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Repeated climatic and vegetation changes during the Pleistocene have shaped biodiversity in Northern Europe including Denmark. The Northern Birch Mouse (

Environmental DNA (eDNA) from water samples is increasingly used to detect the presence and distribution of species in aquatic ecosystems. However, before implementing eDNA in monitoring programmes, various species-specific sampling or analytical issues remain to be resolved in order to minimize frequencies of false-positive and -negative results. For example, empty shells from freshwater pearl mussels (Margaritifera margaritifera) contain extractable DNA (chemical extraction from ground-up shells) suggesting a risk of false-positive samples at stream sites with extinct populations but with empty shell material remaining. The aim of this study was to investigate whether empty and naturally degrading shells from M. margaritifera can cause false-positive eDNA signals in water samples. Water samples were collected from outdoor stream channels (in Lemming, Denmark) with living freshwater pearl mussels or empty shell material (density ~10 individuals m −2) during a 3-week experimental period. Living freshwater pearl mussels were collected from Hemgravs stream in Sweden and transported to Denmark according to permissions granted by the Swedish and Danish authorities. All water samples from stream channels containing empty shells were negative for eDNA indicating that eDNA traces in stream water are most likely to originate from living individuals located upstream of the sampling site. Water samples collected from stream channels containing living individuals of M. margaritifera were consistently positive for eDNA except for one sample (interpreted as a false negative). The study shows that positive eDNA signals for freshwater pearl mussels most likely reflect the presence of living individuals. Consequently, we suggest that eDNA should be used to locate remaining population fragments of M. margaritifera in deep and turbulent streams, providing a platform for faster and more efficient decision making when launching investigative and mitigation initiatives.

In the past the European otter Lutra lutra was distributed throughout most of Europe but since the 1980s its distributional range has been reduced. Currently, the otter population is increasing. Conservation efforts have been implemented, however due to the natural elusiveness of the species it is difficult to monitor. Non-invasive sampling has proven to be the most effective method to derive population parameters such as presence/absence, genetic variability and population structure in European otters. The method to collect non-invasive samples is robust and provides reliable data. This study investigates the validity of the present state-of-The-Art method of identifying otter feces, and suggests modifications and improvements of the method. Results from the comparison of field collected data and data derived from a blind test show that the method is applicable in areas abundant with otters, however the method loses its power in the periphery of the distributional range. In these areas, it would be relevant to supplement traditional sampling with DNA analysis to verify the identification of the sample.

Over the last 20 years, the application of molecular tools in biological and archaeological research has increased concurrently with technological advances. This has provided new possibilities for conducting genetic, stable isotope and fatty acid analyses on modern and historical samples, to improve our understanding of Atlantic walrus biology and demography. The use of palaeontological, archaeological and historical remains, moreover, shows great promise for disentangling the impact of anthropogenic effects and environmental change on Atlantic walrus populations. Such analyses have the potential to provide insights on particular aspects of the behavioural ecology, demography and life-history of Atlantic walruses, including changes in the migratory routes, population size, dispersal rates, choice of haul-out sites, foraging/diet and mating behaviour, in response to particular environmental or climatic conditions, as well as past and present human activities such as hunting. From an archaeological and historical point of view, much can also be learned about the role of walruses to past human societies, and even historical ivory trading networks and their links to regional hunting grounds can be reconstructed through time using molecular data from bones and teeth. Gaining these past insights will play a crucial role in designing future conservation and management strategies for Atlantic walruses and ensuring sustainable futures for humans who still rely upon them.

Identifying genetically different groups of animals, occupying specific geographical areas, is a prerequisite for conservation and management priorities. In the present study, the genetic structure of Atlantic walruses (Odobenus rosmarus rosmarus) occupying the Pechora Sea (PEC) in the western Russian Arctic, including walruses from Svalbard-Franz Josef Land (SVA-FJL) and East Greenland (EGR) regions, was investigated using 14 microsatellites (N = 159) and mtDNA sequences (N = 212). Bayesian-based clustering analysis identified two clusters: EGR and the other Northeast Atlantic areas. Pairwise F-ST analyses based on microsatellites revealed low but significant genetic differences between walruses from the PEC and SVA-FJL groups, which was supported by mtDNA analysis. F-ST was not significant for all sampling years, indicating a temporal effect or male-biased gene flow. Extended Bayesian Skyline Plots suggested a constant female subpopulation size (N-ef) for EGR and an increase for the SVA-FJL and PEC groups that commenced around 40-30 Kyr ago, indicating different demographic histories for walruses in the EGR. Further, the evolutionary phylogenetic relationship between Atlantic and Pacific walruses (O. r. divergence), based on mtDNA sequences, showed a monophyletic Atlantic clade, suggesting that Atlantic and Pacific walruses diverged similar to 949 Kyr. The principal finding suggests that PEC walruses show low, but significant genetic distinction from walruses in SVA-FJL and should be managed conservatively, as a separate, small population.

