SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Hybridisation among wild mammal populations may lead to introgression of genes and genomes over the species barrier. In Sweden, in northern Europe, and on the Iberian Peninsula in southern Europe, mitochondrial DNA from L. timidus occurs among L. europaeus specimens, presumably as a result of interspecific hybridisation. In Russia, the species are believed to hybridise as well, but no investigations have confirmed introgression. Here we develop species diagnostic single nucleotide polymorphisms in the mitochondrial genomes and combine them with analysis of nuclear microsatellite markers to investigate hybridisation and introgression in 71 Lepus specimens from Russia. A total of 58 specimens are typical representatives of either species. An additional nine specimens have slightly intermediate genotypes, potentially as a result of introgression of nuclear genes. Finally, we find three specimens with L. europaeus mitochondrial genome and apparent L. timidus nuclear genome. This indicates that the reciprocal transfer of mtDNA occur among Russian populations of these species. Our observation differs from previous observations of mtDNA introgression in Sweden and Iberia, and provides further support for a reticulated mode of introgression within the genus Lepus.

Hybridisation and subsequent introgression over the species barrier is a common feature of wild populations of many species. Transfer of genes between species may be considered a way to acquire novel genetic material for selection to work upon (Anderson and Stebbins 1954; Arnold 1997), but introgression between native species and introduced, or domesticated, conspecifics may also pose a threat to the genetic integrity of species (Ebenhard 1988; Rhymer and Simberloff 1996). Among mammals, well studies examples of hybridisation and introgression include deer species (Abernethy 1994; Goodman et al. 1999), rodents (Ferris et al. 1983; Tegelström 1987), shrews (Wyttenbach et al. 1999; Andersson et al. 2004) and canids (Wayne and Jenks 1991; Vilà et al. 2003).

The possibility that wild sympatric populations of Lepus timidus (mountain hare) and L. europaeus (brown hare) hybridise has been argued since long (Lönnberg 1905; Fraguglione 1959; Schröder et al. 1987) and hybrids between the species are easily produced in captivity (Gustavsson and Sundt 1965). Attempts to verify the status of suspected wild hybrids have been unsatisfying because of the phenotypic plasticity within the genus Lepus (Lönnberg 1905; Flux and Angerman 1990). Similarly, genetic similarities create difficulties in the search for species diagnostic genetic markers in the nuclear genome (Andersson et al. 1999; Thulin et al. 2006). Nevertheless, the occurrence of subsequent introgression resulting from hybridisation among wild populations has been confirmed using maternally inherited mitochondrial DNA (mtDNA) markers. In Scandinavia, mtDNA lineages of L. timidus origin were detected among L. europaeus in Sweden (Thulin et al. 1997; Thulin and Tegelström 2002). Similarily, introgression of L. timidus mtDNA has also been confirmed among L. europaeus on the Iberian Peninsula (Alves et al. 2003; Melo-Ferreira et al. 2005). Because the current L. timidus distribution does not cover the Iberian Peninsula, the observed introgression must reflect ancestral hybridisation and subsequent preservation of the transferred mtDNA among the currently allopatric L. europaeus populations (Melo-Ferreira et al. 2005). In contrast, transferred mtDNA seems to disappear among L. europaeus in allopatry in southern Sweden (Thulin and Tegelström 2002) and there are no indications of mtDNA introgression in central Europe (Hartl et al. 1993; Fickel et al. 2005).

The occurrence of hybridisation between L. europaeus and L. timidus in Sweden was reported right after the first introductions of L. europaeus in the late 19th century (Lönnberg 1905, 1908). Currently, the range of L. timidus is retreating northwards, possibly due to interspecific competition with L. europaeus (Lind 1963; Wolfe et al. 1996; Thulin 2003). L. europaeus however, seems to expand further northwards in the northern hemisphere (Folitarek 1940; Thenius 1980; Jansson et al. 2003) with a gradual adaptation of pelage coloration to the boreal climate in Russia (Gureev 1964). Apart from Sweden, no investigations of genetic variation, hybridisation and introgression between L. europaeus and L. timidus have been made in the north European sympatric populations of these species. In Russia, the species are believed to hybridise as well, and supposedly L. europaeus gradually replaces L. timidus in some areas by competitive exclusion (Folitarek 1940). In comparison to the semi-natural contact zone between the species in Sweden, the contact zone in Russia is the result of a natural, albeit recent, expansion of L. europaeus (Thenius 1980).

