BACKGROUND
the brown hare  is widely distributed throughout europe, up to 60°n, in asia minor and probably south to israel.
it is a popular game animal and its range has expanded to the east both by natural dispersion and by translocations to central and far-east siberia  <cit> .
worldwide it has also been imported to many other countries, outside its natural distribution range, e.g.
south and north america, australia and new zealand.
the brown hare is common all over denmark and is the only lepus species that exists in the country.
since  <dig> the hare population has declined drastically in europe and in denmark.
the primary cause of the decline is habitat changes in relation to agricultural intensification, whereas effects of climatic changes and predator abundance have increased by loss of year-round access to high-quality food and cover  <cit> .
additional factors like predators , birds of prey and traffic may have influenced the population locally  <cit> .
in denmark this is reflected by a large drop in annual number of hares shot from more than  <dig>  before  <dig> to  <dig>  in 2004/ <dig>  <cit> .
the general range of the mountain hare  covers much of the palaearctic.
in europe it exists in great britain, norway, sweden, finland, parts of the alps and in eastern europe but it does not exist in denmark  <cit> .
during the late nineteenth century the brown hare was introduced in southern sweden as a game animal  <cit> .
in scandinavia the mountain hare populations have been retreating northwards since the introduction of the brown hare, presumably as a result of a gradual competitive exclusion by the latter  <cit> .
mountain hares supposedly colonized scandinavia through repeated immigration waves over two different post-glacial colonization routes; one from the south and one from the northeast  <cit> .
given the sympatric distribution of several hare species, e.g.
in sweden, italy, and spain, a number of studies focused on the possibility of interspecific hybridization.
the first evidence of interspecific hybridization in hares concerns l. timidus and l. europaeus in sweden  <cit>  where unidirectional introgression of the native l. timidus mtdna occurs in the introduced l. europaeus.
however, introgression of mitochondrial dna into other hare species has been demonstrated, e.g.
the introgression of l. timidus mtdna into l. granatensis, l. europaeus and l. castroviejoi in northern iberia  <cit>  and introgression of foreign mtdna is likely to occur in several hare species from asia  <cit> .
these findings have lead to the conclusion that the genetic integrity of many european species largely depends on differences in behaviour and ecology which, at best, offer semi-permeable isolation  <cit> .
in captivity, mountain hare females mate with brown hare males and produce viable offspring, whereas the reverse crosses do not happen spontaneously but can be performed successfully with insemination  <cit> .
the f <dig> hybrids are morphological intermediates between the two species  <cit> .
during the data analysis in a study concerning danish brown hares we identified  <dig> hares with a very divergent haplotype.
here we show that these  <dig> individuals from the danish wild brown hare populations carry mountain hare mtdna haplotypes.
RESULTS
during alignment of  <dig> hare sequences,  <dig> sequences  were clearly different from the rest and collapsed into a single haplotype.
the  <dig> individuals were obtained from five of the eight populations sampled, and these five populations are all located on islands in the eastern part of denmark .
the frequency of sampled individuals with l. timidus mtdna in those five populations averaged 6% .
a blast search against genbank with the divergent haplotype mtdna returned l. timidus as the best hit , but not a single l. europaeus sequence in the first  <dig> hits.
the subsequent phylogenetic analysis  revealed that the divergent danish haplotype is grouped with l. timidus samples from across europe  and most closely related to the swedish l. timidus haplotypes .
the other danish haplotypes included in the phylogenetic analysis, the  <dig> l. europaeus haplotypes, are grouped with the l. europaeus clade as expected, and they are most closely related to german haplotypes .
hence, the danish haplotypes clearly originate from two different species.
discussion
the results show the presence of mountain hare mtdna in danish brown hare populations.
from mtdna alone we cannot decide whether these  <dig> individuals are true mountain hares or introgressed brown hares.
this would require further analysis using nuclear gene sequences  .
however, given a) the frequency of the l. timidus haplotype where it is present , b) that l. timidus is not reported to be a native breeding species in denmark  <cit> , and c) that the two species are able to hybridize  <cit> , these individuals most likely represent introgressed brown hares.
the most likely origins of introgression are; 1) natural migration of mountain hares from sweden followed by introgression, or 2) natural migration or translocation of introgressed brown hares from sweden.
however, since the documented distribution of mountain hares in sweden does not include southern sweden  <cit> , natural migration of mountain hares is less probable than introduction of introgressed brown hares  from sweden.
the fact that the baltic sea occasionally freezes over in very severe winters facilitates natural migration of introgressed brown hares from southern sweden across the sea barrier to the eastern danish islands .
this is also supported by the results from the genetic analyses .