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.

Anthropogenic hybridization is widely perceived as a threat to the conservation of biodiversity. Nevertheless, to date, relevant policy and management interventions are unresolved and highly convoluted. While this is due to the inherent complexity of the issue, we hereby hypothesize that a lack of agreement concerning management goals and approaches, within the scientific community, may explain the lack of social awareness on this phenomenon, and the absence of effective pressure on decision-makers. By focusing on wolf x dog hybridization in Europe, we hereby (a) assess the state of the art of issues on wolf x dog hybridization within the scientific community, (b) assess the conceptual bases for different viewpoints, and (c) provide a conceptual framework aiming at reducing the disagreements. We adopted the Delphi technique, involving a three-round iterative survey addressed to a selected sample of experts who published at Web of Science listed journals, in the last 10 years on wolf x dog hybridization and related topics. Consensus was reached that admixed individuals should always be defined according to their genetic profile, and that a reference threshold for admixture (i.e., q-value in assignment tests) should be formally adopted for their identification. To mitigate hybridization, experts agreed on adopting preventive, proactive and, when concerning small and recovering wolf populations, reactive interventions. Overall, experts' consensus waned as the issues addressed became increasingly practical, including the adoption of lethal removal. We suggest three non-mutually exclusive explanations for this trend: (i) value-laden viewpoints increasingly emerge when addressing practical issues, and are particularly diverging between experts with different disciplinary backgrounds (e.g., ecologists, geneticists); (ii) some experts prefer avoiding the risk of potentially giving carte blanche to wolf opponents to (illegally) remove wolves, based on the wolf x dog hybridization issue; (iii) room for subjective interpretation and opinions result from the paucity of data on the effectiveness of different management interventions. These results have management implications and reveal gaps in the knowledge on a wide spectrum of issues related not only to the management of anthropogenic hybridization, but also to the role of ethical values and real-world management concerns in the scientific debate.

Accurate identification of species and populations is essential for biodiversity conservation and for monitoring of international trade in wild species. Morphological and behavioural traits are not always reliable for species identification of samples of unknown origin, and suspected hybridization augments this challenge. We used mitochondrial DNA (mtDNA) markers to identify 24 canid skins illegally imported from Mongolia to Denmark in 1997. Our results indicated that the skins included both wolf (Canis lupus) and dog (Canis lupus familiaris) haplotypes. One of the wolf haplotypes was new. A phylogenetic analysis revealed that the skins shared haplotypes with wolves in Europe and Mongolia, which appears to support the assumption of a Mongolic origin though a different provenance cannot be excluded. We observed an apparent discrepancy between the skins’ morphological traits and the mtDNA identification for 10 out of 24 individuals. This might suggest the presence of hybrids, but poor quality DNA precluded reliable microsatellite typing to investigate hybridization. At present, the scarcity of published records for wolf mtDNA sequences from central Asia limits more detailed investigation and we recommend increased efforts to document genetic variation in this region.

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.

Following protection measures implemented since the 1970s, large carnivores are currently increasing in number and returning to areas from which they were absent for decades or even centuries. Monitoring programmes for these species rely extensively on non-invasive sampling and genotyping. However, attempts to connect results of such studies at larger spatial or temporal scales often suffer from the incompatibility of genetic markers implemented by researchers in different laboratories. This is particularly critical for long-distance dispersers, revealing the need for harmonized monitoring schemes that would enable the understanding of gene flow and dispersal dynamics. Based on a review of genetic studies on grey wolves Canis lupus from Europe, we provide an overview of the genetic markers currently in use, and identify opportunities and hurdles for studies based on continent-scale datasets. Our results highlight an urgent need for harmonization of methods to enable transnational research based on data that have already been collected, and to allow these data to be linked to material collected in the future. We suggest timely standardization of newly developed genotyping approaches, and propose that action is directed towards the establishment of shared single nucleotide polymorphism panels, next-generation sequencing of microsatellites, a common reference sample collection and an online database for data exchange. Enhanced cooperation among genetic researchers dealing with large carnivores in consortia would facilitate streamlining of methods, their faster and wider adoption, and production of results at the large spatial scales that ultimately matter for the conservation of these charismatic species.

