Varying degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee, Apis mellifera mellifera, in northwest Europe

The natural distribution of honeybee subspecies in Europe has been significantly affected by human activities during the last century. Non-native subspecies of honeybees have been introduced and propagated, so that native black honeybee (Apis mellifera mellifera) populations lost their identity by gene-flow or went extinct. After previous studies investigated the remaining gene-pools of native honeybees in France and Spain, we here assess the genetic composition of eight northwest European populations of the black honeybee, using both mitochondrial (restriction fragment length polymorphisms of the intergenic transfer RNAleu-COII region) and nuclear (11 microsatellite loci) markers. Both data sets show that A. m. mellifera populations still exist in Norway, Sweden, Denmark, England, Scotland and Ireland, but that they are threatened by gene flow from commercial honeybees. Both Bayesian admixture analysis of the microsatellite data and DraI-RFLP (restriction fragment length polymorphism) analysis of the intergenic region indicated that gene-flow had hardly occurred in some populations, whereas almost 10% introgression was observed in other populations. The most introgressed population was found on the Danish Island of Laeso, which is the last remaining native Danish population of A. m. mellifera and the only one of the eight investigated populations that is protected by law. We discuss how individual admixture analysis can be used to monitor the restoration of honeybee populations that suffer from unwanted hybridization with non-native subspecies.     

Postglacial recolonization patterns and genetic relationships among whitefish (Coregonus sp.) populations in Denmark, inferred from mitochondrial DNA and microsatellite markers

The genetic relationships among morphologically and geographically divergent populations of whitefish (genus: Coregonus ) from Denmark and the Baltic Sea region were studied by analysis of microsatellites and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of mitochondrial DNA (mtDNA) segments. The endangered North Sea houting (classified as C. oxyrhynchus ) differs morphologically and physiologically from other Danish whitefish ( C. lavaretus ). However, limited divergence of North Sea houting was observed both at the level of mtDNA and microsatellites. The implications of these results for the conservation status of North Sea houting are discussed in the light of current definitions of evolutionary significant units. Both mtDNA and microsatellite data indicated that postglacial recolonization by C. lavaretus in Denmark was less likely to have taken place from the Baltic Sea. Instead, the data suggested a recent common origin of all Danish whitefish populations, including North Sea houting, probably by recolonization via the postglacial Elbe River system. Estimates of genetic differentiation among populations based on mtDNA and microsatellites were qualitatively different. In addition, for both classes of markers analyses of genetic differentiation yielded different results, depending on whether molecular distances between alleles or haplotypes were included.     

Sharing reference data and including cows in the reference population improve genomic predictions in Danish Jersey

Small reference populations limit the accuracy of genomic prediction in numerically small breeds, such like Danish Jersey. The objective of this study was to investigate two approaches to improve genomic prediction by increasing size of reference population in Danish Jersey. The first approach was to include North American Jersey bulls in Danish Jersey reference population. The second was to genotype cows and use them as reference animals. The validation of genomic prediction was carried out on bulls and cows, respectively. In validation on bulls, about 300 Danish bulls (depending on traits) born in 2005 and later were used as validation data, and the reference populations were: (1) about 1050 Danish bulls, (2) about 1050 Danish bulls and about 1150 US bulls. In validation on cows, about 3000 Danish cows from 87 young half-sib families were used as validation data, and the reference populations were: (1) about 1250 Danish bulls, (2) about 1250 Danish bulls and about 1150 US bulls, (3) about 1250 Danish bulls and about 4800 cows, (4) about 1250 Danish bulls, 1150 US bulls and 4800 Danish cows. Genomic best linear unbiased prediction model was used to predict breeding values. De-regressed proofs were used as response variables. In the validation on bulls for eight traits, the joint DK-US bull reference population led to higher reliability of genomic prediction than the DK bull reference population for six traits, but not for fertility and longevity. Averaged over the eight traits, the gain was 3 percentage points. In the validation on cows for six traits (fertility and longevity were not available), the gain from inclusion of US bull in reference population was 6.6 percentage points in average over the six traits, and the gain from inclusion of cows was 8.2 percentage points. However, the gains from cows and US bulls were not accumulative. The total gain of including both US bulls and Danish cows was 10.5 percentage points. The results indicate that sharing reference data and including cows in reference population are efficient approaches to increase reliability of genomic prediction. Therefore, genomic selection is promising for numerically small population.     

