Understanding spatio-temporal patterns of deer-vehicle collisions to improve roadkill mitigation.

Vehicles collide with hundreds of thousands of deer on European roads each year. This leads to animal deaths and suffering, economic damage and risks for human safety, making the reduction of road mortality a major field in conservation biology. In order to successfully reduce roadkill, we need improved knowledge regarding spatio-temporal patterns of deer-vehicle collisions (DVCs) on a landscape scale. Here, we analyzed >85,000 DVCs collected over 17 years in Denmark to investigate changes in the number of DVCs over time and to find spatio-temporal patterns of DVC occurrence. We used a use-availability design - originally developed for habitat selection analyses - to compare DVCs involving roe deer (Capreolus capreolus), red deer (Cervus elaphus) and fallow deer (Dama dama) with random road locations on a landscape scale. This approach enabled us to combine temporal (seasonal and diel variation), spatial (land cover, road density and type) and other relevant variables (deer population density, traffic, and deer activity) within the same analysis. We found that factors related to infrastructure and land cover were most important in explaining patterns of DVCs, but seasonal and diel changes, deer activity, and population density were also important in predicting the occurrence of DVCs. Importantly, patterns of DVCs were largely similar between the three deer species, with more DVCs occurring at intermediate traffic density, increasing forest cover, during dusk and dawn, and with increasing deer activity and population density. The strong and consistent patterns found here will allow the development of flexible mitigation measures. We propose that our findings could be used to develop a spatio-temporally flexible warning system for smartphones and navigation systems that is based on existing map providers, making it a widely available and cheap mitigation measure.

High exposure rates of anticoagulant rodenticides in predatory bird species in intensively managed landscapes in Denmark.

The extensive use of anticoagulant rodenticides (ARs) for rodent control has led to widespread secondary exposure in nontarget predatory wildlife species. We investigated exposure rates and concentrations of five ARs in liver samples from five raptors and six owls from Denmark. A total of 430 birds were analysed. ARs were detected in 84-100 % of individual birds within each species. Multiple AR exposure was detected in 73 % of all birds. Average number of substances detected in individual birds was 2.2 with no differences between owls and raptors. Difenacoum, bromadiolone, and brodifacoum were the most prevalent substances and occurred in the highest concentrations. Second-generation ARs made up 96 % of the summed AR burden. Among the six core species (sample size >30), summed AR concentrations were lower in rough-legged buzzard (Buteo lagopus) and long-eared owl (Asio otus) than in barn owl (Tyto alba), buzzard (B. buteo), kestrel (Falco tinnunculus), and tawny owl (Strix aluco). There was a strong tendency for seasonal variations in the summed AR concentration with levels being lowest during autumn, which is probably related to an influx of less-exposed migrating birds from northern Scandinavia during autumn. High hepatic AR residue concentrations (>100 ng/g wet weight), which have been associated with symptoms of rodenticide poisoning and increased mortality, were recorded high frequencies (12.9-37.4 %) in five of the six core species. The results suggest that the present use of ARs in Denmark, at least locally, may have adverse effects on reproduction and, ultimately, population status in some raptors and owls.

Mercury exposure and risk assessment for Eurasian otters (Lutra lutra) in Denmark.

Mercury (Hg) exposure may cause a wide range of adverse effects in mammals. A piscivorous apex predator, as the Eurasian otters (Lutra lutra) may accumulate and biomagnify heavy metals and pollutants. Here we investigate the Hg burden in 117 otters from Denmark to evaluate Hg concentrations and risks of health effects. Mercury concentrations ranged between 0.02 and 10.1 µg/g wet weight (ww) in liver tissue and 0.30-40.0 µg/g dry weight in fur. Concentrations in liver and fur in individual otters were highly correlated (P < 0.001). Generalized linear modelling showed that mercury concentrations in both liver and fur increased with age (liver: P < 0.002; fur P < 0.05) and with distance to the marine coastline i.e. towards freshwater habitats (P = 0.0526), and δ13C in muscle tissues (P < 0.001). No differences were detected between males and females (P > 0.59) and no correlation were detected between Hg concentration and trophic level (δ15N) (liver: P = 0.35; fur: P = 0.54). Mercury concentrations in the liver exceeded the No Risk threshold in 15% of the otters, and 25% had levels above the lowest observed effect level of 3.4 µg/g ww that may be associated with adverse effects on the central nervous system. The proportion exceeding these thresholds should therefore be monitored to document possible temporal and spatial trends in Hg exposure and the potential risk to the conservation status of the otter population.

