BACKGROUND
widely distributed species face different selection pressures along climatic and ecological gradients.
in this respect, basal metabolic rate  is particularly prone to evolve adaptively, since it has been shown to be heritable and correlates with fitness components in endotherms  <cit> .
several comparative studies have proposed that patterns of variation in metabolic rate match certain climatic variables in widely distributed groups of species  <cit> , suggesting that the level of bmr responds to different selection regimes generated by local conditions.
bmr is generated mainly in physiologically important internal organs including the kidneys, brain and liver  <cit>  with most of the physiological activity localized in certain compartments of cells in these organs.
mitochondria are the main cellular energy "factories", supplying organisms with energy stored in atp molecules.
the rate of energy production by mitochondria is crucial for an individual's fitness and is thought to be determined by natural selection  <cit> .
mitochondria are characterized by their own genome and harbour enzymatic elements of aerobic metabolic pathways.
however, oxidative phosphorylation , the process responsible for energy production, depends not only on the enzymes encoded in the internal organelle genome , but also on nuclear-encoded polypeptides which interact to enable the enzymatic reactions.
therefore, selection may act on and constrain the co-evolution of mitochondrial and nuclear genes  <cit> .
interestingly, between-species transfer of mtdna has been frequently observed among many group of animals and plants  <cit> .
such transfer has been traditionally considered neutral, however, it may be maladaptive, if introgression breaks the coevolved mito-nuclear complexes  <cit> , or adaptive, as mtdna function may represent important selective value  <cit> .
interspecific mtdna transfer observed among the broadly distributed rodent genus myodes  <cit>  offers an ideal situation to test 1) if reticulate evolution has an important and recognizable phenotypic effect and 2) whether natural selection may influence mtdna introgression.
the bank vole, myodes glareolus, is dispersed across different climatic and geographic zones, from the mediterranean to beyond the arctic circle  <cit>  .
the distribution of its close relative, the northern red-backed vole , m. rutilus, is restricted to northern parts of the circum-boreal zone, extending to the arctic zone  <cit> .
the red vole occurs from northern fennoscandia and northern eurasia, where it is partly sympatric with the bank vole, to the northern latitudes in north america.
introgressive hybridization between the bank vole and the red vole has been documented across a vast geographic area, from sweden to central and southern russia  <cit>  including northern finland  <cit> .
all these cases showed introgression of mtdna alone and no introgression of the nuclear genome has been reported  <cit> .
in this work we revisited a geographic region where mtdna introgression between m. glareolus and m. rutilus has been previously described  <cit> .
first, using sequence data both from mtdna and nuclear dna, we confirm that mtdna introgression from m. rutilus into m. glareolus occurs naturally in finland, with the introgressed haplotype being fixed in the northernmost populations.
since the mitochondria house the energy machinery of the cells, we analyzed variation in two morpho-physiological traits  among m. glareolus populations, and found differences between females from populations with different mtdna types, although the effect of geography could not be excluded.
the underpinnings and consequences of this variation are discussed.
RESULTS
genetic variation
in total,  <dig> voles trapped during summer  <dig> in  <dig> localities in finland  were analyzed for variation in mitochondrial and nuclear markers .
after allelic phase determination  the number of alleles present in m. glareolus nuclear genes varied between  <dig> and  <dig> .
the phylogenies reconstructed for each of the nuclear genes were consistent across methods and coincided with the expected assignment of individuals to the species , since both m. glareolus and m. rutilus  were recovered as monophyletic with high statistical support .
the cyt b sequences obtained were most likely of mitochondrial origin and not of nuclear integrated copies, as the reading frame was unbroken  and the composition of the third codon position was typical  compared to the average in mammals .
polymorphism was found in  <dig> sites  which defined  <dig> haplotypes  within m. glareolus .
contrary to inferences based on the sequences of the four nuclear genes, the cyt b haplotypes sampled in m. glareolus did not form a monophyletic clade, and was instead split into two clades, only partially overlapping with species assignment.
one clade, the m. rutilus-type mtdna , grouped  <dig>  individuals of m. glareolus together with m. rutilus specimens, suggesting mtdna introgression.
the second clade, m. glareolus-type mtdna , only grouped haplotypes specific for m. glareolus .
the geographic distribution of each of these haplogroups was partly disjoint, with southern and central finland  being occupied by the gla type mtdna, whereas the north and central-east  populations had the rut type.
one population located in central-eastern finland exhibited both mitotypes .
ni, number of sampled chromosomes; nh, number of haplotypes; np, number of polymorphic sites; h, haplotype diversity; π, nucleotide diversity; Θ, computed from the number of segregating sites.
standard deviations are shown in brackets.