Genetic structure within and among regional populations of the Eurasian badger (Meles meles) from Denmark and the Netherlands

The Eurasian badger Meles meles has a wide distribution area ranging from Japan to Ireland. In western Europe badger habitats are severely disturbed by anthropogenic factors, leading to fragmentation into subpopulations and formation of a metapopulation substructuring of once continuous panmictic populations. We have examined the genetic structure of Dutch and Danish badger populations on a relatively small scale (within countries) and a larger scale (between countries). The levels of genetic variation of populations were moderate and did not differ significantly among populations (overall H _O=0.30, overall H _E=0.34). Considerable genetic differentiation between the Dutch and Danish populations was found (overall F _ST=0.32, mean pairwise Dutch–Danish F _ST=0.42), indicating a large-scale substructuring of these western European badger populations. Further analysis showed that the Danish badger population can be substructured into three clusters [ P _{( k =3)}=0.99], but the Dutch populations cluster into one more or less panmictic population [ P _{( k =1)}=0.99] with little or no substructuring. The presence of migration barriers, such as roads, together with the peninsular geography of Denmark, may have led to this structuring of badger populations. In contrast, measures that improve migration and connection to other populations from neighboring countries may have prevented substructuring of the Dutch badger population.     

Late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae): variation patterns and taxonomic inferences

Principal components analysis (PCA) was applied to summarize variation in 23 morphological characters scored from nine late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae) in Wales, The Netherlands, and Denmark. The Dutch and Danish groups of populations were vaguely separated in a PCA conducted on populations, and a very broad overlap between them was found in a PCA conducted on individual specimens. On the other hand, a clear morphological distinction between the Welsh and continental groups of populations appeared from both PCAs. The probability of each character to distinguish correctly between the main groups of populations was estimated. Three characters were found to distinguish reliably (success ≥ 90 %) between the Welsh and continental groups of populations, while no character was found to distinguish reliably between the Dutch and Danish groups of populations. It is concluded that the Welsh and Dutch/Danish populations represent two distinct taxa distributed in the British Isles and in continental Northwestern Europe, respectively. The British taxon is recognized as D. incarnata subsp. coccinea and the continental one as D. incarnata subsp. lobelii (stat. nov.). Brief taxonomic accounts are provided.     

Genetic diversity and phenotypic variation in marginal populations of the locally endangered speciesHordeum secalinum (Poaceae)

Electrophoresis was used to compare variation in hordein polypeptide patterns and isozymes in five marginal populations ofHordeum secalinum (four Swedish and one Danish population) and four populations along the Atlantic coast from Denmark to Spain. Hordein patterns were uniform within and among Swedish and Danish populations whereas the materials from France and Spain were divergent. The banding patterns for nine isozymes (AAT, IDH, MDH, PGD, GPI, ME, ACP, EST, and PRX) agreed with those reported for otherHordeum species. All putative loci were monomorphic with the exception of PRX, which showed three different phenotypes. The five marginal populations varied in seed set and cultivated plants differed in phenological traits.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Low level of gene flow from cultivated beets (Beta vulgaris L. ssp. vulgaris) into Danish populations of sea beet (Beta vulgaris L. ssp. maritima (L.) Arcangeli)

Gene flow from sugar beets to sea beets occurs in the seed propagation areas in southern Europe. Some seed propagation also takes place in Denmark, but here the crop-wild gene flow has not been investigated. Hence, we studied gene flow to sea beet populations from sugar beet lines used in Danish seed propagation areas. A set of 12 Danish, two Swedish, one French, one Italian, one Dutch, and one Irish populations of sea beets, and four lines of sugar beet were analysed. To evaluate the genetic variation and gene flow, eight microsatellite loci were screened. This analysis revealed hybridization with cultivated beet in one of the sea beet populations from the centre of the Danish seed propagation area. Triploid hybrids found in this population were verified with flow cytometry. Possible hybrids or introgressed plants were also found in the French and Italian populations. However, individual assignment test using a Bayesian method provided 100% assignment success of diploid individuals into their correct subspecies of origin, and a Bayesian Markov chain Monte Carlo (MC MC) approach revealed clear distinction of individuals into groups according to their subspecies of origin, with a zero level of genetic admixture among subspecies. This underlines that introgression beyond the first hybridization is not extensive. The overall pattern of genetic distance and structure showed that Danish and Swedish sea beet populations were closely related to each other, and they are both more closely related to the population from Ireland than to the populations from France, the Netherlands, and Italy.     