The Eurasian woodcock is a highly valued game bird in Western Europe from which c. 2.7 million individuals are harvested annually from an estimated population of 20-26 million birds. The population size and status remains uncertain due to the cryptic behaviour and widespread and solitary occurrence of woodcock, on breeding and wintering areas, making reliable population surveys difficult. Hunting bag records provide age ratios amongst bagged birds, but sex ratios remain poorly known because of the sexually monomorphic nature of this species. We used DNA analysis to determine sex ratios amongst 327 shot woodcocks from two hunting seasons in Denmark (1 October-31 January, 2012/ 13 and 2013/ 14). Based on bag totals, age ratios and sex ratios, juvenile females constituted 37%, juvenile males 27%, adult females 16% and adult males 20% of the annual woodcock bag. The female bias was related to a significant deviation from parity in the sex ratio amongst juvenile birds in October, although no such deviation was found at other times or amongst adults. Compared to limited data from other European countries, our data suggest that autumn migration of woodcock involves an initial wave of juvenile females followed by juvenile males and adults, and perhaps that males stay further north in Europe than females during autumn and winter. This migratory pattern would suggest that postponing the opening of the hunting season could reduce the hunting bag on reproductively valuable females in this polygamous species.

This study reports on the first successful identification of the site of origin of an extralimital walrus in Europe. On 24 February 2010 an adult male Atlantic walrus (Odobenus rosmarus rosmarus) migrant was instrumented with a SPOT-5 satellite-linked transmitter (SLT) while hauled out on a beach on the Faroe Islands at 62° 15' N/06° 32' W. This SLT transmitted until 5 March during which period the walrus made local movements, likely for feeding. Transmissions were not received during 6-25 March, however, visual observations during this time indicated that the walrus remained at the Faroe Islands. A second transmitter was deployed on the same animal on 25 March 2010 at another site on the islands (62° 16' N/07° 04' W). Activity data collected over 13 days indicated that the walrus hauled out in three different places in the Faroe Islands and used a total of 24% of its time resting on land. On 29 March 2010 the walrus left the Faroe Islands and headed WNW towards NE Iceland. On 2 April it took a NNE course and swam towards Svalbard where the last location was received from a sea ice covered area on 25 April 2010 at 78° 27' N/09° 20' E (i.e. ca. 40 km west of the island of Prins Karls Forland in the western Svalbard archipelago). During 29 March-22 April the walrus swam a minimum distance of 2216 km between the last location at the Faroe Islands and the first location at Svalbard, with an average swimming speed of 4.5 km/h. A genetic analysis indicated that this walrus belonged to the Svalbard-Franz Josef Land subpopulation, thereby confirming that it returned to its site of origin.

Determining the mechanisms that generate population structure is essential to the understanding of speciation and the evolution of biodiversity. Here, we investigate a geographical range that transects two habitat gradients, the North Sea to North Atlantic transition, and the temperate to subpolar regions. We studied the harbour porpoise (Phocoena phocoena), a small odontocete inhabiting both subpolar and temperate waters. To assess differentiation among putative populations, we measured morphological variation at cranial traits (N = 462 individuals) and variation at eight microsatellite loci for 338 of the same individuals from Norwegian, British and Danish waters. Significant morphological differentiation reflected the size of the buccal cavity. Porpoises forage in relatively shallow waters preying mainly on benthic species in British and Danish waters, and on mesopelagic and pelagic fish off the coast of Norway. We suggest that the observed differentiation may be explained by resource specialization and either adaptation or developmental responses to different local habitats.

Non-local population stocking can have adverse genetic effects through loss of genetic diversity and introgressive hybridisation in wild populations exploited by man. The grey partridge (Perdix perdix) has been an important European game species for centuries, widely subject to translocation and stocking. After ca 80 years releasing reared grey partridges into Danish wild partridge areas, this study investigated whether an indigenous Danish grey partridge still existed. We predicted that (i) an indigenous Danish partridge would belong to the western European clade (W1) and (ii) would be more closely related to the historical, indigenous grey partridges than to farm bred partridges. These predictions were tested analysing the variation in the mitochondrial control region (CR1) and microsatellite markers in museum samples representing the ancestral indigenous Danish grey partridge, contemporary (recent) wild grey partridges and farmed grey partridges from the five largest farms in Denmark. Phylogeography- and population structure analyses showed traces of the indigenous Danish grey partridges amongst recent wild partridges in certain areas and significant genetic differences between reared, farmed partridges and historical- and recent partridges. The results also showed that the indigenous Danish grey partridges belonged to the western European clade (W1 haplotype). A foreign stocking effect was detected on the remote island of Bornholm, where the current population originated from introduced Danish and Bohemian grey partridges. The loss of haplotype diversity over time in certain geographical areas probably results from serious declines in wild Danish grey partridge numbers in recent decades. This combined with the 2 observation that hybridisation between released stocked and wild partridges can occur may complicate recovery of partridge populations.