during the mid 1980'ies a network of hare breeding facilities was established in denmark, and it was initiated with imported hares primarily from italy, france, hungary and sweden  <cit> .
no combined record is kept of the amount and origin of hares imported to denmark.
in  <dig>  when farming hares were prohibited in denmark, there were  <dig> hare-farms in denmark, with an annual export of up to  <dig>  hares  <cit> .
as approximately 15% of swedish l. europaeus specimens from sympatric areas carried transmitted l. timidus mtdna  <cit> , it is possible that phenotypic brown hare specimens imported from sweden for breeding purposes have carried the l. timidus mtdna and later escaped to the natural hare population.
the detection of only one haplotype in the  <dig> individuals shows low mtdna diversity in the total population of hybrids in denmark.
the geographical distribution of the  <dig> intogressed brown hares  and the lack of haplotype diversity support a common origin in southern sweden.
the implications of introgressed brown hares in the danish hare populations are currently unclear.
it has been suggested that hares with an alien mtdna have a lowered fitness as a result of a functional incompatibility between the cytoplasmatic mitochondrial genes and the cell nucleus  <cit> .
in opposition to this, due to its observed high frequency in brown hares  melo-ferreira et al.
<cit>  suggested, that the ancient l. timidus mtdna observed in the iberian peninsular might have some selective advantage depending on the nuclear background.
thulin et al.
<cit>  raised the question as to whether any 'pure' population of brown hares exists anywhere.
our findings give further support to this statement.
this has important consequences for conservation and population genetics, e.g.
problematic definition of management units, unclear fitness effects, genetically mixed populations and the inclusion of nuclear markers will be necessary for future hare studies.
CONCLUSIONS
contrary to what is known, the danish hare populations are not pure l. europaeus populations but include introgressed brown hares with swedish l. timidus mtdna.
the most probable explanation is natural migration or translocation of introgressed brown hares from sweden.
the impurity of hare populations has implications for conservation and population genetics.

 the brown hare is widely distributed throughout europe, up to 60°n, in asia minor and probably south to israel. it is a popular game animal and its range has expanded to the east both by natural dispersion and by translocations to central and far-east siberia. worldwide it has also been imported to many other countries, outside its natural distribution range, e.g. south and north america, australia and new zealand. the brown hare is common all over denmark and is the only lepus species that exists in the country. since the hare population has declined drastically in europe and in denmark. the primary cause of the decline is habitat changes in relation to agricultural intensification, whereas effects of climatic changes and predator abundance have increased by loss of year-round access to high-quality food and cover. additional factors like predators, birds of prey and traffic may have influenced the population locally. in denmark this is reflected by a large drop in annual number of hares shot from more than before to in 2004/. the general range of the mountain hare covers much of the palaearctic. in europe it exists in great britain, norway, sweden, finland, parts of the alps and in eastern europe but it does not exist in denmark. during the late nineteenth century the brown hare was introduced in southern sweden as a game animal. in scandinavia the mountain hare populations have been retreating northwards since the introduction of the brown hare, presumably as a result of a gradual competitive exclusion by the latter. mountain hares supposedly colonized scandinavia through repeated immigration waves over two different post-glacial colonization routes; one from the south and one from the northeast. given the sympatric distribution of several hare species, e.g. in sweden, italy, and spain, a number of studies focused on the possibility of interspecific hybridization. the first evidence of interspecific hybridization in hares concerns l. timidus and l. europaeus in sweden where unidirectional introgression of the native l. timidus mtdna occurs in the introduced l. europaeus. however, introgression of mitochondrial dna into other hare species has been demonstrated, e.g. the introgression of l. timidus mtdna into l. granatensis, l. europaeus and l. castroviejoi in northern iberia and introgression of foreign mtdna is likely to occur in several hare species from asia. these findings have lead to the conclusion that the genetic integrity of many european species largely depends on differences in behaviour and ecology which, at best, offer semi-permeable isolation. in captivity, mountain hare females mate with brown hare males and produce viable offspring, whereas the reverse crosses do not happen spontaneously but can be performed successfully with insemination. the f hybrids are morphological intermediates between the two species. during the data analysis in a study concerning danish brown hares we identified hares with a very divergent haplotype. here we show that these individuals from the danish wild brown hare populations carry mountain hare mtdna haplotypes.  