Geographic variation of freeze-tolerance in the earthworm Dendrobaena octaedra

Freeze-tolerance and some of the underlying biochemical defence mechanisms in the earthworm Dendrobaena octaedra was investigated. Survival after slow cooling to -2 degrees C, -4 degrees C, or -6 degrees C was analysed in D. octaedra from three geographic regions representing large differences in winter temperature (Denmark, Finland and Greenland). A large variation in freeze-tolerance between the three populations of D. octaedra was found. Earthworms from the northern populations (Finland and Greenland) tolerated lower temperatures (-6 degrees C) than earthworms from the Danish population (poor survival at -4 degrees C and -2 degrees C). In the Finnish population, freezing led to the production of high concentrations of glucose, which reached values much higher than controls (94 mg g(-1) vs. 2 mg g(-1) dry weight). Other potential cryoprotectants were not elevated after freezing. The Danish and Greenlandic populations had substantially lower mean glucose levels after freezing than the Finnish population (about 15 mg g(-1)). Danish earthworms rapidly frozen did not accumulate glucose, and did not survive freezing at -2 degrees C. Danish earthworms exposed to osmotic stress in Ringer's solutions, containing different concentrations of glycerol, showed significantly elevated glucose levels, but did not survive rapid freezing. It was determined if freezing had an influence on the reproduction of the earthworms. After warming to summer temperatures (15 degrees C), survivors of freezing produced viable cocoons. In a field experiment it was tested if natural acclimatization during autumn and winter months had an effect on freeze-tolerance in the Danish population. There was a significant increase of post-freeze survival during this period. The results of the freezing experiments are discussed in relation to the general ecology of D. octaedra.     

Molecular analysis of PKU haplotypes in the population of southern Poland

Summary Out of a population of 138598 infants born in southern Poland between 1987 and 1989, and screened for phenylketonuria (PKU), 28 cases were ascertained giving an incidence of 1 in 5000. DNA from 22 of these probands and their parents was isolated and eight polymorphic restriction sites were analyzed within the phenylalanine hydroxylase gene region. Twenty-one different haplotypes (HT) were revealed, five of them representing new categories. The most common haplotypes among those carrying normal alleles were: HT1 (27.3%) and HT4 (11.4%). Within the group of haplotypes with mutant alleles the most frequent was HT2 (56.8%), whereas the frequency of this haplotype in other European populations, such as French, Danish and German, ranged from 12% to 24%. HT3, being the most common in Danish (38%), and relatively frequent in the other western European (13–14%) populations, appeared to be very rare in our sample (2.3%). The mutation of codon 408 (exon 12, CT, ArgTrp), which has been described to be tightly linked to HT2, was tested on amplified DNA by dot-blot hybridization. This mutation was found in 25 out of 44 proband chromosomes. In one case it was linked to HT5, in the remaining 24 to HT2. Our results confirm molecular heterogeneity of PKU haplotypes, as well as their significant interpopulation variation.     

Primula farinosa in Denmark; genetic diversity and population management

During the past centuries Danish populations of Primula farinosa have seriously declined in number. We investigated the genetic structure and genetic diversity of plants of seven populations from two different regions, Zealand and Bornholm in Denmark, using three AFLP markers. Two populations from nearby Scania, Sweden were included as reference. We found 54 unambiguously polymorphic loci. The genetic structure analysis suggested division of the 268 plants into three distinct groups, to a large extent matching the geographical distribution of the populations. Analysis of molecular variance (AMOVA) indicated significant genetic differentiation of 67% within populations and 33% among the populations. Our results suggest that genetic differentiation among regions and unique local genetic diversity should carefully be considered in future conservation attempts if we are to maintain as much genetic variation as possible. We present a historical overview of the decline in Danish populations and discuss conservation management and restoration strategies.     

Matrilinear phylogeography of Atlantic salmon (Salmo salar L.) in Europe and postglacial colonization of the Baltic Sea area

Sixty‐four samples from 46 salmon populations totalling 2369 specimens were used for polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of the mitochondrial ND1 region. The final analyses included 3095 specimens from 60 populations in Northern Europe. A subsample was analysed by RFLP of ND3/4/5/6. Representative RFLP haplotypes from different parts of the distribution area were sequenced and the phylogeny of European haplotypes and their relations to the North American lineage was described. The four common European haplotypes derive from the ancestral ND1‐BBBA (rooting the European clade to the North American) by one‐step substitutions: AAAA < AABA < BBBA > BBBB. The Swedish west‐coast populations differ from the geographically close southern Baltic, indicating absence of inward and limited outward gene flow through the Danish straits during the last 8000 years. Within the Baltic Sea, only three ND1 haplotypes were detected and there was no variation for ND3/4/5/6. In the whole southern Baltic and in lakes Vänern, Ladoga and Onega the haplotype AABA dominated. Proposed postglacial colonization routes to the Baltic Sea are discussed in relation to the haplotype distribution pattern.     