It is crucial to examine the genetic diversity and structure of small, isolated populations, especially those at the edge of their distribution range, because they are vulnerable to stochastic processes if genetic diversity is low and the isolation level is high, and because such populations provide insight into the consequences of population declines in a broader conservation context. The harbour seal (Phoca vitulina) population in Svalbard is the world's northernmost P. vitulina population. Nothing is known about the genetic diversity, distinctiveness, or origin of this small, marginalized mammalian population. Thus, the present study investigated its genetic status in the context of nearby P. vitulina in Iceland, south-east Greenland, and northern Norway: this species is depleted/threatened in all of these regions. A total of 174 samples distributed between the four locations were analysed using 15 polymorphic microsatellites and variation in the displacement loop region (D-loop). Each of the four locations was a genetically distinct population. The Svalbard population was highly genetically distinct, had reduced genetic diversity, received limited gene flow, had a rather low effective population size, and showed an indication of having experienced a bottleneck resulting from a recent population decline. The significant heterozygote excess observed in the Svalbard sample might be attributed to the low effective population size, which could initiate future population inbreeding effects. This phenomenon has not been reported earlier from other P. vitulina populations, but if the Svalbard population is experiencing inbreeding, this could reduce its resilience to climate change, disease outbreaks, or other perturbations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102, 420–439.

The population status of the harbour porpoise (Phocoena phocoena) in the Baltic area has been a continuous matter of debate. Here we present the by far most comprehensive genetic population structure assessment to date for this region, both with regard to geographic coverage and sample size: 497 porpoise samples from North Sea, Skagerrak, Kattegat, Belt Sea, and Inner Baltic Sea were sequenced at the mitochondrial Control Region and 305 of these specimens were typed at 15 polymorphic microsatellite loci. Samples were stratified according to sample type (stranding vs. by-caught), sex, and season (breeding vs. non-breeding season). Our data provide ample evidence for a population split between the Skagerrak and the Belt Sea, with a transition zone in the Kattegat area. Among other measures, this was particularly visible in significant frequency shifts of the most abundant mitochondrial haplotypes. A particular haplotype almost absent in the North Sea was the most abundant in Belt Sea and Inner Baltic Sea. Microsatellites yielded a similar pattern (i.e., turnover in occurrence of clusters identified by STRUCTURE). Moreover, a highly significant association between microsatellite assignment and unlinked mitochondrial haplotypes further indicates a split between North Sea and Baltic porpoises. For the Inner Baltic Sea, we consistently recovered a small, but significant separation from the Belt Sea population. Despite recent arguments that separation should exceed a predefined threshold before populations shall be managed separately, we argue in favour of precautionary acknowledging the Inner Baltic porpoises as a separate management unit, which should receive particular attention, as it is threatened by various factors, in particular local fishery measures.

Defining sub-populations and determining migration rates between them is crucial for sound management and conservation. This also applies to the Atlantic walrus Odobenus rosmarus rosmarus, which is still exploited although population levels are low in some cases and information on abundance is lacking in others. Analysis of muscle and skin tissues using 11 nuclear microsatellite markers from a total of 297 Atlantic walruses from the Hudson Strait across W, NW and E Greenland to Svalbard and Franz Josef Land was undertaken to determine the number of sub-populations, their ancestral origin and the contemporary rates and directions of migration (gene flow) between the var- ious areas. The study indicated the existence of 5 sub-populations in the Hudson Strait, W Greenland, NW Greenland, E Greenland and Franz Josef Land-Svalbard. Identification of the Hudson Strait sub- population was novel; although differences between animals here and in W Greenland were small, they were statistically significant and indicated that walruses in the Hudson Strait could be a popu- lation source for walruses for the W Greenland sub-population. The direction of migration (Hudson Strait to W Greenland) suggested a genetic signal from a historical large-scale counter-clockwise perennial migration pattern in the Baffin Bay region. The study provides essential information on population sub-structuring that is a prerequisite for management of Atlantic walruses at sustainable levels.

Denmark lies on the edge of the distributional range of the brown hareLepus europaeus Pallas, 1778, where population differentiation is most likely to occur. A total of 369 brown hares from eight geographically distinct Danish European brown hare populations were used to study the genetic population structure. In all, 480bp of the mitochondrial D-loop were sequenced in both directions. Observed genetic diversity (π) was relatively low (π=0.41%) while haplotype diversity (h=0.808) and the number of unique haplotypes (19) were similar to levels found in other European brown hare populations. The observed population structure was pronounced (pairwise conventionalF ST and ϕ st ranged between 6.9–57% and 5–69.8%, respectively). There was no correlation between the geographic and the genetic distance. Population structure was influenced by genetic drift, anthropogenic effects (eg translocation and escapes from hare-farms) and by post-glacial recolonization from southern refuges or refuges north east of the Black Sea. Analysis of historical population expansion/fluctuation events indicated that the populations have experienced different demographic events in the recent past. Relatively high sequence divergence between some populations might be explained by multiple recolonization events after the last Pleistocene glaciations or by stocking effects. Colonization from southern refuges was supported by the observation that haplotype 2 in the Danish brown hare was identical to the central European ancestral haplotype c07.