A comparison of microsatellites and genome-wide SNPs for the detection of admixture brings the first molecular evidence for hybridization between Mustela eversmanii and M. putorius (Mustelidae, Carnivora).

Introgressive hybridization can pose a serious threat to endangered species which have an overlapping distribution such as in the case of two polecat species, Mustela eversmanii and M. putorius, in Europe. The population size of steppe polecat is known to continuously shrink, whereas its sister species, the European polecat, is still somehow widespread. In this study, we perform an analysis using microsatellite (SSR) and genomic (SNP) data sets to identify natural hybrids between polecats. Four populations were genotyped for eight polymorphic SSR loci, and thousands of unlinked SNPs were generated using a reduced-representation sequencing approach, RADseq, to characterize the genetic make-up of allopatric populations and to identify hybrids in the sympatric area. We applied standard population genetic analyses to characterize the populations based on their SSR allelic frequency. Only a single sample out of 48 sympatric samples showed exact intermediacy that we identified as an F1 hybrid. Additionally, one specimen was indicated in the genomic data sets as backcrossed. Other backcrosses, indicated by SSRs, were not validated by SNPs, which highlights the higher efficacy of the genomic method to identify backcrossed individuals. The low frequency of hybridization suggests that the difference in habitat preference of the two species may act as a barrier to admixture. Therefore, it is apparently unlikely that polecat populations are threatened by significant introgression. The two species showed a clear genetic differentiation using both techniques. We found higher genetic diversity values in the sympatric steppe polecat population than in the other studies on polecat populations. Although M. putorius is a hunted species in most countries, genetic diversity values indicate worse conditions in Europe than in the protected sibling species M. eversmanii. Suspending hunting and providing protected status of the former seems to be reasonable and timely.

Different increase rate in body mass of two marten species due to climate warming potentially reinforces interspecific competition.

Many species show spatial variation in body size, often associated with climatic patterns. Studying species with contrasting geographical patterns related to climate might help elucidate the role of different drivers. We analysed changes in the body mass of two sympatric medium-sized carnivores-pine marten (Martes martes) and stone marten (Martes foina)-across Europe over 59 years. The body mass of pine marten increased with decreasing latitude, whereas stone marten body mass varied in a more complex pattern across its geographic range. Over time, the average body mass of pine martens increased by 255 g (24%), while stone marten by 86 g (6%). The greatest increase of body mass along both martens' geographic range was observed in central and southern Europe, where both species occur in sympatry. The body mass increase slowed down over time, especially in allopatric regions. The average pine/stone marten body mass ratio increased from 0.87 in 1960 to 0.99 in 2019, potentially strengthening the competition between them. Thus, a differential response in body size to several drivers over time might have led to an adaptive advantage for pine martens. This highlights the importance of considering different responses among interacting species when studying animal adaptation to climate change.

The potential of VKORC1 polymorphisms in Mustelidae for evolving anticoagulant resistance through selection along the food chain.

In response to strong selection, new mutations can arise quickly and sweep through populations, particularly, if survival and reproduction depend on certain allele copies for adaptation to rapidly changing environments, like resistance against deadly diseases or strong toxins. Since the 1950s, resistance to anticoagulant rodenticides in several rodents has emerged through single nucleotide mutations in the vitamin-K-epoxid-reductase-complex-subunit-1 (VKORC1) gene, often located in its exon 3. Detection of high prevalence and concentrations of anticoagulant rodenticides in non-target vertebrates, including carnivorous Mustelidae, let us assume that secondary exposure by feeding on poisoned prey may also cause selection along the food chain and we hypothesized that VKORC1-based resistance might also have evolved in rodents' predators. Using newly-developed mustelid-specific primers for direct sequencing of genomic DNA, we studied VKORC1-DNA-polymorphisms in 115 mustelids of five species (Martes martes, M. foina, Mustela nivalis, M. erminea, M. putorius), obtained from northern Denmark, yielding six sites with nonsynonymous and several synonymous amino acid polymorphisms in exon 3. Comparison of these VKORC1-genotypes with hepatic rodenticide residues (obtained by HPLC combined with fluorescence or mass spectrometry) in 83 individuals (except M. martes), using generalized linear models, suggested that anticoagulant levels depended on species and specific polymorphisms. Although most VKORC-1 polymorphisms may present standing genetic variation, some are situated in resistance-mediating membrane parts of the VKORC1-encoded protein, and might be a result of selection due to exposure to anticoagulant poisons. Our new molecular markers might allow detecting indirect effects of anticoagulant rodenticides on rodent predator populations in the future.

Exposure of non-target small mammals to anticoagulant rodenticide during chemical rodent control operations.