Single nucleotide polymorphisms (SNPs) have relatively recently been applied as genetic markers in population studies (Brumfield et al. 2003; Morin et al. 2004). The utility of SNPs may reduce costs and efforts in many aspects of molecular ecology (Morin et al. 2004). Screening for SNPs typically involves detection of polymorphic nucleotide positions in a known sequence followed by routine screenings for this particular polymorphism. Thus, to find usable SNPs, it is necessary to possess polymorphic DNA sequence data and a reference population to test the degree of polymorphism and frequency of different alleles or haplotypes. At most, each position may provide four different polymorphisms (four DNA nucleotides). The deterministic appearance of SNPs at certain nucleotide positions is particularly useful for allele identification in the nuclear genome or haplotype identification in mtDNA.

Here, we develop SNPs from diagnostic PCR-RFLPs (restriction fragment length polymorphisms) for L. timidus and L. europaeus mitochondrial DNA, respectively. The accuracy of these SNPs are tested in a previously investigated sample of Scandinavian hares. Then we determine mtDNA haplotypes among L. europaeus and L. timidus from Russia to look for mtDNA introgression. In addition, we use a panel of seven microsatellite markers to test the species identity of samples. The results are discussed in relation to previous investigations of hybridisation and introgression within genus Lepus.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Samples

Tissue samples (muscle) from 25 L. europaeus, 45 L. timidus and one putative L. europaeus×L. timidus hybrid (total of 71 Lepus specimens), were collected in Russia from Karelia in the west to Baikal in south Siberia in the east (Fig. 1). The hunter who bagged the hare determined the species identity of each sample. For the development of the mtDNA SNP analysis, a reference panel including 10 L. europaeus and 15 L. timidus from a previous investigation in Scandinavia (Thulin and Tegelström 2002) was used to verify that the nucleotide polymorphism is species diagnostic. Samples were transported in 95% ethanol and stored in freezers upon arrival. Whole genomic DNA was extracted using a standard salt (NaCl) isolation protocol with proteinase treatment and ethanol precipitation (Miller et al. 1988)

image

Figure 1. Map of sampling localities, where each dot represents one specific locality in the region. Numbers are total amount of hares sampled for each region. The 25 reference samples from Sweden are represented by one large dot. Sample localities in Buryatia in the Baikal region (5 samples), south Siberia, are not shown. The approximate location of Moscow is included to facilitate orientation.

Download figure to PowerPoint

SNP development and MtDNA scoring

Two previously documented diagnostic restriction enzyme sites in the mtDNA cytochrome b gene were tested for SNPs (for details on the restrictions sites see Thulin and Tegelström 2002). The primers “U67” (5′-C TAC ACA TCA GAC ACA GC-3′) and “L207” (5′-A TGA GCC GTA GTA GAT TC-3′) were designed based on the published sequence from one L. europaeus specimen (EMBL accession number AJ250143) and one L. timidus specimen (EMBL accession number AJ250144). The primers amplify a 158 base pair (bp) region including the targeted restriction sites (Fig. 2). Primer U67 was 5′-biotinylated to allow capture of the PCR products onto streptavidin-coated beads (Syvanen et al. 1990, 1993). The PCR was optimized for 12.5 μl reactions composed of 1 μl of template (5–100 ng μl−1), 1×PCR buffer (Mg2+ free), 3 mM MgCl2, 0.8 mM dNTP, 0.4 mM of each primer, 800 mM Betain (Sigma) and 1 unit of polymerase (AmpliTaq GoldTM, Applied Biosystems). The PCR cycle started with 12 min at 94°C, then 30 s at 94°C, 30 s at 53°C and 30 s at 72°C, repeated 35 times and a final elongation for 7 min at 72°C.

image

Figure 2. Sequence motif with the inner and outer primers and the diagnostic single nucleotide polymorphisms. The 307 base pair sequence is from one L. europaeus specimen (upper sequence, EMBL accession number AJ250143) and one L. timidus specimen (lower sequence, EMBL accession number AJ250144) previously published (Thulin and Tegelström 2002). The asterisk (*) indicate that the actual primer sequence is the reversed complementary of the shown sequence. Upper outer primer (U67) is tagged with a biotin molecule to facilitate the PyrosequencingTM.