&and * indicate p <  <dig>  and <  <dig> , respectively.
ni, number of individuals; nh, number of mtdna haplotypes; np, number of polymorphic sites; h, haplotypes diversity; π, nucleotide diversity; Θ, computed from the number of segregating sites.
standard deviations are shown in brackets.
+, &and * indicate p <  <dig> , <  <dig>  and <  <dig> , respectively.
nuclear dna sequence variation within m. glareolus was also detected .
the frequency spectrum of mutations did not significantly differ from the neutral mutation-drift expectations for most genes, as determined by tajima's d and fu's fs .
for cyt b, sequence diversity of individuals with the gla and rut mitotype was comparable and high .
gla and rut type cyt b sequences differed by  <dig>   and  <dig>   substitutions per site, respectively, from m. rufocanus sequences, while the divergence between gla and rut mitotypes was  <dig>   .
the mismatch analysis of cyt b sequences from m. glareolus showed a bimodal distribution of the number of pairwise differences , which illustrates the existence of two divergent lineages.
similar results were obtained for the population from east-central finland , where the mtdna lineages coexist.
the mismatch distributions analyzed separately for each of the mtdna type  and population were unimodal .
the goodness of fit test for deviation from the expectation under the sudden expansion model rejected the model for the data set including all bank voles with their own mtdna .
all significant tajima's d and fu's fs values were negative .
both the unimodality of the cyt b mismatch distribution within clades/populations and negative and significant values of tests of selective neutrality are expected for populations undergoing recent demographic growth or/and under positive selection.
given the average rate of cytochrome b divergence for rodents  <cit>  of  <dig> % per site per myr and the parameters derived from the mismatch distribution  for the introgressed haplogroup, this translates to an expansion time of approximately <dig>  <dig>  years ago.
the unimodal distribution and star-like network may imply that introgression of mtdna happened only once into the finnish populations of bank voles .
despite the excess of rare alleles that were generally detected in m. glareolus in the cyt b and not in the nuclear genes, the multilocus hka test  <cit>  did not reject the null neutral model of evolution .
phenotypic variation
all traits measured were positively correlated  and all repeatability estimates  were high and statistically significant .
as body mass and bmr were significantly influenced by a latitude-sex interaction in the initial analyses of variance , further tests were performed separately within sexes.
whole body bmr was higher in the gla than in the rut mitotype in m. glareolus females .
for males, whole body and mass-corrected bmr decreased toward the north  but did not differ between mitotypes .
female m. glareolus with the gla mitotype were significantly heavier than females with the rut mitotype .
longitude, latitude and their interaction did not affect variation in females' body mass and were sequentially excluded from the model .
within the sympatric population of two mitotypes ,  <dig> females and  <dig> males of m. glareolus were detected with m. rutilus mtdna .
the introgressed females had significantly lower values of whole-body bmr than sympatric females with native mtdna .
mass corrected bmr showed the same trend but was not significant .
these effects were also similar for males and in the same direction, but were also insignificant.
no significant differences between sympatric mitotypes in body mass were detected in this population .
τ, coefficient of intraclass correlation based on two repeated measurements across in average  <dig>  days , based on variance components from anova with study individual as categorical predictors, or from ancova with body mass included as covariate .
variation in body mass , head width  and basal metabolic rate  is presented for bank voles from "all populations" and from sotkamo "sympatric population" where mitotypes coexist.
significance of the effects: type of the mtdna , latitude  and longitude  of population  were tested in anova or ancova  models on log transformed traits as dependent variables.
marginal means  for mtdna types for ancova models were calculated accounting for variation in bm.
the insignificant effects  were hierarchically reduced from the analyses of variances "-" and are not presented in the table.
latitude×longitude interactions affected only males log transformed and residual values of hw , not included in the table.