Comparison of human papillomavirus genotypes in archival cervical cancer specimens from Alaska natives, Greenland natives and Danish Caucasians

Archival, formalin-fixed, paraffin-embedded cervical cancer specimens from 53 Alaska natives, 32 Greenland natives and 34 Danish Caucasians were analyzed for human papillomavirus (HPV) genotypes 16, 18, 31, 33, 35 and 45 and unidentified genotypes (HPV X) using PCR. The specimens were from the time period 1980-1989. No significant differences were observed in the overall HPV detection rates among cases from Alaska (98.1%), Greenland (84.4%) and Denmark (85.3%). HPV genotype 16 was the most prevalent type: 78.8% in Alaska natives, 96.3% in Greenland natives and 82.8% in Danish Caucasians. A prevalence of 21.2% HPV 31 and 30.8% HPV 33 was found in Alaska natives, of which most were coinfections with HPV 16. Only 3.7% HPV 31 and 3.7% HPV 33 were found in Greenland natives and no HPV 31 and 6.9% HPV 33 were found in Danish Caucasians. HPV 18 was only detected in Alaska natives and HPV 35 and 45 were not detected in any of the three populations. Infections with multiple genotypes were prevalent in Alaskan (36.5%) but not in Greenland natives (3. 7%) and Danish Caucasians (6.9%). The Eskimo subgroup of the Alaska native population has a significantly higher prevalence of HPV genotypes 31 and 33 associated with mixed infections in invasive cancer than the two other native subgroups (P = 0.04) and Greenland and Danish populations, reflecting genotype distributions in dysplasia and normal cervical cytology. The reason for HPV genotype diversity, although unknown, may be relevant to the current development of HPV vaccines.     

Genetic indications of translocated and stocked grey partridges (Perdix perdix): does the indigenous Danish grey partridge still exist?

Non‐local population stocking can have adverse genetic effects on wild populations through loss of genetic diversity and introgressive hybridization. The grey partridge (Perdix perdix) has been an important European game species for centuries, widely subject to translocation and stocking. After c. 80 years of releasing reared grey partridges in Denmark, this study investigated whether an indigenous Danish grey partridge still existed. If so, they would (1) belong to the western European clade (W1) and (2) be more closely related to the historical, indigenous grey partridges than to farm‐bred partridges. These predictions were tested by analysing the variation in both the mitochondrial control region (CR1) and microsatellite markers in museum samples representing the ancestral indigenous Danish grey partridge, contemporary 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 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 observation that hybridization between released stocked and wild partridges can occur, may complicate recovery of partridge populations. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 694–710.     

Dactylorhiza majalis s.l. (Orchidaceae) in acid habitats: variation patterns, taxonomy, and evolution

Morphometric and allozyme data were collected from 11 populations of Dactylorhiza majalis s.l. in the Ardennes (Belgium, France) and North Jutland (Denmark). All study populations were growing in obviously acid habitats. Genetic distances were calculated and illustrated by UPGMA dendrograms, while principal components analyses (PCAs) were used to summarize morphological variation patterns. Based on the PCAs, the probability of each character to distinguish correctly between the main groups of populations was estimated. The study populations from the Ardennes and from the Danish island of Læsø seem to agree with populations from Sweden previously studied by Hedrén—and to belong to D. majalis subsp. sphagnicola (comb. nov.). It should be noted that allozyme data suggest a polytopic origin of this taxon. The study populations from the Danish region of Thy differ morphologically from the others and exhibit much less genetic variation. They are described as a new subspecies, D. majalis subsp. calciñigiens. The evolution and biogeography of the two taxa are discussed, and brief taxonomic accounts are provided.     

Heritability of eleven metabolic phenotypes in Danish and Chinese twins: A cross‐population comparison

Objectives : A twin‐based comparative study on the genetic influences in metabolic endophenotypes in two populations of substantial ethnic, environmental, and cultural differences was performed. Design and Methods : Data on 11 metabolic phenotypes including anthropometric measures, blood glucose, and lipids levels as well as blood pressure were available from 756 pairs of Danish twins (309 monozygotic and 447 dizygotic twin pairs) with a mean age of 38 years (range: 18‐67) and from 325 pairs of Chinese twins (183 monozygotic and 142 dizygotic twin pairs) with a mean age of 40.5 years (range: 18‐69). Twin modeling was performed on full and nested models with the best fitting models selected. Results : Heritability estimates were compared between Danish and Chinese samples to identify differential genetic influences on each of the phenotypes. Except for hip circumference, all other body measures exhibited similar heritability patterns in the two samples with body weight showing only a slight difference. Higher genetic influences were estimated for fasting blood glucose level in Chinese twins, whereas the Danish twins showed more genetic regulation over most lipids phenotypes. Systolic blood pressure was more genetically controlled in Danish than in Chinese twins. Conclusions : Metabolic endophenotypes show disparity in their genetic determinants in populations under distinct environmental conditions.     

