooporation with the Black Forest National Park. Having worked with adders in the previous years, we decided to devote most of our theses to the genetic constitution of adder populations in Baden-Württemberg, mainly the Black Forest. These populations have suffered heavily from anthropogenic pressure in the last decades, leaving us with the question, weather this was already visible in the genetic consitution of the populations. Despite expecting to find generally high genetic differentiation, we were surprised by the low, nearly absent gene flow between populations. We furthermore found two sampling sites to be substructured into subclusters, indicating isolation by anthropogenic structures. Genetic diversity displayed a high variability with some sites having very high assessments of genetic diversity, while others displayed multiple signs of genetic erosion. Our study in the end shows how sensitive adder populations react to anthropogenic disruptions and how decades of urbanization, forestry and agriculture have isolated adder populations in Baden-Württemberg. 

For the genetic analyses, we selected eight sites, seven of which were located in the Black Forest and one in the Swabian Jura (see map on the left). Adder habitats have suffered from changes in forestry and agriculture as well as urbanization over decades. Since large adder populations have become rare in Baden-Württemberg, sampling site selection was limited to the few sites left. We nevertheless did our best to choose sites with different habitat compostions, some being located in the highands, some in the valleys, some in natural areas and others in anthropogenically impacted habitats.

We sampled all sites during a similar amount of visits and (whenever possible) similar weather conditions. For sampling, we aquired permits by the regional councils of Karlsruhe, Freiburg and Tübingen. Further measurements of the snakes such as body length, tail length, weight, temperature (at three locations on the underside of the snakes) and of microhabitat, e.g. soil moisture and soil temperature were taken during the sampling. The genetic samples were taken via buccal swab. Generally, this procedure took about 3-7 minutes per snake, which was then released in the exact position it was found in. Habitat measurements were taken after release and dataloggers were installed at all sites.

In total, we aquired 139 samples from the sites NBF (15), NP1 (15), NP2 (19), NP3 (27), MV (12), CBF (14), SBF (19) and SA (10) as well as eight individuals between the sites NP2 - NP3.

We carried out our labwork at the Institute for Organismis and Molecular Evolution (imoE) at the University of Mainz. This mainly concerned DNA extraction and PCR. The samples were then brought to StarSeq for sequencing. For the analyses, we selected 13 microsatellite markers, out of which 10 proved suitable for the final dataset. Microsatellites (msats), also known as short tandem repeats (STRs) are non-coding areas of the genome, that consist of tandem like base pairs, the repeat units, that are repeated multiple times. The number of these repetitions determines the alleles and becomes visible as a peak in an electropherogramm (see picture on the right). As adders are diploid, they have a double set of chromosomes, one from the mother and one from the father. If the alleles in a marker are different, it is heterozygous, displaying two peaks (see lines 1-3, 5 & 7 in the picture). If the allele is the same in both parents, it is homozygous in the offspring, resulting in only one peak (see lines 4 & 6 in the picture). The frequency of heterozygosity/homozygosity is curcial for determining the genetic diversity within a population. 

We conducted several analyses on our final dataset, mainly concerning:

• Assessments of genetic diversity (including inbreeding)
• Assessments of genetic differentiation (Fixation indices, isolation by distance)
• Bottleneck analysis
• Structure analysis (an assessment of the true number of populations, independet from the number of sites)
• Sibship analysis

Despite expecting to find a certain level of genetic differentiation, the results of our analyses were severe. In the end, we had little to no indication of gene flow between sampling sites. On top of that, we detected signs of genetic erosion at all sampling sites:

• NBF: Average genetic diversity, historical bottleneck
• NP1: Average genetic diversity, historical bottleneck
• NP2: High genetic diversity, historical bottleneck
• NP3: Spacial substructuring, inbreeding, historical bottleneck
• MV: Low genetic diversity, historical & recent bottleneck, high relatedness
• CBF: Spacial substructuring, recent bottleneck
• SBF: Average genetic diversity, high relatedness
• SA: Low genetic diversity, inbreeding

We investigated the spacial substructuring of the CBF and NP3 more in-depth as these indicated that our sampling sites, assumed as single populations, were actually divided into several sub-populations. 

For the CBF site, we identified a small town as the main barrier to gene flow, separating the site into two genetically distinct sub populations. For NP3 however, the barrier to gene flow was a single road, crossing the site. Despite sampling the snakes on both roadsides, only 5 m apart from one another, the sub populations appeared to be genetically different. Additionally, the sub population north of the road had the highest measured inbreeding coefficient in our study (FIS = 0.213). This indicates clearly how negatively anthropogenic barriers can impact adder populations. Monitoring of this site will determine if an inbreeding depression has already taken place and weather genetic rescue should be considered. 

Generally, our results show how the historical and recent changes in forestry and agriculture as well as the increasing pressure from urbanization have isolated adder populations. As all sites display signs of genetic erosion, some more some less severe, it is highly important to re-establish gene flow between populations for the long-term survival of the species. This becomes especially important in the face of global warming, where high genetic diversity is crucial for adaptation to changing climates.