* effects significant after implemented bonferroni correction.
discussion
although mitochondria serve important physiological functions in organisms, its evolution has traditionally been considered neutral.
accordingly, mtdna variation should have little phenotypic consequences in natural populations  <cit> .
however, during the last decade evidence is accumulating that the evolution of mtdna is often subject to natural selection  <cit> .
over  <dig> years ago, mtdna flow between two species of myodes voles, the bank vole, myodes glareolus, and the red vole, m. rutilus, was first described  <cit> , providing a potentially good model to test whether naturally occurring mtdna introgression may influence phenotypic traits.
here, in addition to sampling some m. rutilus specimens, we sampled m. glareolus in  <dig> finnish populations, including the northern range of the species where it overlaps with m. rutilus, where mtdna introgression might occur.
we assessed mtdna introgression by comparing the mtdna-based phylogeny with that of  <dig> nuclear dna markers.
as expected, we detected congruence between species assignment of the individual voles with the variation in four nuclear markers , and found that most of the mtdna haplotypes sampled in m. glareolus from northern finland  cluster within the clade of m. rutilus , which is a typical consequence of mtdna introgression  <cit> .
indeed, it is striking to note that the mtdna haplotypes from m. rutilus are fixed in the northern populations of m. glareolus and only in one population did we find both the native and the introgressed mitotypes .
having identified among the sampled specimens those with native and introgressed mtdna, we aimed to test whether introgression explains differences in two key phenotypic measures: body size and basal metabolic rate.
these traits were studied because size and energetic physiology are important predictors for life history traits in animals  <cit> .
also, some of the most recent data and theoretical predictions link variation in those traits with different fitness components  <cit> .
we found that the repeatability of size and metabolism was significant  and that phenotypes varied markedly between distantly located populations of bank voles.
it has been postulated that the geographic variation in morpho-physiological traits might be caused by climatic differences among distant localities  <cit> .
if so, the phenotypic differences are predicted to be noticeable and caused by phenotypic plasticity or adaptation to different habitats along environmental gradients, which here could include information inherited in mtdna as a possible mechanism of physiological adaptation  <cit> .
an interesting observation from our results is that the northern  myodes glareolus voles were smaller and had lower bmr values than voles from the southern populations .
sex specific tests showed that females with different mtdna types differed in body mass and absolute  metabolic rate but not in mass-corrected bmr, whereas males from northern populations had lower values of absolute and mass-corrected bmr, but not body mass, and their traits did not vary significantly between mtdna types .
whether the inferred differences across populations are due to mtdna introgression or related to genome reticulation is difficult to assess because bank voles from northern populations are also exclusively introgressed.
to circumvent this problem we analyzed a subsample of the data from the single population where both mitotypes coexist, thus eliminating the geographic factor.
although we did not find a significant difference in body size between introgressed and non-introgressed voles , we found that introgressed females had lower absolute bmr values than non-introgressed ones .
however, this association was not significant when bmr was mass-corrected.
though, only  <dig> females with m. rutilus mtdna type were sampled in this population, which yields little power to detect effects on body size and mass corrected bmr.
the absence of a strong relationship between mtdna type and variation in phenotypic characters could have resulted from relatively recent divergence between vole species, and consequently, the functional similarity between mitochondrial and nuclear genes, as shown in recent experimental transitions of whole genomes in drosophila species  <cit> .
otherwise, the co-introgression of some nuclear genes that have not been included in current analyses, but may be important for energetic physiology and body growth rate, may have balanced the incompatibility and influenced particular phenotypes  <cit> .
the observed sex-specific effects of mtdna on phenotypes, visible at least in the between-population analyses carried out here and in another study  <cit> , could be explained by different requirements between sexes connected to their reproductive biology  <cit> .
in fact, recent models predict e.g.
that the level of the rate of basal metabolism in endotherms is a consequence of directional selection operating on reproductive performance  <cit> .
accordingly, such processes are related to two energetically-costly female reproductive processes: pregnancy and lactation.
it has been also experimentally confirmed that directional selection can perform differentially on size and metabolic rate between sexes in mammals  <cit> .
however, the most important prediction for differential effects of mtdna on phenotype comes from the fact that in mammals, mitochondria are almost exclusively transmitted through females.
consequently, mtdna can only have direct adaptive effect for females and the mtdna effects on male phenotype are expected to result from counterbalancing effects of nuclear genes  <cit> .
the prevalence of the introgressed mtdna haplotype in the northern populations of m. glareolus in extreme frequencies - the foreign haplotype seems fixed in these populations - is striking, and we thus raised the hypothesis that mtdna introgression could have been promoted by natural selection.