The walrus (Odobenus rosmarus) is in some current systematic schemes divided into three subspecies: O. r. rosmarus in the North Atlantic, O. r. divergens in the North Pacific and O. r. laptevi in the Laptev Sea. These three subspecies have been described as differing in body size, but the taxonomic status of O. r. laptevi is disputed. The current study applies molecular and morphometric methods to assess the taxonomic status of O. r. laptevi and to analyse the systematic and phylogeographic relationships between the three purported walrus subspecies. Tusk length and tusk circumference were measured from the few skulls available of O. r. laptevi, and the obtained values were within the ranges reported for Pacific walruses. Thus, morphologically, subspecies status for O. r. laptevi is not supported according to the Amadon Mayr '75% rule'. Phylogenetic analyses and haplotype networks based on mitochondrial nucleotide sequence data of NADH dehydrogenase 1, 16S rRNA, cytochrome oxidase I and the D-loop of the control region of the historic O. r. laptevi bone material and contemporary O. r. rosmarus and O. r. divergens showed that the Laptev Sea walrus groups with individuals from the North Pacific. Thus, the mitochondrial sequence data do not support the recognition of three walrus subspecies as reciprocally monophyletic evolutionary units with independent evolutionary histories. Only O. r. rosmarus and O. r. divergens meet this criterion with the present sampling. Accordingly, we recommend that Odobenus r. laptevi be abandoned and the Laptev walrus instead be recognized as the westernmost population of the Pacific walrus, Odobenus r. divergens. However, further research is recommended to assess whether the Laptev walrus could be considered as a significant unit in terms of conservation and management, since it is unique in several ecological parameters. (Less)

We evaluated the ability of a set of published trans-species molecular sexing primers and a set of walrus-specific primers, which we developed, to accurately identify sex of 235 Pacific walruses (Odobenus rosmarus divergens). The trans-species primers were developed for mammals and targeted the X- and Y-gametologs of the zinc finger protein genes (ZFX, ZFY). We extended this method by using these primers to obtain sequence from Pacific and Atlantic walrus (O. r. rosmarus) ZFX and ZFY genes to develop new walrus-specific primers, which yield polymerase chain reaction products of distinct lengths (327 and 288 base pairs from the X- and Y-chromosome, respectively), allowing them to be used for sex determination. Both methods yielded a determination of sex in all but 1–2% of samples with an accuracy of 99.6–100%. Our walrus-specific primers offer the advantage of small fragment size and facile application to automated electrophoresis and visualization.

The chromosomes obtained by culturing lymphocytes from blood samples of the long-finned pilot whale Globicephala melaena, were studied by Q- and C-banding. The culture method was very difficult to handle. The karyotype of the long-finned pilot whale has a typical cetacean 2n = 44 chromosome number. The O-band revealed a characteristic banding pattern, which showed a heteromorphism between individuals but not within individuals. These observations were supported by the C-banding, which disclosed clear size variation of heterochromatin in some chromosome pairs. A species specific C-band pattern was observed in comparison between Globicephala melaena and Globicephala macrorhynchus, the short-finned pilot whale. O- and C-band markers can be used in family- and population studies if a good technical quality can be achieved. The finding of an inactivation of one of the X-chromosomes in females opens the possibility of using the method of staining the sex chromatin to determine the sex of small foetuses (

Several mammal species have recolonized their historical ranges across Europe during the last decades. In November 2012, a wolf-looking canid was found dead in Thy National Park (56° 56′ N, 8° 25′ E) in Jutland, Denmark. DNA from this individual and nine German wolves were genotyped using a genome-wide panel of 22,163 canine single nucleotide polymorphism (SNP) markers and compared to existing profiles based on the same marker panel obtained from northeastern Polish (n = 13) wolves, domestic dogs (n = 13) and known wolf-dog hybrids (n = 4). The Thy canid was confirmed to be a wolf from the German-western Polish population, approximately 800 km to the southeast. Access to the German reference database on DNA profiles based on 13 autosomal microsatellites of German wolves made it possible to pinpoint the exact pack origin of the Thy wolf in Saxony, Germany. This was the first documented observation of a wolf in Denmark in 200 years and another example of long-distance dispersal of a carnivore.

Species from the steppe region of Eastern Europe likely colonized northwestern Europe in connection with agriculture after 6500 BP. The striped field mouse ( Apodemus agrarius Pallas, 1783), is a steppe-derived species often found in human crops. It is common on the southern Danish islands of Lolland and Falster, which have been isolated from mainland Europe since approximately 10 300–8000 BP. Thus, this species could have been brought in with humans in connection with agriculture, or it could be an earlier natural invader. We sequenced 86 full mitochondrial genomes from the northwestern range of the striped field mouse, analysed phylogenetic relationships and estimated divergence time. The results supported human-induced colonization of Denmark in the Subatlantic or Subboreal period. A newly discovered population from Central Jutland in Denmark diverged from Falster approximately 100–670 years ago, again favouring human introduction. One individual from Sweden turned out to be a recent introduction from Central Jutland.