The extensive use of anticoagulant rodenticides (ARs) results in widespread unintentional exposure of non-target rodents and secondary poisoning of predators despite regulatory measures to manage and reduce exposure risk. To elucidate on the potential vectoring of ARs into surrounding habitats by non-target small mammals, we determined bromadiolone prevalence and concentrations in rodents and shrews near bait boxes during an experimental application of the poison for 2 weeks. Overall, bromadiolone was detected in 12.6% of all small rodents and insectivores. Less than 20 m from bait boxes, 48.6% of small mammals had detectable levels of bromadiolone. The prevalence of poisoned small mammals decreased with distance to bait boxes, but bromadiolone concentration in the rodenticide positive individuals did not. Poisoned small mammals were trapped up to 89 m from bait boxes. Bromadiolone concentrations in yellow-necked mice (Apodemus flavicollis) were higher than concentrations in bank vole (Myodes glareolus), field vole (Microtus agrestis), harvest mouse (Micromys minutus), and common shrew (Sorex araneus). Our field trials documents that chemical rodent control results in widespread exposure of non-target small mammals and that AR poisoned small mammals disperse away from bating sites to become available to predators and scavengers in large areas of the landscape. The results suggest that the unintentional secondary exposure of predators and scavengers is an unavoidable consequence of chemical rodent control outside buildings and infrastructures.

Conservation genetics of the pond bat (Myotis dasycneme) with special focus on the populations in northwestern Germany and in Jutland, Denmark.

Conservation genetics is important in the management of endangered species, helping to understand their connectivity and long-term viability, thus identifying populations of importance for conservation. The pond bat (Myotis dasycneme) is a rare species classified as "Near Threatened" with a wide but patchy Palearctic distribution. A total of 277 samples representing populations in Denmark, Germany, Latvia, Hungary, and Russia were used in the genetic analyses; 224 samples representing Denmark, Germany, and Russia were analyzed at 10 microsatellite loci; 241 samples representing all areas were analyzed using mitochondrial D-loop and cytochrome B sequences. A Bayesian clustering approach revealed two poorly resolved clusters, one representing the Danish and German groups and the other the Russian group. However, significantly different pairwise F ST and D EST estimates were observed between the Danish and German groups and between the Danish and Russian groups suggesting a recent population structure. These conflicting results might be attributed to the effect of migration or low resolution due to the number of microsatellite markers used. After concatenating the two mitochondrial sequences, analysis detected significant genetic differentiation between all populations, probably due to genetic drift combined with a founder event. The phylogenetic tree suggested a closer relationship between the Russian and Northern European populations compared to the Hungarian population, implying that the latter belongs to an older ancestral population. This was supported by the observed haplotype network and higher nucleotide diversity in this population. The genetic structuring observed in the Danish/German pond bat stresses the need for a cross-border management between the two countries. Further, the pronounced mtDNA structuring, together with the indicated migration between nearby populations suggest philopatric female behavior but male migration, emphasizes the importance of protecting suitable habitat mosaics to maintain a continuum of patches with dense pond bat populations across the species' distribution range.

Exposure of stone marten (Martes foina) and polecat (Mustela putorius) to anticoagulant rodenticides: Effects of regulatory restrictions of rodenticide use.

When anticoagulant rodenticides (ARs) are used to control rodent populations there is also a widespread secondary exposure of non-target predators to ARs. To reduce secondary exposure, regulatory restrictions in AR usage were tightened in Denmark in 2011. The restrictions included the cessation of AR use for plant protection and any use away from buildings, as well as limitations in private consumers' access to ARs. To quantify and evaluate the efficiency of the regulatory measures to reduce secondary exposure, we analysed ARs in liver tissue from 40 stone martens (Martes foina) and 40 polecats (Mustela putorius) collected before and 31 stone martens and 29 polecats collected after the restrictions were imposed. No declines in the prevalence ARs were detected following the regulatory restrictions in either stone marten (Before: 98%, After: 100%) or polecat (Before: 93%, After: 97%). The total AR concentration was higher in stone martens than in polecats in both sampling periods. Between the two sampling periods, the total AR concentrations in the mustelids increased (P<0.001). The increase was significant for stone marten (Before: 419ng/g ww, After: 1116ng/g ww, P<0.001), but not for polecat (Before: 170ng/g ww, After: 339ng/g ww). Overall, the total AR concentration was positively correlated to the urban area and the area used for Christmas tree production in which ARs were regularly used before 2011. The regulatory restrictions in AR usage did not reduce exposure of non-target stone martens and polecats. The temporal and spatial patterns of AR concentrations in predators indicate that chemical rodent control in and around buildings is the dominant source for the exposure of non-target predators in intensively human-dominated landscapes in Denmark. The results suggest that non-chemical methods for rodents control at buildings are necessary to prevent widespread secondary AR exposure of predators in human modified landscapes.