we assessed if the frequency spectrum of mutations of the introgressed haplotypes could be biased towards an excess of rare alleles, a pattern compatible with post-introgression positive selection.
our analysis of sequence variation among the introgressed cyt b haplotypes indeed shows a pattern that is not compatible with a model of population equilibrium .
this could result from adaptive introgression of mtdna, promoting the expansion of the novel haplotype northward.
however, selection is expected to affect solely the locus in question, while the effect of population expansion and other demographic phenomena should affect the whole genome equally, on average.
as the hka test did not indicate any deviation from neutral expectations of the multilocus ratios of divergence to polymorphism  and both mitotypes identified in m. glareolus  showed similar expansion signals, our results suggest that the species itself may have undergone recent population expansion, spreading the traces of mtdna introgression throughout the northern range of m. glareolus.
indeed, if hybridization occurs between a resident and an invading species, introgression is expected to occur into the spatially expanding one, in a stochastically neutral phenomenon  <cit> .
this scenario has been used to explain massive mtdna introgression among hares  <cit>  and  in other organisms  <cit> .
in this demographic replacement process, markers with lower intraspecific migration rates, as is often the case with mtdna, are expected to introgress more easily because the influx of native alleles into the invasion front is lower  <cit> .
thus, the northern expansion of m. glareolus into the range of m. rutilus could have favoured mtdna introgression in a purely neutral manner, and could also explain the asymmetry of the observed introgression.
such asymmetry could be explained by female-biased assortative mating  <cit> .
in a situation of interspecific contact with imbalanced frequencies of the interacting species, the females of the rare species  tend to mate more easily with the heterospecific males, i.e., the expanding m. glareolus.
the continuous backcrossing of hybrids with the same frequency-dependence over generations would cause introgression of the maternally transmitted mtdna in the direction of the more abundant species .
this asymmetry could also occur if the invasion of the range of m. rutilus by m. glareolus was pioneered by males, which are known to disperse farther than females in this species  <cit> .
CONCLUSIONS
this study uncovered marked differences in body mass and bmr across finnish populations of m. glareolus along a north-south axis.
these differences correspond with massive mtdna introgression from m. rutilus into the populations of m. glareolus, which suggests that genome reticulation may presumably be related to the differences in phenotype.
given its role in energy processing, mtdna is an obvious candidate to underlie physiological phenotypic differences more directly.
although a signal of association between mtdna introgression and whole-body bmr was detected from the analysis of the population where the native and foreign mtdna haplotypes coexist, the results failed to show a relationship between mass-corrected bmr or body mass alone and the mitochondrial dna.
further analyses using increased sample sizes and more populations where mtdna types exist in sympatry will help to clarify this result.
also, the co-introgression of nuclear elements may be responsible for the altered phenotype and counterbalance eventual incompatibilities between nuclear and mitochondrial genomes.
obvious candidates for co-introgression with mtdna are nuclear genes involved in physiological pathways, most notably those involved in mitochondrial function, which are known to co-evolve with mtdna  <cit> .
the construction of congenic strains in breeding experiments  <cit> , where the mtdna form m. rutilus is fixed in a nuclear background of m. glareolus, would allow testing if mtdna alone alters phenotype, and ultimately, whether the mtdna introgression has any significant fitness effects.