Identification of populations and management units is an essential step in the study of natural systems. Still, there is limited consensus regarding how to define populations and management units, and whether genetic methods allow for inference at the relevant spatial and temporal scale. Here, we present a novel approach, integrating genetic, life history and demographic data to identify populations and management units in southern Scandinavian harbour seals. First, 15 microsatellite markers and model- and distance-based genetic clustering methods were used to determine the population genetic structure in harbour seals. Second, we used harbour seal demographic and life history data to conduct population viability analyses (PVAs) in the VORTEX simulation model in order to determine whether the inferred genetic units could be classified as management units according to Lowe and Allendorf’s (Molecular Ecology, 19, 2010, 3038) ‘population viability criterion’ for demographic independence. The genetic analyses revealed fine-scale population structuring in southern Scandinavian harbour seals and pointed to the existence of several genetic units. The PVAs indicated that the census population size of each of these genetic units was sufficiently large for long-term population viability, and hence that the units could be classified as demographically independent management units. Our study suggests that population genetic inference can offer the same degree of temporal and spatial resolution as ‘nongenetic’ methods and that the combined use of genetic data and PVAs constitutes a promising approach for delineating populations and management units.

We examined polymorphism at 12 microsatelliteloci in 807 harbour porpoises , Phocoenaphocoena, collected from throughout thecentral and eastern North Atlantic to theBaltic Sea. Multilocus tests for allelefrequency differences, assignment tests,population structure estimates (FST) andgenetic distance measures (DLR andDC) all indicate six geneticallydifferentiated populations/sub-populationsafter pooling sub-samples within regions.Harbour porpoises from West Greenland, theNorwegian Westcoast, Ireland, the British NorthSea, the Danish North Sea and the inland watersof Denmark (IDW) are all geneticallydistinguishable from each other. A sample ofharbour porpoises collected off the Dutch coast(mainly during winter) was geneticallyheterogeneous and likely comprised a mixture ofindividuals of diverse origin. A mixed stockanalysis indicated that most of the individualsin this sample (∼77%) were likely migrantsfrom the British and Danish North Sea.

Analyses of nuclear and mitochondrial DNA in walruses from northwestern Greenland (76°30'-78°30'N; i.e., in the area of the North Water polynya of northern Baffin Bay and Smith Sound) and west-central Greenland ( 67°-68°N) revealed two genetically distinct subpopulations. The studied sample consisted of tissues from 91 Atlantic walruses (Odobenus rosmarus rosmarus) from northwestern Greenland (1989-1990) and 33 Atlantic walruses from western Greenland (1988-1997). The analyses were based upon 12 nuclear microsatellite loci and restriction length polymorphisms observed in the ND1, ND2, and ND3/4 segments of mtDNA. Evolutionary factors creating the observed genetic differences were mainly drift and gene flow, even though a more pronounced mutational effect was observed at the mitochondrial level. Hence, there appears to be some male-mediated gene flow between the two subpopulations, whereas female-mediated gene flow apparently has been restricted for a considerable time. No temporal variation in population structure was detected in the sample from northwestern Greenland. Females collected in the summer season in this area were shown to be philopatric, meaning that closely related females stay and (or) travel together with a mean relatedness value close to the expected relatedness value for half siblings.

The population structure of the Atlantic walrus, Odobenus rosmarus rosmarus, was studied using 11 polymorphic microsatellites and restriction fragment length polymorphism detected in the NADH-dehydrogenase ND1, ND2 and ND3/4 segments in mtDNA. A total of 105 walrus samples were analysed from northwest (NW) Greenland, east (E) Greenland, Svalbard and Franz Joseph Land. Two of the 10 haplotypes detected in the four samples were diagnostic for the NW Greenland sample, which implied that the group of walruses in this area is evolutionary distinct from walruses in the other three areas. One individual sampled in E Greenland exhibited a Pacific haplotype, which proved a connection between the Pacific walrus and walruses in eastern Greenland. The Franz Joseph Land, Svalbard and E Greenland samples shared the most common haplotype, indicating very little differentiation at the mtDNA level. Gene flow (Nm) estimates among the four areas indicated a very restricted exchange of female genes between NW Greenland and the more eastern Atlantic Arctic samples, and a closer relationship between the three samples composing the eastern Atlantic Arctic. The genetic variation at 11 polymorphic microsatellite loci grouped individuals into three populations, NW Greenland, E Greenland and a common Franz Joseph Land-Svalbard population, which were connected by moderate gene flow.