Download figure to PowerPoint

For the SNP analysis, we used the specifically designed minisequencing method (Syvanen et al. 1990, 1993) and the PyrosequencingTM technique (Ronaghi et al. 1996, 1998). Specific detection primers “Pyro1” (5′-CA AAT ATG TGT RAC TGA – 3′) and “Pyro2” (5′-TGC TCC ATT RGC GTG – 3′) were designed to read the sequence over the diagnostic SNPs toward the biotinylated upper primer U67 (Fig. 2). Because of polymorphism within the targeted DNA sequences, both PyrosequencingTM primers were degenerate at one position each (R=A or G). The pyrosequencing reactions were performed on a PSQ HS 96QTM apparatus and analysed with the PSQTM HS 96A software (Biotage).

Microsatellite scoring

Seven previously reported microsatellite loci (Sol8 (Rico et al. 1994); Sat5, Sat12 and Sat13 (Mougel et al. 1997) and Lsa1, Lsa2 and Lsa6 (Kryger et al. 2002)) were used to score species origin with regard to nuclear markers in our Lepus sample. The upper primers for each locus were labelled with the fluorescent dye FAM (Lsa2, Lsa6, Sat5, Sol8), HEX (Sat12) and TET (Lsa1, Sat13). The PCR was optimized for 10 μl reactions composed of 1 μl of template (5–100 ng μl−1), 1×PCR buffer (Mg2+ free), 1.5 or 2.5 mM MgCl2, 0.2 mM dNTP, 0.3 mM of each primer and 0.5 unit of polymerase. Two touchdown PCR cycles with different annealing temperature (Table 1) started with denaturation step at 94°C for 5 min, then followed by 20 cycles with 30 s denaturation at 94°C, 30 s annealing at 65/60-55/50°C (lowered 0.5° cycle−1) and 30 s elongation at 72°C. The last cycle with annealing at 55/50°C was repeated 15 times, and the whole cycle was ended with a 7 min elongation step at 72°C. The PCR products were denatured 2 min before electrophoresis in 4% polyacrylamide gels using a MegaBase™ capillary instrument and subsequently scored with the software Genetic Analyzer and analysed with the software Genetic Profiler (Amersham Biosciences).

Table 1.  The seven microsatellite loci investigated, with their respective PCR annealing temperature (Ta) in °C, number of alleles detected (NA), expected (HE) and observed heterozygosity (HO) and references.
LocusTaNAHEHOreference
      
Lsa165–55 50.7140.746Kryger et al. 2002
Lsa265–55160.9090.552Kryger et al. 2002
Lsa665–55 20.0150.015Kryger et al. 2002
Sat565–55150.6050.431Mougel et al. 1997
Sat1260–50 90.8250.562Mougel et al. 1997
Sat1360–50 50.6960.531Mougel et al. 1997
Sol865–55100.7710.623Rico et al. 1994

Data analysis

To determine which mtDNA haplotype each specimen carried (i.e. L. europaeus or L. timidus type), all specimens were scored for SNPs at the previously documented restriction enzyme cutting sites. The reference specimens from Scandinavia were scored first to assure the accuracy of the SNPs. Apart from the previously known restriction sites, one additional diagnostic polymorphism was detected within the Pyro1 sequencing frame and two within the Pyro2 frame (Fig. 2). Thus, after screening, each specimen was given a two base pair code for the Pyro1 SNPs and a three base pair code for Pyro2 that were summarized as a five letter code (Table 2). After the accuracy was tested, we scored the 71 samples from Russia.

Table 2.  The single nucleotide polymorphisms detected in mtDNA cytochrome b region from Russian L. timidus and L. europaeus using primers Pyro1 and Pyro2. The “tag” is the consensus sequence.
MtDNA ancestrypyro1pyro2tag
L. timidusAACGAAACGA
L. timidus (new)AATGAAATGA
L. europaeus (new)GGTAAGGTAA
L. europaeusGGTAGGGTAG

For microsatellite markers, basic data like number of alleles, allele frequencies, exact tests of Hardy Weinberg equilibrium and linkage disequilibrium was calculated in Genepop on the Web (http://wbiomed.curtin.edu.au/genepop/), versions 3.1c-3.4 (Raymond and Rousset 1995). To subdivide the Russian specimens into two groups according to species, we used the model-based program Structure 2.1 (Pritchard et al. 2000). The settings for the subdivision was a burn-in period of 10 000 and 10 000 repetitions, assuming k=2 (i.e. two “populations”), using the admixture model and correlated allele frequencies. Each specimen was assigned to one of two groups if the probability of belonging this groups was >0.9. For probabilities >0.5, the specimen was considered “most likely” belonging to that particular group.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Single nucleotide polymorphisms