 widely distributed species face different selection pressures along climatic and ecological gradients. in this respect, basal metabolic rate is particularly prone to evolve adaptively, since it has been shown to be heritable and correlates with fitness components in endotherms. several comparative studies have proposed that patterns of variation in metabolic rate match certain climatic variables in widely distributed groups of species, suggesting that the level of bmr responds to different selection regimes generated by local conditions. bmr is generated mainly in physiologically important internal organs including the kidneys, brain and liver with most of the physiological activity localized in certain compartments of cells in these organs. mitochondria are the main cellular energy "factories", supplying organisms with energy stored in atp molecules. the rate of energy production by mitochondria is crucial for an individual's fitness and is thought to be determined by natural selection. mitochondria are characterized by their own genome and harbour enzymatic elements of aerobic metabolic pathways. however, oxidative phosphorylation, the process responsible for energy production, depends not only on the enzymes encoded in the internal organelle genome, but also on nuclear-encoded polypeptides which interact to enable the enzymatic reactions. therefore, selection may act on and constrain the co-evolution of mitochondrial and nuclear genes. interestingly, between-species transfer of mtdna has been frequently observed among many group of animals and plants. such transfer has been traditionally considered neutral, however, it may be maladaptive, if introgression breaks the coevolved mito-nuclear complexes, or adaptive, as mtdna function may represent important selective value. interspecific mtdna transfer observed among the broadly distributed rodent genus myodes offers an ideal situation to test 1) if reticulate evolution has an important and recognizable phenotypic effect and 2) whether natural selection may influence mtdna introgression. the bank vole, myodes glareolus, is dispersed across different climatic and geographic zones, from the mediterranean to beyond the arctic circle. the distribution of its close relative, the northern red-backed vole, m. rutilus, is restricted to northern parts of the circum-boreal zone, extending to the arctic zone. the red vole occurs from northern fennoscandia and northern eurasia, where it is partly sympatric with the bank vole, to the northern latitudes in north america. introgressive hybridization between the bank vole and the red vole has been documented across a vast geographic area, from sweden to central and southern russia including northern finland. all these cases showed introgression of mtdna alone and no introgression of the nuclear genome has been reported. in this work we revisited a geographic region where mtdna introgression between m. glareolus and m. rutilus has been previously described. first, using sequence data both from mtdna and nuclear dna, we confirm that mtdna introgression from m. rutilus into m. glareolus occurs naturally in finland, with the introgressed haplotype being fixed in the northernmost populations. since the mitochondria house the energy machinery of the cells, we analyzed variation in two morpho-physiological traits among m. glareolus populations, and found differences between females from populations with different mtdna types, although the effect of geography could not be excluded. the underpinnings and consequences of this variation are discussed.  genetic variation in total, voles trapped during summer in localities in finland were analyzed for variation in mitochondrial and nuclear markers. after allelic phase determination the number of alleles present in m. glareolus nuclear genes varied between and. the phylogenies reconstructed for each of the nuclear genes were consistent across methods and coincided with the expected assignment of individuals to the species, since both m. glareolus and m. rutilus were recovered as monophyletic with high statistical support. the cyt b sequences obtained were most likely of mitochondrial origin and not of nuclear integrated copies, as the reading frame was unbroken and the composition of the third codon position was typical compared to the average in mammals. polymorphism was found in sites which defined haplotypes within m. glareolus. contrary to inferences based on the sequences of the four nuclear genes, the cyt b haplotypes sampled in m. glareolus did not form a monophyletic clade, and was instead split into two clades, only partially overlapping with species assignment. one clade, the m. rutilus-type mtdna, grouped individuals of m. glareolus together with m. rutilus specimens, suggesting mtdna introgression. the second clade, m. glareolus-type mtdna, only grouped haplotypes specific for m. glareolus. the geographic distribution of each of these haplogroups was partly disjoint, with southern and central finland being occupied by the gla type mtdna, whereas the north and central-east populations had the rut type. one population located in central-eastern finland exhibited both mitotypes. ni, number of sampled chromosomes; nh, number of haplotypes; np, number of polymorphic sites; h, haplotype diversity; π, nucleotide diversity; Θ, computed from the number of segregating sites. standard deviations are shown in brackets. &and * indicate p < and <, respectively. ni, number of individuals; nh, number of mtdna haplotypes; np, number of polymorphic sites; h, haplotypes diversity; π, nucleotide diversity; Θ, computed from the number of segregating sites. standard deviations are shown in brackets. +, &and * indicate p <, < and <, respectively. nuclear dna sequence variation within m. glareolus was also detected. the frequency spectrum of mutations did not significantly differ from the neutral mutation-drift expectations for most genes, as determined by tajima's d and fu's fs. for cyt b, sequence diversity of individuals with the gla and rut mitotype was comparable and high. gla and rut type cyt b sequences differed by and substitutions per site, respectively, from m. rufocanus sequences, while the divergence between gla and rut mitotypes was. the mismatch analysis of cyt b sequences from m. glareolus showed a bimodal distribution of the number of pairwise differences, which illustrates the existence of two divergent lineages. similar results were obtained for the population from east-central finland, where the mtdna lineages coexist. the mismatch distributions analyzed separately