One hundred and twenty-four specimens of the harbour porpoise, Phocoena phocoena, occurring in inner Danish waters (IDW), the North Sea and West Greenland were analysed to study subdivision into genetically differentiated subpopulations using PCR-amplified DNA-microsatellites and isozyme markers. Three polymorphic microsatellites, 415/416, 417/418 and Igf-I (insulin-like growth factor I) were detected with nine, eight and 15 alleles, respectively, and from a former study two polymorphic isozymes, Mpi-1 and Pgm, with three and two alleles, respectively, were used in the analysis. Overall deviations from the expected Hardy-Weinberg distribution were only observed in the total sample and at a single locus in the North Sea-summer sample and at two loci in the West Greenland sample. Whenever this occurred a surplus of homozygotes was observed, suggesting a Wahlund effect, a null allele or nonrandom mating. The analysis of the genetical population structure showed that harbour porpoises from West Greenland, the North Sea and IDW were three geographically, genetically differentiated populations even though connected through some degree of gene flow. A tendency for females to be more stationary than males was suggested. Furthermore, the population structure suggested a closer relationship between IDW and the North Sea.

Several arthropods are known to be highly beneficial to agricultural production. Consequently it is of great relevance to study the importance of land management and land composition for the conservation of beneficial aphid-predator arthropod species in agricultural areas. Therefore our study focusing on the beneficial arthropod Bembidion lampros had two main purposes: I) identifying the physical barriers to the species' dispersal in the agricultural landscape, and II) assessing the effect of different land management strategies (i.e. use of pesticides and intensiveness) on the dispersal patterns. The study was conducted using genetic analysis (microsatellite markers) applied to samples from two agricultural areas (in Denmark) with different agricultural intensity. Land management effects on dispersal patterns were investigated with particular focus on: physical barriers, use of pesticide and intensity of cultivation. The results showed that Bembidion lampros disperse preferably through hedges rather than fields, which act as physical barriers to gene flow. Moreover the results support the hypothesis that organic fields act as reservoirs for the re-colonization of conventional fields, but only when cultivation intensity is low. These results show the importance of non-cultivated areas and of low intensity organic managed areas within the agricultural landscape as corridors for dispersal (also for a species typically found within fields). Hence, the hypothesis that pesticide use cannot be used as the sole predictor of agriculture's effect on wild species is supported as land structure and agricultural intensity can be just as important.

The impact of landscape structure and land management on dispersal of populations of wild species inhabiting the agricultural landscape was investigated focusing on the field vole (Microtus agrestis) in three different areas in Denmark using molecular genetic markers. The main hypotheses were the following: (i) organic farms act as genetic sources and diversity reservoirs for species living in agricultural areas and (ii) gene flow and genetic structure in the agricultural landscape are influenced by the degree of landscape complexity and connectivity. A total of 443 individual voles were sampled within 2 consecutive years from two agricultural areas and one relatively undisturbed grassland area. As genetic markers, 15 polymorphic microsatellite loci (nuclear markers) and the central part of the cytochrome-b (mitochondrial sequence) were analysed for all samples. The results indicate that management (that is, organic or conventional management) was important for genetic population structure across the landscape, but that landscape structure was the main factor shaping gene flow and genetic diversity. More importantly, the presence of organically managed areas did not act as a genetic reservoir for conventional areas, instead the most important predictor of effective population size was the amount of unmanaged available habitat (core area). The relatively undisturbed natural area showed a lower level of genetic structuring and genetic diversity compared with the two agricultural areas. These findings altogether suggest that political decisions for supporting wildlife friendly land management should take into account both management and landscape structure factors.

The development in sequencing methods has made it possible to perform whole genome de novo sequencing of species without large commercial interests. Within the EU-financed QUANTOMICS project (KBBE-2A-222664), we have performed de novo sequencing of quail (Coturnix coturnix) and grey partridge (Perdix perdix) on a Genome Analyzer GAII (Illumina) using paired-end sequencing. The amount of generated sequences amounts to 8 to 9 Gb for each species. The analysis and assembly of the generated sequences is ongoing. Access to the whole genome sequence from these two species will enable enhanced comparative studies towards the chicken genome and will aid in identifying evolutionarily conserved sequences within the Galliformes. The obtained sequences from quail and partridge represent a beginning of generating the whole genome sequence for these species. The continuation of establishing the genome sequence of these species will require generation of more sequences from more diverse libraries. The QUANTOMICS group encourages other research groups to participate in a joint effort to establish the genome sequence of these two Galliformes species.