The reference specimens showed that all SNPs were diagnostic for species-specific mtDNA from Scandinavian L. timidus and L. europaeus, respectively. For Pyro1, L. timidus was AA and L. europaeus GG, and for Pyro2, L. timidus was CGA and L. europaeus TAG, resulting in the five-letter tag AACGA for L. timidus and GGTAG for L. europaeus (Table 2). Among the Russian sample, two novel polymorphisms were detected at the Pyro2 segment, which resulted in a “timidus like” code TGA, differing at position one (C/T) from the diagnostic L. timidus code CGA. The other polymorphism was a “europaeus like” code TAA, differing at the last position (G/A) from the diagnostic L. europaeus TAG code. The resulting five letter tags for these additional polymorphisms are AATGA and GGTAA (Table 2).

Genetic analysis

Within the Russian sample, mtDNA haplotype scored with Pyro1 SNPs revealed 45 specimens with L. timidus code (AA) and 26 with L. europaeus code (GG). Thus, 44 of the 45 L. timidus specimens along with the putative hybrid had the L. timidus code (AA), while one L. timidus and all of the L. europaeus specimens had the L. europaeus code (GG). As indicated above, Pyro2 revealed two new polymorphisms. A total of 41 specimens had the expected L. timidus code (CGA), of which 40 were L. timidus and one was the putative hybrid. Four L. timidus specimens had the “timidus-like” code TGA (Table 2). Surprisingly, only three L. europaeus specimens had the L. europaeus type (TAG), while a total of 22 L. europaeus specimens and one L. timidus had the “europaeus like” code TAA. In comparison, none of the Swedish reference specimens carried this mtDNA haplotype that dominated among Russian L. europaeus.

Significant linkage disequilibrium was observed for Lsa2 and Sol8 among the Russian specimens (investigations of Scandinavian hares do however not indicate that these loci are linked; G. Jansson, Å. Pehrson and C.-G. Thulin, unpubl.). Homozygous excess was observed within the Russian sample for the loci Lsa2, Sat12, Sat5 and Sol8. A potential explanation is a Wahlund effect (Wahlund 1928), because the samples were collected over a large geographical area (Fig. 1). Thus, our samples most likely represent several independent populations and therefore violate the expectations from Hardy-Weinberg distribution of genetic variation (Hardy 1908, Weinberg 1908). The species origin assignment of each Russian Lepus specimen as inferred from the nuclear microsatellite data resulted in the following sorting:

  • 40 L. timidus assigned to the “timidus” group

  • 19 L. europaeus assigned to the “europaeus” group

  • 5 L. timidus assigned intermediate, but mostly to the “timidus” group

  • 4 L. europaeus assigned intermediate, but mostly to the “europaeus” group

  • 2 L. europaeus assigned intermediate, but mostly to the “timidus” group

  • 1 putative hybrid assigned to the “timidus” group

When summarising genetic data from mtDNA and microsatellites and phenotypic information as documented by hunters, we find that our sample consists of 39 L. timidus specimens and 19 L. europaeus with species-specific genotypic and phenotypic characteristics. The remaining 13 specimens with deviating genotype and/or phenotype (six L. timidus, six L. europaeus and one putative hybrid) are presented individually in Table 3.