for each of the mtdna type and population were unimodal. the goodness of fit test for deviation from the expectation under the sudden expansion model rejected the model for the data set including all bank voles with their own mtdna. all significant tajima's d and fu's fs values were negative. both the unimodality of the cyt b mismatch distribution within clades/populations and negative and significant values of tests of selective neutrality are expected for populations undergoing recent demographic growth or/and under positive selection. given the average rate of cytochrome b divergence for rodents of % per site per myr and the parameters derived from the mismatch distribution for the introgressed haplogroup, this translates to an expansion time of approximately years ago. the unimodal distribution and star-like network may imply that introgression of mtdna happened only once into the finnish populations of bank voles. despite the excess of rare alleles that were generally detected in m. glareolus in the cyt b and not in the nuclear genes, the multilocus hka test did not reject the null neutral model of evolution. phenotypic variation all traits measured were positively correlated and all repeatability estimates were high and statistically significant. as body mass and bmr were significantly influenced by a latitude-sex interaction in the initial analyses of variance, further tests were performed separately within sexes. whole body bmr was higher in the gla than in the rut mitotype in m. glareolus females. for males, whole body and mass-corrected bmr decreased toward the north but did not differ between mitotypes. female m. glareolus with the gla mitotype were significantly heavier than females with the rut mitotype. longitude, latitude and their interaction did not affect variation in females' body mass and were sequentially excluded from the model. within the sympatric population of two mitotypes, females and males of m. glareolus were detected with m. rutilus mtdna. the introgressed females had significantly lower values of whole-body bmr than sympatric females with native mtdna. mass corrected bmr showed the same trend but was not significant. these effects were also similar for males and in the same direction, but were also insignificant. no significant differences between sympatric mitotypes in body mass were detected in this population. τ, coefficient of intraclass correlation based on two repeated measurements across in average days, based on variance components from anova with study individual as categorical predictors, or from ancova with body mass included as covariate. variation in body mass, head width and basal metabolic rate is presented for bank voles from "all populations" and from sotkamo "sympatric population" where mitotypes coexist. significance of the effects: type of the mtdna, latitude and longitude of population were tested in anova or ancova models on log transformed traits as dependent variables. marginal means for mtdna types for ancova models were calculated accounting for variation in bm. the insignificant effects were hierarchically reduced from the analyses of variances "-" and are not presented in the table. latitude×longitude interactions affected only males log transformed and residual values of hw, not included in the table. * effects significant after implemented bonferroni correction. discussion although mitochondria serve important physiological functions in organisms, its evolution has traditionally been considered neutral. accordingly, mtdna variation should have little phenotypic consequences in natural populations. however, during the last decade evidence is accumulating that the evolution of mtdna is often subject to natural selection. over years ago, mtdna flow between two species of myodes voles, the bank vole, myodes glareolus, and the red vole, m. rutilus, was first described, providing a potentially good model to test whether naturally occurring mtdna introgression may influence phenotypic traits. here, in addition to sampling some m. rutilus specimens, we sampled m. glareolus in finnish populations, including the northern range of the species where it overlaps with m. rutilus, where mtdna introgression might occur. we assessed mtdna introgression by comparing the mtdna-based phylogeny with that of nuclear dna markers. as expected, we detected congruence between species assignment of the individual voles with the variation in four nuclear markers, and found that most of the mtdna haplotypes sampled in m. glareolus from northern finland cluster within the clade of m. rutilus, which is a typical consequence of mtdna introgression. indeed, it is striking to note that the mtdna haplotypes from m. rutilus are fixed in the northern populations of m. glareolus and only in one population did we find both the native and the introgressed mitotypes. having identified among the sampled specimens those with native and introgressed mtdna, we aimed to test whether introgression explains differences in two key phenotypic measures: body size and basal metabolic rate. these traits were studied because size and energetic physiology are important predictors for life history traits in animals. also, some of the most recent data and theoretical predictions link variation in those traits with different fitness components. we found that the repeatability of size and metabolism was significant and that phenotypes varied markedly between distantly located populations of bank voles. it has been postulated that the geographic variation in morpho-physiological traits might be caused by climatic differences among distant localities. if so, the phenotypic differences are predicted to be noticeable and caused by phenotypic plasticity or adaptation to different habitats along environmental gradients, which here could include information inherited in mtdna as a possible mechanism of physiological adaptation. an interesting observation from our results is that the northern myodes glareolus voles were smaller and had lower bmr values than voles from the southern populations. sex specific tests showed that females with different mtdna types differed in body mass and absolute metabolic rate but not in mass-corrected bmr, whereas males from northern populations had lower values of absolute and mass-corrected bmr, but not body mass, and their traits did not vary significantly between mtdna types. whether the inferred differences across populations are due to mtdna introgression or related to genome reticulation is difficult to assess because bank voles from northern populations are also