Linkage disequilibrium between mutation and RFLP haplotype at the phenylalanine hydroxylase locus in the German population

Summary Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase (PAH) locus have been determined in 60 German families with PAH deficiency. Similar to the Danish population, about 90% of the mutant alleles are confined to four distinct haplotypes. There are however, differences in the frequency distributiion of these haplotypes among the mutant alleles between the two populations. Using an oligonucleotide probe for the splicing mutation associated with mutant haplotype 3 in the Danish population, a tight association between the mutation and the RFLP haplotype has also been observed in Germany. The results provide strong evidence that the splicing mutation occurred on a haplotype 3 chromosome and that the mutant allele has spread into different populations smong Caucasians.     

Genetic diversity of Saccharina latissima (Phaeophyceae) along a salinity gradient in the North Sea–Baltic Sea transition zone

The North Sea–Baltic Sea transition zone constitutes a boundary area for the kelp species Saccharina latissima due to a strong salinity gradient operating in the area. Furthermore, the existence of S. latissima there, along Danish waters, is fairly patchy as hard bottom is scarce. In this study, patterns of genetic diversity of S. latissima populations were evaluated along the salinity gradient area of Danish waters (here designated brackish) and were compared to reference sites (here designated marine) outside the gradient area, using microsatellite markers. The results showed that the S. latissima populations were structured into two clusters corresponding to brackish versus marine sites, and that gene flow was reduced both between clusters and between populations within clusters. In addition, results provided empirical evidence that marginal populations of S. latissima in the salinity gradient area exhibited a distinct genetic structure when compared to marine ones. Brackish populations were less diverse, more related, and showed increased differentiation over distance compared to marine populations. The isolation of the brackish S. latissima populations within the salinity gradient area of Danish waters in conjunction with their general low genetic diversity makes these populations vulnerable to ongoing environmental and climate change, predicted to result in declining salinity in the Baltic Sea area that may alter the future distribution and performance of S. latissima in the area.     

Pathways of spread of the introduced ascidian Styela clava (Tunicata) in Northern Europe, as revealed by microsatellite markers

Styela clava, a solitary ascidian native to the NW Pacific, has become a conspicuous member of fouling communities in NW European waters. As its natural dispersal appears to be limited, the wide distribution of S. clava along coasts within its introduced range may be attributed to secondary spread assisted by human activities. Here, we used six microsatellite loci to examine the genetic diversity and extent of gene flow among S. clava populations in its European introduced range. Samples were collected from 21 populations within Europe (N = 808), 4 populations within the USA and two populations within the native range (Japan). Large variation in genetic diversity was observed among the European populations but were not explained either by the geographic distance from the first introduction area (i.e. Plymouth, UK) nor by the time elapsed since the introduction. No founder effect was observed in the introduced populations, except possibly in Puget Sound (USA). At least two different introductions occurred in Europe, identified as distinct genetic clusters: northern Danish populations (resembling one Japanese population), and the rest of Europe; a sample from Shoreham (England) possibly represents a third introduction. In North America, the population from the Atlantic was genetically similar to the majority of European populations, suggesting a European origin for populations on this seaboard, while populations from the Pacific coast were genetically similar to the same Japanese population as the Danish populations.     

Life history parameters of two geographically separated populations of <Emphasis Type="Italic">Spalangia cameroni</Emphasis>, a microhymenopteran pupal parasitoid of muscoid flies

Spalangia cameroni Perkins (Hymenoptera: Pteromalidae) is used as a biological control agent against house flies and stable flies in livestock farms in Northern Europe. In the present study, the juvenile development, survival, and sex ratio of one Danish and one Norwegian population of S. cameroni were investigated at 15, 20, 25, 30, and 35°C to determine the best adapted strain for release programs. The Danish population developed 50 days faster at 15°C, whereas the Norwegian developed 3–4 days faster at 25°C. The difference was smaller at 20°C and 30°C. Only a few Danish female parasitoids emerged at 35°C. Both populations required 381 degree days to complete development and exhibited optimal juvenile survival at 23.7°C. The Norwegian population produced more females than the Danish population at all temperatures (average of 67.5% vs. 55.5%, respectively). The overall differences between the populations were small, but might still be important for inundative release programs.