Table 3.  The 13 specimens that deviate from expectations from their inferred species status in the resulting assignment from microsatellite data using the software Structure 2.1 (Pritchard et al. 2000), with a burn-in period of 10 000, 10 000 repetitions, k=2 assumed, using the admixture model and correlated allele frequencies. Assignment probabilities higher than 0.9 were considered as conclusive assignments (e.g. “timidus”), while lower probabilities were considered intermediate but most likely belonging to the group with the higher assignment probability (e.g. “timidus-like”, P>0.5). Sample region refers to the area in Russia where the sample was collected. The final column present our consensus species status. Three specimens are L. timidus with L. europaeus mtDNA (marked by asterisk*) or hybrids between the species.
Inferred species (ID)sample regionMicrosat. assignmentsMtDNAConsensus
  tim.-groupeur.-groupinferred ancestrySNP taginferred ancestryspecies status
L. timidus (R4)Baikal (South Siberia)0.8940.106timdius-likeAATGAtimidusL. timidus
L. timidus (R5)St Petersburg (West Russia)0.8820.118timidus-likeAACGAtimidusL. timidus
L. europaeus (R15)Chelyabinsk (South Ural)0.5480.452timidus-likeGGTAAeuropaeus (new)L. timidus*/hybrid?
L. europaeus (R16)Chelyabinsk (South Ural)0.7510.249timidus-likeGGTAAeuropaeus (new)L. timidus*/hybrid?
L. timidus (R19)St Petersburg (West Russia)0.6210.379timidus-likeAACGAtimidusL. timidus
L. timidus (R24)Baikal (South Siberia)0.8330.167timidus-likeAATGAtimidus (new)L. timidus
L. timidus (R37)Chelyabinsk (South Ural)0.9830.017timdiusGGTAAeuropaeus (new)L. timidus*
L. timidus (R39)Chelyabinsk (South Ural)0.8570.143timidus-likeAACGAtimidusL. timidus
L. europaeus (R41)St Petersburg (West Russia)0.1230.877europaeus-likeGGTAAeuropaeus (new)L. europaeus
L. europaeus (R52)Chelyabinsk(South Ural)0.2140.786europaeus-likeGGTAAeuropaeus (new)L. europaeus
Putative hybrid (R55)Chelyabinsk (South Ural)0.9900.010timdiusAACGAtimidusL. timidus
L. europaeus (R57)Tver (near Moscow)0.2090.791europaeus-likeGGTAGeuropaeusL. europaeus
L. europaeus (R63)Tver (near Moscow)0.1250.875europaeus-likeGGTAAeuropaeus (new)L. europaeus

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Advancements and refinements of molecular techniques constantly provide novel methods for rapid and reliable genotyping of terrestrial animal populations. The modern techniques also enable non-invasive monitoring of endangered species with numerous applications in conservation biology (Kohn and Wayne 1997; Taberlet et al. 1999) as well as in management (Solberg et al. 2006). There are, however, serious limitations with non-invasive methods, most of them related to the low quality of the template DNA, that may increase the susceptibility for contamination and cause erroneous genotyping (Taberlet et al. 1999; Maudet et al. 2004). Screening of SNPs in natural populations provide yet another possibility to produce data from low quality template DNA. Here we show that development of a SNP assay is plausible for detecting mtDNA origin in natural Lepus populations, possibly also from faecal pellets (Maudet et al. 2004). With a minimalistic approach, the method could enable detection of SNPs in fragments down to 61 bp long if one biotinylated and two reversed primers of 20 bp length are used in addition to the target nucleotide position. In addition to the smaller intact fragment required than in microsatellite screening, the descriptive status of each polymorphic nucleotide position may increase the accuracy of the genotyping. Thus, we believe that SNPs may replace microsatellite screening for many aspects of conservation biology and non-invasive applications in particular.

The genus Lepus has proven a challenge for investigations of dynamics of mtDNA within natural populations. In Sweden, mtDNA lineages of L. timidus origin have been detected among 10% (51) of 522 investigated L. europaeus (Thulin and Tegelström 2002). In addition, recent investigations on the Iberian Peninsula indicate that as much as 93% (75 of 81) of the L. europaeus carry L. timidus mtDNA (Melo-Ferreira et al. 2005). Here we combine mtDNA data with analyses of seven microsatellite loci to study potential introgression among L. europaeus and L. timidus from the wide contact zone between the species in Russia. Our analyses show that 39 L. timidus specimens and 19 L. europaeus did not deviate from what we expected from the phenotype (as documented by hunters). Thus, we consider them to be true representatives of their respective species. The remaining 13 specimens had deviating nuclear and/or mitochondrial genotypes: six L. timidus, six L. europaeus and one putative hybrid (Table 3). It is our interpretation that five of the L. timidus specimens and four of the L. europaeus also are pure representatives of their species, albeit their intermediate nuclear genotype may indicate recent introgression of nuclear genes (Andersson et al. 1999; Thulin et al. 2006). The putative hybrid is most likely a L. timidus specimen with an unusual pelage/morphology: Investigations of hare hybridisation in Scandinavia showed that the morphological plasticity within the species often confuses the species identification (Thulin et al. 2006). One of the L. timidus specimens (sample ID “R37”) assigned to the L. timidus group according to nuclear microsatellite markers but had L. europaeus type mtDNA (tag GGTAA, Table 2). This specimen clearly indicates the occurrence of mtDNA introgression from L. europaeus to L. timidus in Russia, as opposed to the previously documented reciprocal, mtDNA introgression among Scandinavian and Iberian hares (Thulin et al. 1997; Thulin and Tegelström 2002; Alves et al. 2003; Melo-Ferreira et al. 2005). In addition, microsatellite markers also revealed that two L. europaeus specimens (sample ID “R15” and “R16”) had their inferred ancestry to L.timidus group of with respect to their nuclear genotypes (Table 3). Thus, it seems that within our sample we may have as many as three specimens with L. europaeus mitochondrial genome and L. timidus nuclear genome.