during alignment of hare sequences, sequences were clearly different from the rest and collapsed into a single haplotype. the individuals were obtained from five of the eight populations sampled, and these five populations are all located on islands in the eastern part of denmark. the frequency of sampled individuals with l. timidus mtdna in those five populations averaged 6%. a blast search against genbank with the divergent haplotype mtdna returned l. timidus as the best hit, but not a single l. europaeus sequence in the first hits. the subsequent phylogenetic analysis revealed that the divergent danish haplotype is grouped with l. timidus samples from across europe and most closely related to the swedish l. timidus haplotypes. the other danish haplotypes included in the phylogenetic analysis, the l. europaeus haplotypes, are grouped with the l. europaeus clade as expected, and they are most closely related to german haplotypes. hence, the danish haplotypes clearly originate from two different species. discussion the results show the presence of mountain hare mtdna in danish brown hare populations. from mtdna alone we cannot decide whether these individuals are true mountain hares or introgressed brown hares. this would require further analysis using nuclear gene sequences. however, given a) the frequency of the l. timidus haplotype where it is present, b) that l. timidus is not reported to be a native breeding species in denmark, and c) that the two species are able to hybridize, these individuals most likely represent introgressed brown hares. the most likely origins of introgression are; 1) natural migration of mountain hares from sweden followed by introgression, or 2) natural migration or translocation of introgressed brown hares from sweden. however, since the documented distribution of mountain hares in sweden does not include southern sweden, natural migration of mountain hares is less probable than introduction of introgressed brown hares from sweden. the fact that the baltic sea occasionally freezes over in very severe winters facilitates natural migration of introgressed brown hares from southern sweden across the sea barrier to the eastern danish islands. this is also supported by the results from the genetic analyses. during the mid 1980'ies a network of hare breeding facilities was established in denmark, and it was initiated with imported hares primarily from italy, france, hungary and sweden. no combined record is kept of the amount and origin of hares imported to denmark. in when farming hares were prohibited in denmark, there were hare-farms in denmark, with an annual export of up to hares. as approximately 15% of swedish l. europaeus specimens from sympatric areas carried transmitted l. timidus mtdna, it is possible that phenotypic brown hare specimens imported from sweden for breeding purposes have carried the l. timidus mtdna and later escaped to the natural hare population. the detection of only one haplotype in the individuals shows low mtdna diversity in the total population of hybrids in denmark. the geographical distribution of the intogressed brown hares and the lack of haplotype diversity support a common origin in southern sweden. the implications of introgressed brown hares in the danish hare populations are currently unclear. it has been suggested that hares with an alien mtdna have a lowered fitness as a result of a functional incompatibility between the cytoplasmatic mitochondrial genes and the cell nucleus. in opposition to this, due to its observed high frequency in brown hares melo-ferreira et al. suggested, that the ancient l. timidus mtdna observed in the iberian peninsular might have some selective advantage depending on the nuclear background. thulin et al. raised the question as to whether any'pure' population of brown hares exists anywhere. our findings give further support to this statement. this has important consequences for conservation and population genetics, e.g. problematic definition of management units, unclear fitness effects, genetically mixed populations and the inclusion of nuclear markers will be necessary for future hare studies.  contrary to what is known, the danish hare populations are not pure l. europaeus populations but include introgressed brown hares with swedish l. timidus mtdna. the most probable explanation is natural migration or translocation of introgressed brown hares from sweden. the impurity of hare populations has implications for conservation and population genetics. 
in contrast to what is known, the danish hare populations are not pure l. europaeus populations but include introgressed brown hares with swedish l. timidus mtdna. in europe the mountain hare exists in great britain, norway, sweden, finland, parts of the alps and in eastern europe, but not in denmark. the frequency of lepus timidus mtdna haplotype in the eastern danish hare populations is estimated to 6%. the impurity of hare populations has implications for conservation and population genetics. phylogenetic analysis shows that the divergent danish haplotype is most closely related to the swedish mountain hare. during the data collection in a study concerning danish brown hares we identified hares with a single very divergent haplotype. interspecific hybridization has been demonstrated between native swedish mountain hares and introduced brown hares. the most probable explanation of this is natural migration or translocation of introgressed brown hares from sweden.
 in europe the mountain hare exists in great britain, norway, sweden, finland, parts of the alps and in eastern europe, but not in denmark. interspecific hybridization has been demonstrated between native swedish mountain hares and introduced brown hares. during the data collection in a study concerning danish brown hares we identified hares with a single very divergent haplotype.  phylogenetic analysis shows that the divergent danish haplotype is most closely related to the swedish mountain hare. the frequency of lepus timidus mtdna haplotype in the eastern danish hare populations is estimated to 6%.  in contrast to what is known, the danish hare populations are not pure l. europaeus populations but include introgressed brown hares with swedish l. timidus mtdna. the most probable explanation of this is natural migration or translocation of introgressed brown hares from sweden. the impurity of hare populations has implications for conservation and population genetics. 