A study of the homogeneity between putative stocks of Phocoena phocoena in the North Sea and inner Danish waters was performed using isozyme electrophoresis. Two polymorphic systems, mannose phosphate isomerase (MPI) and phosphoglucomutase (PGM) were used in the analysis. A two-locus homogeneity test showed an effect of season and locality on the genotypic distribution. This led to a division of the total sample into season and into the two localities, the inner Danish waters (IDW) and the North Sea. The samples were then compared to the Hardy-Weinberg expectations where a deficit in heterozygotes was observed in the North Sea sample in both systems and at the Pgm locus in the total sample, indicating a mixing of sub-populations. A hierarchical contingency table analysis, also based on the genotypic distribution, showed a combined effect of season and locality and only an insignificant effect of sampling period, thus supporting the division into season and locatily. The seasonal effect is further supported by earlier observations of a seasonal migration of harbour porpoises out of the Baltic in winter. A significant difference was observed between the two supposed sub-populations, IDW-summer and North Sea-summer samples, based on the genotypic distribution. This could also be a reflection of the sample sizes. On the basis of samples obtained from Canada, West Greenland and Holland a preliminary study of the population structure on a larger scale, comprising the North East and North West Atlantic, was performed. The results so far accord well with the hitherto accepted sub-populations of P. phocoena in the North Atlantic.

The European natterjack toad (Bufo calamita) has declined rapidly in recent years, primarily due to loss of habitat, and in Denmark it is estimated that 50% of the isolated populations are lost each decade. To efficiently manage and conserve this species and its genetic diversity, knowledge of the genetic structure is crucial. Based on nine polymorphic microsatellite loci, the genetic diversity, genetic structure and gene flow were investigated at 12 sites representing 5-10% of the natterjack toad localities presently known in Denmark. The expected heterozygosity (H E) within each locality was generally low (range: 0.18-0.43). Further analyses failed to significantly correlate genetic diversity with population size, degree of isolation and increasing northern latitude, indicating a more complex combination of factors in determining the present genetic profile. Genetic differentiation was high (overall θ = 0.29) and analyses based on a Bayesian clustering method revealed that the dataset constituted 11 genetic clusters, defining nearly all sampling sites as distinct populations. Contemporary gene flow among populations was undetectable in nearly all cases, and the failure to detect a pattern of isolation by distance within major regions supported this apparent lack of a gene flow continuum. Indications of a genetic bottleneck were found in three populations. The analyses suggest that the remaining Bufo calamita populations in Denmark are genetically isolated, and represent independent units in a highly fragmented gene pool. Future conservation management of this species is discussed in light of these results.

Tissue-samples from 161 bank voles (Clethrionomys glareolus) collected in three forests (five sampling localities) situated in eastern Jutland (Denmark) were analysed by nine microsatellite loci. The genetic diversity found within the populations was high (He=0.753–0.806). Bank voles have specific habitat requirements favouring woodlots, hedgerows and deciduous forests as their prime living area. Hence, a natural or human-induced fragmentation of the forest may cause a sub-structuring of the populations and thereby a restriction of dispersal among populations. The sub-structuring indicated by the observed significant genetic differentiation among the five geographically distinct localities (Fst =0.033, P

A 23-year-old female polar bear (Ursus maritimus) killed in an Inuit hunt in East Greenland on July 9, 1999 had a significantly enlarged clitoris resembling, in size, form and colour, those of previously reported ‘pseudohermaphroditic’ polar bears from Svalbard. It has been suggested that an enzyme defect (21-hydroxylase deficiency), androgen producing tumour or high exposure to organochlorines during the foetal stage or early development could be the reason for the supposed pseudohermaphroditism observed for Svalbard bears. Except for the enlarged clitoris, all dimensions of the external and internal reproductive organs of the present were similar to a reference group of 23 normal adult female polar bears from East Greenland collected in 1999–2002. The aberrant bear was a female genotype, and macroscopic examination of her internal reproductive organs indicated that she was reproductively functional. A histological examination of the clitoral enlargement in the present East Greenland specimen allows a first-time histological evaluation of the earlier macroscopic field diagnosis from Svalbard. This examination revealed intense chronic ulcerative and perivascular clitoriditis similar to “acral lick dermatitis” frequently seen in domestic dogs (i.e., we did not find any signs of pseudohermaphroditic hyperplasia of clitoral tissue due to androgenic or antiestrogenic endocrine disruption). The levels of organohalogens and TEQ values were lower than concentration thresholds of toxicological risk. It is hence possible that the previously reported adult female polar bear pseudohermaphrodites from Svalbard are in fact misdiagnoses. Therefore, future studies examining pseudohermaphrodism in wildlife should consider that certain occurrences are natural events, e.g., enlarged clitoris in the present East Greenland polar bear. Furthermore, caution should be exercised in suggesting linkages of such inflammatory abnormalities with correlations to anthropogenic pollutant exposures.