Previously, the detection of one specimen in Sweden is the only support to date for introgression of L. europaeus mtDNA into L. timidus (Thulin et al. 2006). Interestingly, this specimen was collected on the northernmost margin of the present L. europaeus distribution in Sweden, where L. europaeus density is expected to be low in relation to local L. timidus populations because of the recent colonisation (Jansson et al. 2003). This result indicates that hybridisation between L. europaeus and L. timidus might be frequency-dependent, where males from a population with high density hybridise with females from a population with low density (Wirtz 1999). Supposedly, investigations that focus on populations in areas recently colonised by L. europaeus (e.g. the last five to ten years) are needed to verify potential bi-directional gene flow between L. europaeus and L. timidus (Thulin et al. 2006). L. europaeus has colonized west and central Russia gradually since early 19th century (Thenius 1980), so this vast area is not in all aspects a recent contact zone. Nevertheless, the criteria for frequency dependent hybridisation may still withhold. L. europaeus has colonised the region naturally, and gradually, which also implies the requirement of a gradual adaptation to local conditions. Potentially, the L. europaeus populations in Russia have a lower density than sympatric L. timidus and, in addition, their numbers may vary considerably over time. Thus, in the margin of L. europaeus natural distribution, where they are in minority, interspecific hybridisation between L. timidus males and L. europaeus females is perhaps more common.

