Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA‐based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 μm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor‐prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

Environmental DNA (eDNA) has evolved into a valuable asset of the ecologists' toolkit, enabling time‐ and cost‐efficient biodiversity assessments in a wide variety of ecosystems. Since eDNA can be isolated from a broad range of environmental substrates, its persistence times in those media are of decisive importance for drawing inferences about species presence. For the first time, we characterize eDNA persistence in water and sediment samples of seminatural waterholes in a savanna system in South Africa to gain a better understanding of eDNA decay in these waterbodies. Using mesocosm experiments, we tracked eDNA decay in two different DNA states (extracellular and membrane bound), during two different seasons (wet and dry), and from two different substrates (surface water and sediment). Extracellular DNA degraded rapidly in a first‐order exponential decay fashion and membrane‐bound DNA exhibited a slower decline with more intricate patterns, involving initial reduction followed by a subsequent increase in measured DNA concentrations. The latter we attribute to cell disassociation and cell lysis at 24–48 h after introduction into the environment. Higher stochasticity of membrane‐bound DNA capture in the dry season highlights the need for higher sampling efforts in natural systems in which eDNA is presumably more patchily distributed. Additionally, we observed longer eDNA persistence in sediments than in water samples, presumably due to better protection from nucleases. We could not reveal any effects of environmental parameters on eDNA decay, emphasizing that further research is needed to better understand eDNA dynamics in those waterbodies in order to exploit their full potential for eDNA‐based bioassessments in those systems.

Applying environmental DNA (eDNA) metabarcoding to samples from waterholes and their surroundings offers a promising approach for monitoring terrestrial vertebrates in semi‐arid and arid ecosystems, such as the southern African savannas. However, minimal guidance exists on key sampling design parameters for terrestrial ecosystems, which can significantly influence species detection. This study investigated the effects of sampled substrate, sampling season, and metabarcoding primer pair on species richness and taxonomic group detection in terrestrial vertebrates, with a focus on mammals, using eDNA samples from waterholes in Botsalano Game Reserve, South Africa. A total of 725 eDNA samples were collected from 94 sampling events across wet and dry seasons, detecting 95 species (45 birds, 42 mammals, 4 amphibians, 3 reptiles, and 1 fish). Sediment samples provided more reliable detection of abundant taxa, whereas water samples had higher detection frequencies of rare taxa. A mixed sampling approach yielded the highest species richness. Sampling during the wet season yielded higher species richness overall, while more mammal species were detected from dry season sampling. Overlap in species detection between the two metabarcoding primers tested was low (47%). We formulate recommendations for future eDNA metabarcoding study designs in similar systems, including remote sampling logistics and discuss potential sources of false positives in eDNA metabarcoding, including (1) secondary eDNA input, (2) incomplete genetic reference databases, and (3) the low genetic resolution of metabarcoding markers.

The Alpine salamander (Salamandra atra) is a cold-adapted amphibian with low dispersal capacity, endemic to the Alps and Dinarides. Isolated populations at the range’s margins are especially vulnerable to habitat fragmentation and genetic erosion. We investigated the population genetic structure of S. atra in the Koralpe, a biogeographically important mountain range in southeastern Austria. Using mitochondrial DNA, we found that the Koralpe population harbors unique genetic variants not shared with other known populations. Strong genetic differentiation and low connectivity among Koralpe subpopulations, inferred from microsatellites, indicate long-term isolation, likely caused by unsuitable intervening habitats and the species’ limited dispersal capacity. Although the estimated effective population size (Ne = 245) is moderate and no severe genetic bottlenecks were detected, subpopulation sizes are likely small. These findings highlight the conservation value of this peripheral population and support its recognition as a distinct management unit. In situ protection, improved landscape connectivity, and continued (genetic) monitoring are essential for the population’s long-term survival. Given its unique genetic signature and pronounced structuring, targeted conservation measures are critical—especially under increasing pressure from climate change and habitat degradation. Preserving this isolated lineage will contribute to local biodiversity and help safeguard the evolutionary potential of S. atra as a whole.

Applying environmental DNA (eDNA) metabarcoding to samples from waterholes and their surroundings offers a promising approach for monitoring terrestrial vertebrates in semi-arid and arid ecosystems, such as the southern African savannas. However, minimal guidance exists on key sampling design parameters for terrestrial ecosystems, which can significantly influence species detection. This study investigated the effects of sampled substrate, sampling season, and metabarcoding primer pair on species richness and taxonomic group detection in terrestrial vertebrates, with a focus on mammals, using eDNA samples from waterholes in Botsalano Game Reserve, South Africa. A total of 725 eDNA samples were collected from 94 sampling events across wet and dry seasons, detecting 95 species (45 birds, 42 mammals, 4 amphibians, 3 reptiles, and 1 fish). Sediment samples provided more reliable detection of abundant taxa, whereas water samples had higher detection frequencies of rare taxa. A mixed sampling approach yielded the highest species richness. Sampling during the wet season yielded higher species richness overall, while more mammal species were detected from dry season sampling. Overlap in species detection between the two metabarcoding primers tested was low (47%). We formulate recommendations for future eDNA metabarcoding study designs in similar systems, including remote sampling logistics and discuss potential sources of false positives in eDNA metabarcoding, including (1) secondary eDNA input, (2) incomplete genetic reference databases, and (3) the low genetic resolution of metabarcoding markers.

Environmental DNA (eDNA) is increasingly used to monitor terrestrial mammal biodiversity, yet factors that influence detection probability in such systems remain poorly understood. Substrates in and around freshwater drinking areas represent particularly promising eDNA sampling points to capture terrestrial biodiversity, especially in arid and semi-arid landscapes. However, eDNA detection probabilities are expected to depend strongly on the frequency and intensity of animal interaction with the sampled substrate at these watering holes. We combined camera trapping with eDNA metabarcoding to investigate drivers of mammal eDNA detection at savanna waterholes in South Africa (Botsalano Game Reserve) over several weeks during both wet and dry seasons and evaluated the effects of species behavior (interaction intensity with water), species traits, environmental covariates and substrate type (water versus sediment) on eDNA detection probabilities. Camera trapping detected 30 mammal species, whereas eDNA metabarcoding detected 40 species, with 25 species being shared between methods. Body size increased detection probability using camera traps but not using eDNA metabarcoding. eDNA detection was strongly influenced by trophic level, with herbivores exhibiting significantly higher eDNA detection rates than omnivores and carnivores. eDNA detection probability further increased with behavioral water interaction intensity, social group size, visitation frequency, the number of observed individuals, and declined with time elapsed since the last visit of the respective species. A fine-scale spatial analysis within the water bodies revealed that eDNA deposition is highly localized. Substrate type had a pronounced effect, with sediment samples reflecting more long-term mammal activity, whereas water samples reflected more recent interactions and were more sensitive to environmental conditions. Samples collected at wildlife trails exhibited similar patterns, with trophic level and spatial proximity to the trail both increasing detection rates.