Homogenous Population Genetic Structure of the Non-Native Raccoon Dog (Nyctereutes procyonoides) in Europe as a Result of Rapid Population Expansion.

The extent of gene flow during the range expansion of non-native species influences the amount of genetic diversity retained in expanding populations. Here, we analyse the population genetic structure of the raccoon dog (Nyctereutes procyonoides) in north-eastern and central Europe. This invasive species is of management concern because it is highly susceptible to fox rabies and an important secondary host of the virus. We hypothesized that the large number of introduced animals and the species' dispersal capabilities led to high population connectivity and maintenance of genetic diversity throughout the invaded range. We genotyped 332 tissue samples from seven European countries using 16 microsatellite loci. Different algorithms identified three genetic clusters corresponding to Finland, Denmark and a large 'central' population that reached from introduction areas in western Russia to northern Germany. Cluster assignments provided evidence of long-distance dispersal. The results of an Approximate Bayesian Computation analysis supported a scenario of equal effective population sizes among different pre-defined populations in the large central cluster. Our results are in line with strong gene flow and secondary admixture between neighbouring demes leading to reduced genetic structuring, probably a result of its fairly rapid population expansion after introduction. The results presented here are remarkable in the sense that we identified a homogenous genetic cluster inhabiting an area stretching over more than 1500km. They are also relevant for disease management, as in the event of a significant rabies outbreak, there is a great risk of a rapid virus spread among raccoon dog populations.

Distribution and molecular phylogeny of biliary trematodes (Opisthorchiidae) infecting native Lutra lutra and alien Neovison vison across Europe.

The recent identification of Pseudamphistomum truncatum, (Rudolphi, 1819) (Trematoda: Opisthorchiidae) and Metorchis bilis (Braun, 1790) Odening, 1962 (synonymous with Metorchis albidus (Braun, 1893) Loos, 1899 and Metorchis crassiusculus (Rudolphi, 1809) Looss, 1899 (Trematoda: Opisthorchiidae)) in otters from Britain caused concern because of associated biliary damage, coupled with speculation over their alien status. Here, we investigate the presence, intensity and phylogeny of these trematodes in mustelids (principally otters) across Europe (Czech Republic, Denmark, France, Germany, Norway, Poland and Sweden and Britain). The trematodes were identified to species using the internal transcribed spacer II (ITS2) locus. Both parasites were found across Europe but at unequal frequency. In the German state of Saxony, eight out of eleven (73%) otters examined were infected with P. truncatum whilst this parasite was not found in either mink from Scotland (n=40) or otters from Norway (n=21). Differences in the phylogenies between the two species suggest divergent demographic histories possibly reflecting contrasting host diet or competitive exclusion, with M. bilis exhibiting greater mitochondrial diversity than P. truncatum. Shared haplotypes within the ranges of both parasite species probably reflect relatively unrestricted movements (both natural and anthropogenic) of intermediate and definitive hosts across Europe.

Concentrations of anticoagulant rodenticides in stoats Mustela erminea and weasels Mustela nivalis from Denmark.

Anticoagulant rodenticides are widely used to control rodent populations but they also pose a risk of secondary poisoning in non-target predators. Studies on anticoagulant rodenticide exposure of non-target species have mainly reported on frequency of occurrence. They have rarely analyzed variations in residue concentrations. We examine the occurrence and concentrations of five anticoagulant rodenticides in liver tissue from 61 stoats (Mustela erminea) and 69 weasels (Mustela nivalis) from Denmark. Anticoagulant rodenticides were detected in 97% of stoats and 95% of weasels. 79% of the animals had detectable levels of more than one substance. Difenacoum had the highest prevalence (82% in stoats and 88% in weasels) but bromadiolone was detected in the highest concentrations in both stoat (1.290 µg/g ww) and weasel (1.610 µg/g ww). Anticoagulant rodenticide concentrations were highest during autumn and winter and varied with sampling method. Anticoagulant rodenticide concentrations were higher in stoats and weasels with unknown cause of death than in specimens killed by physical trauma. There was a negative correlation between anticoagulant rodenticide concentrations and body condition. Our results suggest that chemical rodent control in Denmark results in an extensive exposure of non-target species and may adversely affect the fitness of some stoats and weasels.