In general, the specific status of species within the genus Lepus is debated (Flux and Angerman 1990). Molecular tools may shed some light, but since there is interbreeding between as highly differentiated species as L. europaeus and L. timidus, introgression may confuse any potential conclusions. For example, mtDNA from two half-sibling L. europaeus in Sweden can differ with 10% sequence divergence (Thulin et al. 1997). In Iberia, 695 specimens belonging to three different species (L. europaeus, L. granatensis and L. castroviejoi) were investigated with respect to their mtDNA haplotypes (Melo-Ferreira et al. 2005). A total of 269 specimens (39%) from all three species carry L. timidus mtDNA, although postglacial occurrence of L. timidus in the area has not been confirmed. Thus, L. timidus mtDNA has been conserved in related species, potentially because of a selective advantage as compared to other mtDNA lineages (Melo-Ferreira et al. 2005). Our observations of reciprocal introgression from L. europaeus to L. timidus in Russia differ from the previous observations of mtDNA introgression in Sweden and Iberia, and definitely add to the overall complexity that seems to be characteristic for the genus Lepus. The reticulated mode of evolution, the repeated hybridisation and introgression in contact zones, competitive exclusion and morphological plasticity certainly provide challenges for evolutionary biologists as well as conservationists.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are grateful to Russian hunters for providing samples. This work was supported by research grants to C.-G. Thulin from Carl Tryggers Foundation, Swedish Hunters Association and Nilsson-Ehle Foundation.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Abernethy K., (1994). The establishment of a hybrid zone between red and sika deer (genus Cervus). Mol. Ecol. 3: 551562.
  • Alves P. C., Ferrand N., Suchentrunk F. et al., (2003). Ancient introgression of Lepus timidus mtDNA into L. granatensis and L. europaeus in the Iberian Peninsula. Mol. Phylogenet. Evol. 27: 7080.
  • Anderson E. and Stebbins G. L., (1954). Hybridization as an evolutionary stimulus. Evolution 8: 378388.
  • Andersson A.-C., Thulin C.-G. and Tegelström H., (1999). Applicability of rabbit microsatellite primers for studies of hybridisation between an introduced and a native hare species. Hereditas 130: 309315.
  • Andersson A.-C., Narain Y., Tegelström H. et al., (2004). No apparent reduction of gene flow in a hybrid zone between the West and North European karyotypic groups of the common shrew, Sorex araneus. Mol. Ecol. 13: 12051215.
  • Arnold, M. L. 1997. Natural hybridization and evolution. – Oxford Univ. Press.
  • Brumfield R. T., Beerli P., Nickerson D. A. et al., (2003). The utility of single nucleotide polymorphisms in inferences of population history. Trends Ecol. Evol. 18: 249256.
  • Ebenhard T., (1988). Introduced birds and mammals and their ecological effects. Swed. Wildlife Res. (Viltrevy) 4: 5107.
  • Ferris S. D., Sage R. D., Huang C.-M. et al., (1983). Flow of mitochondrial DNA across a species boundary. Proc. Natl Acad. Sci. USA 80: 22902294.
  • Fickel J., Schmidt A., Putze M. et al., (2005). Genetic structure of populations of European brown hare: Implications for management. J. Wildlife Manage. 69: 770780.
  • Flux, J. E. C. and Angerman, R. 1990. The hares and jackrabbits. – In: Chapman, J. A. and Flux, J. E. C. (eds), Rabbits, hares and pikas: status survey and conservation action plan. IUCN, p. 61–94.
  • Folitarek S. S., (1940). Geographic distribution of the European hare Lepus europaeus Pall. in the USSR. Proc. Severetsov's Inst. Evol. Morphol., Acad. Sci. USSR 1: 7997.
  • Fraguglione, D. 1959. Les anomalies du pelage chez les lièvres commun et variable. – Diana 4: 57–59. (in French)
  • Goodman S. J., Barton N. H., Swanson G. et al., (1999). Introgression through rare hybridization: a genetic study of a hybrid zone between red and sika deer (Genus Cervus) in Argyll, Scotland. Genetics 152: 355371.
  • Gureev, A. A. 1964. Fauna USSR. III. – Lagomorpha. Izvestii Akademii Nauk SSSR. (in Russian)
  • Gustavsson, I. and Sundt, C. O. 1965. Anwendung von künstlicher Befruchtung bei der Hybridisierung von zwei Hasenarten. – Z. Jagdwiss. 11: 155–158. (in German)
  • Hardy G. H., (1908). Mendelian proportions in a mixed population. Science 28: 4150.
  • Hartl G. B., Suchentrunk F., Nadlinger K. et al., (1993). An integrative analysis of genetic differentiation in the brown hare Lepus europaeus based on morphology, allozymes and mitochondrial DNA. Acta Theriol. 38: 3357.
  • Jansson, G., Pehrsson, Å. and Helldin, J.-O. 2003. Fäthararna vinner terräng-med hybrider i släptåg. – Svensk Jakt 2/3: 52–55. (in Swedish)
  • Kohn M. H. and Wayne. R. K., (1997). Facts from feces revisited. Trends Ecol. Evol. 12: 223227.
  • Kryger U., Robinson T. J. and Bloomer P., (2002). Isolation and characterization of six polymorphic microsatellite loci in South African hares (Lepus saxatilis F. Cuvier, 1823 and Lepus capensis Linnaeus, 1758). Mol. Ecol. Notes 2: 422424.