exclusively introgressed. to circumvent this problem we analyzed a subsample of the data from the single population where both mitotypes coexist, thus eliminating the geographic factor. although we did not find a significant difference in body size between introgressed and non-introgressed voles, we found that introgressed females had lower absolute bmr values than non-introgressed ones. however, this association was not significant when bmr was mass-corrected. though, only females with m. rutilus mtdna type were sampled in this population, which yields little power to detect effects on body size and mass corrected bmr. the absence of a strong relationship between mtdna type and variation in phenotypic characters could have resulted from relatively recent divergence between vole species, and consequently, the functional similarity between mitochondrial and nuclear genes, as shown in recent experimental transitions of whole genomes in drosophila species. otherwise, the co-introgression of some nuclear genes that have not been included in current analyses, but may be important for energetic physiology and body growth rate, may have balanced the incompatibility and influenced particular phenotypes. the observed sex-specific effects of mtdna on phenotypes, visible at least in the between-population analyses carried out here and in another study, could be explained by different requirements between sexes connected to their reproductive biology. in fact, recent models predict e.g. that the level of the rate of basal metabolism in endotherms is a consequence of directional selection operating on reproductive performance. accordingly, such processes are related to two energetically-costly female reproductive processes: pregnancy and lactation. it has been also experimentally confirmed that directional selection can perform differentially on size and metabolic rate between sexes in mammals. however, the most important prediction for differential effects of mtdna on phenotype comes from the fact that in mammals, mitochondria are almost exclusively transmitted through females. consequently, mtdna can only have direct adaptive effect for females and the mtdna effects on male phenotype are expected to result from counterbalancing effects of nuclear genes. the prevalence of the introgressed mtdna haplotype in the northern populations of m. glareolus in extreme frequencies - the foreign haplotype seems fixed in these populations - is striking, and we thus raised the hypothesis that mtdna introgression could have been promoted by natural selection. we assessed if the frequency spectrum of mutations of the introgressed haplotypes could be biased towards an excess of rare alleles, a pattern compatible with post-introgression positive selection. our analysis of sequence variation among the introgressed cyt b haplotypes indeed shows a pattern that is not compatible with a model of population equilibrium. this could result from adaptive introgression of mtdna, promoting the expansion of the novel haplotype northward. however, selection is expected to affect solely the locus in question, while the effect of population expansion and other demographic phenomena should affect the whole genome equally, on average. as the hka test did not indicate any deviation from neutral expectations of the multilocus ratios of divergence to polymorphism and both mitotypes identified in m. glareolus showed similar expansion signals, our results suggest that the species itself may have undergone recent population expansion, spreading the traces of mtdna introgression throughout the northern range of m. glareolus. indeed, if hybridization occurs between a resident and an invading species, introgression is expected to occur into the spatially expanding one, in a stochastically neutral phenomenon. this scenario has been used to explain massive mtdna introgression among hares and in other organisms. in this demographic replacement process, markers with lower intraspecific migration rates, as is often the case with mtdna, are expected to introgress more easily because the influx of native alleles into the invasion front is lower. thus, the northern expansion of m. glareolus into the range of m. rutilus could have favoured mtdna introgression in a purely neutral manner, and could also explain the asymmetry of the observed introgression. such asymmetry could be explained by female-biased assortative mating. in a situation of interspecific contact with imbalanced frequencies of the interacting species, the females of the rare species tend to mate more easily with the heterospecific males, i.e., the expanding m. glareolus. the continuous backcrossing of hybrids with the same frequency-dependence over generations would cause introgression of the maternally transmitted mtdna in the direction of the more abundant species. this asymmetry could also occur if the invasion of the range of m. rutilus by m. glareolus was pioneered by males, which are known to disperse farther than females in this species.  this study uncovered marked differences in body mass and bmr across finnish populations of m. glareolus along a north-south axis. these differences correspond with massive mtdna introgression from m. rutilus into the populations of m. glareolus, which suggests that genome reticulation may presumably be related to the differences in phenotype. given its role in energy processing, mtdna is an obvious candidate to underlie physiological phenotypic differences more directly. although a signal of association between mtdna introgression and whole-body bmr was detected from the analysis of the population where the native and foreign mtdna haplotypes coexist, the results failed to show a relationship between mass-corrected bmr or body mass alone and the mitochondrial dna. further analyses using increased sample sizes and more populations where mtdna types exist in sympatry will help to clarify this result. also, the co-introgression of nuclear elements may be responsible for the altered phenotype and counterbalance eventual incompatibilities between nuclear and mitochondrial genomes. obvious candidates for co-introgression with mtdna are nuclear genes involved in physiological pathways, most notably those involved in mitochondrial function, which are known to co-evolve with mtdna. the construction of congenic strains in breeding experiments, where the mtdna form m. rutilus is fixed in a nuclear background of m. glareolus, would allow testing if mtdna alone alters phenotype, and ultimately, whether the mtdna introgression has any significant fitness effects. 