  • Lind E. A., (1963). Observations on the mutual relationship between the snow hare (Lepus timidus) and the field hare (L. europaeus). Suomen Riista 16: 128135.
  • Lönnberg E., (1905). On hybrids between Lepus timidus L. and Lepus europeus Pall. from southern Sweden. Proc. Zool. Soc. Lond. 1: 278287.
  • Lönnberg, E. 1908. Några villebrådsarters nutida utbredning i Skåne. – Svenska Jägareförbundets Tidskrift 46: 7–16. (in Swedish)
  • Maudet C., Luikart G., Dubray D. et al., (2004). Low genotyping error rates in wild ungulate faeces sampled in winter. Mol. Ecol. Notes 4: 77775.
  • Melo-Ferreira J., Boursot P., Suchentrunk F. et al., (2005). Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia. Mol. Ecol. 14: 24592464.
  • Miller S. A., Dykes D. D. and Polesky H. F., (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1215.
  • Morin P. A., Luikart G., Wayne R. K. et al., (2004). SNPs in ecology, evolution and conservation. Trends Ecol. Evol. 19: 208216.
  • Mougel F., Mounolou J.-C. and Monnerot M., (1997). Nine polymorphic microsatellite loci in the rabbit Oryctolagus cuniculus. Anim. Genet. 28: 5871.
  • Pritchard J. K., Stephens M. and Donelly P., (2000). Inference of population structure using mulitlocus genotype data. Genetics 155: 945959.
  • Raymond M. and Rousset F., (1995). GENEPOP (version 1.2), population genetics software for exact tests and ecumenicism. J. Hered. 86: 248249.
  • Rhymer J. M. and Simberloff D., (1996). Extinction by hybridisation and introgression. Annu. Rev. Ecol. Syst. 27: 83109.
  • Rico C., Rico I., Webb N. et al., (1994). Four polymorphic microsatellite loci for the European wild rabbit Oryctolagus cuniculus. Anim. Genet. 25: 367.
  • Ronaghi M., Karamohamed S., Pettersson B. et al., (1996). Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242: 8489.
  • Ronaghi M., Uhlen M. and Nyren P., (1998). A sequencing method based on real-time pyrophosphate. Science 281: 363365.
  • Schröder J., Soveri T., Suomalainen H. A. et al., (1987). Hybrids between Lepus timidus and Lepus europeus are rare although fertile. Hereditas 107: 185189.
  • Solberg K. H., Bellemain E., Drageset O.-M. et al., (2006). An evaluation of field and non-invasive genetic methods to estimate brown bear (Ursus arctos) population size. Biol. Conserv. 128: 158168.
  • Syvanen A. C., Aalto-Setala K., Harju L. et al., (1990). A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8: 684692.
  • Syvanen A. C., Sajantila A. and Lukka M., (1993). Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing. Am. J. Human Genet. 52: 4659.
  • Taberlet P., Waits L. P. and Luikart G., (1999). Noninvasive genetic sampling: look before you leap. Trends Ecol. Evol. 14: 323327.
  • Tegelström H., (1987). Transfer of mitchondrial DNA from the northern red-backed vole (Clethrionomys rutilus) to the bank vole (C. glareolus). J. Mol. Evol. 24: 218227.
  • Thenius, E. 1980. Grundzüge der Faunen- und Verbreitungsgesichte der Säugetiere. – Gustav Fisher Verlag. (in German)
  • Thulin C.-G., (2003). The distribution of mountain hares (Lepus timidus, L. 1758) in Europe: a challenge from brown hares (L. europaeus, Pall. 1778)?. Mammal Rev. 33: 2942.
  • Thulin C.-G., Jaarola M. and Tegelström H., (1997). The occurrence of mountain hare mitochondrial DNA in wild brown hares. Mol. Ecol. 6: 463467.
  • Thulin C.-G. and Tegelström H., (2002). Biased geographical distribution of mitochondrial DNA that passed the species barrier from mountain hares to brown hares (genus Lepus), an effect of genetic incompatibility and mating behaviour?. J. Zool. 258: 299306.
  • Thulin C.-G., Stone J., Tegelström H. et al., (2006). Species assignment and hybrid identification among Scandinavian hares. Wildlife Biol. 12: 2938.
  • Wahlund, S. 1928. Zusammensetzung von Populationen und Korrelationserscheinungen vom Standpunkt der Vererbungslehre aus betrachtet.-Hereditas 11: 65–106. (in German)
  • Wayne R. K. and Jenks S. M., (1991). Mitochondrial DNA analysis implying extensive hybridization of the endangered red wolf, Canis rufus. Nature 351: 565568.
  • Weinberg, W. 1908. On the demonstration of heredity in man. (translated by SH Boyer 1963 in Papers on Human Genetics. Prentice-Hall, Englewood Cliffs, NJ).
  • Vilà C., Walker C., Sundqvist A.-K. et al., (2003). Combined use of maternal and bi-parental genetic markers for identification of wolf-dog hybrids. Heredity 90: 1724.
  • Wirtz P., (1999). Mother species-father species: unidirectional hybridization in animals with female choice. Anim. Behav. 58: 112.
  • Wolfe A., Whelan J. and Hayden T. J., (1996). The diet of the mountain hare (Lepus timidus hibernicus) on coastal grassland. J. Zool. Soc. London 240: 804810.
  • Wyttenbach A., Narain Y. and Fredga K., (1999). Genetic structuring and gene flow in a hybrid zone between two chromosome races of the common shrew (Sorex araneus, Insectivora) revealed by microsatellites. Heredity 82: 7988.