in contrast, the two northernmost populations of m. glareolus have a fixed mitotype that is shared with m. rutilus, likely due to introgressive hybridization. our results show that the phenotypic traits vary markedly along the north-south axis of populations of m. glareolus. we discuss the causes and biological meaning of our results and the means to clarify these questions in future research. introgression of mitochondrial dna is among the most frequently described cases of reticulate evolution. first, we confirmed that m. glareolus harbour mtdna introgressed from m. rutilus by analyzing mtdna and nuclear dna sequence variation and reconstructing loci phylogenies among six natural populations in finland. we then studied geographic variation in body size and basal metabolic rate among the populations of m. glareolus and tested its relationship with mtdna type. the analyses of phenotypic traits revealed that the body mass and whole-body, but not mass corrected, bmr are significantly reduced in m. glareolus females from northern finland that also have the introgressed mitotype. this variation may be related to adaptation to local environments and coincides with the gradient of genome reticulation between m. glareolus and m. rutilus, which was assessed by mtdna introgression. myodes glareolus and its arctic neighbour, m. rutilus, are reciprocally monophyletic at the analyzed nuclear dna loci. here we investigated how morpho-physiological traits vary in natural populations of a common rodent and whether this variation could be associated with mtdna introgression. introgression of mtdna may have affected morpho-physiological traits but do not show strong effects on either body mass or basal metabolic rate alone. restricting the analysis to the single population where the mitotypes coexist, the association of mtdna type with whole-body bmr remained but those with mass corrected bmr and body mass did not. mitochondrial sequence variation in the introgressed haplotypes is compatible with demographic growth of the populations, but may also be a result of positive selection.
 introgression of mitochondrial dna is among the most frequently described cases of reticulate evolution. the tendency of mtdna to cross interspecific barriers is somewhat counter-intuitive considering the key function of enzymes that it encodes in the oxidative-phosphorylation process, which could give rise to hybrid dysfunction. how mtdna reticulation affects the evolution of metabolic functions is, however, uncertain. here we investigated how morpho-physiological traits vary in natural populations of a common rodent and whether this variation could be associated with mtdna introgression. first, we confirmed that m. glareolus harbour mtdna introgressed from m. rutilus by analyzing mtdna and nuclear dna sequence variation and reconstructing loci phylogenies among six natural populations in finland. we then studied geographic variation in body size and basal metabolic rate among the populations of m. glareolus and tested its relationship with mtdna type.  myodes glareolus and its arctic neighbour, m. rutilus, are reciprocally monophyletic at the analyzed nuclear dna loci. in contrast, the two northernmost populations of m. glareolus have a fixed mitotype that is shared with m. rutilus, likely due to introgressive hybridization. the analyses of phenotypic traits revealed that the body mass and whole-body, but not mass corrected, bmr are significantly reduced in m. glareolus females from northern finland that also have the introgressed mitotype. restricting the analysis to the single population where the mitotypes coexist, the association of mtdna type with whole-body bmr remained but those with mass corrected bmr and body mass did not. mitochondrial sequence variation in the introgressed haplotypes is compatible with demographic growth of the populations, but may also be a result of positive selection.  our results show that the phenotypic traits vary markedly along the north-south axis of populations of m. glareolus. this variation may be related to adaptation to local environments and coincides with the gradient of genome reticulation between m. glareolus and m. rutilus, which was assessed by mtdna introgression. introgression of mtdna may have affected morpho-physiological traits but do not show strong effects on either body mass or basal metabolic rate alone. we discuss the causes and biological meaning of our results and the means to clarify these questions in future research. 