Introgression from Lepus europaeus to L ... - Wiley Online Library

Hereditas 143: 68 76 (2006)

Introgression from Lepus europaeus to L. timidus in Russia revealed by mitochondrial single nucleotide polymorphisms and nuclear microsatellites CARL-GUSTAF THULIN1,2, MEIYING FANG3 and ALEXANDER O. AVERIANOV4 1

Population Biology and Conservation Biology, Department of Ecology and Evolution, EBC, Uppsala University, Sweden 2 Department of Animal Ecology, Swedish University of Agricultural Sciences, Umea˚, Sweden 3 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Sweden 4 Zoological Institute, Russian Academy of Sciences, St Petersburg, Russia Thulin, C.-G., Fang, M. and Averianov, A. O. 2006. Introgression from Lepus europaeus to L. timidus in Russia revealed by mitochondrial single nucleotide polymorphisms and nuclear microsatellites. * Hereditas 143: 68 76. Lund, Sweden. eISSN 1601-5223. Received March 31, 2006. Accepted April 5, 2006 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. Carl-Gustaf Thulin, Population Biology and Conservation Biology, Department of Ecology and Evolution, EBC, Uppsala University, Norbyva ¨ gen 18 D, SE-752 36 Uppsala, Sweden. E-mail: [email protected]

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; TEGELSTRO¨M 1987), shrews (WYTTENBACH et al. 1999; ANDERSSON et al. 2004) and canids (WAYNE and JENKS 1991; VILA` 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 (LO¨NNBERG 1905; FRAGUGLIONE 1959; SCHRO¨DER 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 pheno-

typic plasticity within the genus Lepus (LO¨NNBERG 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 TEGELSTRO¨M 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 TEGELSTRO¨M 2002) and there are no indications

Introgression in Russian Lepus

Hereditas 143 (2006)

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 (LO¨NNBERG 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 PCRRFLPs (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

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previous investigations of hybridisation and introgression within genus Lepus. MATERIAL AND METHODS 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 TEGELSTRO¨M 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) 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 TEGELSTRO¨M 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 ml reactions composed of 1 ml of template (5 100 ng ml 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 948C, then 30 s at 948C, 30 s at 538C and 30 s at 728C, repeated 35 times and a final elongation for 7 min at 728C. 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

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RUSSIA

SWEDEN

Karelia region (4) Chelyabinsk region (33)

St Petersburg region (22) Tver region (7)

Moscow

Fig. 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.

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 ml reactions composed of 1 ml of template (5 100 ng ml 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 948C for 5 min, then followed by 20 cycles with 30 s denaturation at 948C, 30 s annealing at 65/60-55/508C (lowered 0.58 cycle1) and 30 s elongation at 728C. The last cycle with annealing at 55/508C was repeated 15 times, and the whole cycle was ended with a 7 min elongation step U67 (biotinylated)

Le 5´ C TTC GGC TCT CTA TTA GGA TTA TGC CTA ATA ATC CAG ATC CTA ACT GGC TTA TTT CTA GCC ATA CAC TAC ACA TCA GAC ACA GCT ACA GCA TTC TCC TCA Lt 5´ . ..T ..T ..C ... ... ... C.. ... ... ... ... ..A ... ... ... ... C.. ..C ... ..T ... ... ... ... ... ... ... ..A ... ... ..T ..T ...

Pyro1*

Pyro2*

GTT ACA CAT ATT TGC CGA GAT GTA AAC TAC GGC TGA CTC ATT CGT TAC TTA CAC GCT AAT GGA GCA TCA ATA TTC TTT ATT TGC TTA TAT ATA CAT GTA GGC CGT ..C ... ... ... ... ... ..C ... ... ..T ... ... ..T ... ... ... C.G ... ..C ... ... ... ... ... ..T ... ..C ... ... ... ... ... ... ..T ...

U207* GGA ATC TAC TAC GGC TCA TAT ACT TAC CTA GAA ACC TGA AAC ATT GGC ATT ATT CTA CTA TTC GCA GTA ATG GCT ACA GCA TTC ATA GGC TAC GTC CTC CCA 3´ ... ... ... ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... ... ..T ... ... ..A ..C ... ... ..T ... ... ..T ..T ... ... 3´

Fig. 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 Tegelstro¨m 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.



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Table 1. The seven microsatellite loci investigated, with their respective PCR annealing temperature (Ta) in 8C, number of alleles detected (NA), expected (HE) and observed heterozygosity (HO) and references. Locus

Ta

NA

HE

HO

Lsa1 Lsa2 Lsa6 Sat5 Sat12 Sat13 Sol8

65 55 65 55 65 55 65 55 60 50 60 50 65 55

5 16 2 15 9 5 10

0.714 0.909 0.015 0.605 0.825 0.696 0.771

0.746 0.552 0.015 0.431 0.562 0.531 0.623

at 728C. The PCR products were denatured 2 min before electrophoresis in 4% polyacrylamide gels using a MegaBaseTM capillary instrument and subsequently scored with the software Genetic Analyzer and analysed with the software Genetic Profiler (Amersham Biosciences). 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. 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. 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 ancestry

pyro1

pyro2

tag

L. L. L. L.

AA AA GG GG

CGA TGA TAA TAG

AACGA AATGA GGTAA GGTAG

timidus timidus (new) europaeus (new) europaeus

reference KRYGER et al. 2002 KRYGER et al. 2002 KRYGER et al. 2002 MOUGEL et al. 1997 MOUGEL et al. 1997 MOUGEL et al. 1997 RICO et al. 1994

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 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 speci-

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mens 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, A 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.

DISCUSSION 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 TEGELSTRO¨M 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


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)

Baikal (South Siberia) St Petersburg (West Russia) Chelyabinsk (South Ural) Chelyabinsk (South Ural) St Petersburg (West Russia) Baikal (South Siberia) Chelyabinsk (South Ural) Chelyabinsk (South Ural) St Petersburg (West Russia) Chelyabinsk(South Ural) Chelyabinsk (South Ural) Tver (near Moscow) Tver (near Moscow)

Microsat. assignments

MtDNA

tim.-group

eur.-group

inferred ancestry

SNP tag

inferred ancestry

0.894 0.882 0.548 0.751 0.621 0.833 0.983 0.857 0.123 0.214 0.990 0.209 0.125

0.106 0.118 0.452 0.249 0.379 0.167 0.017 0.143 0.877 0.786 0.010 0.791 0.875

timdius-like timidus-like timidus-like timidus-like timidus-like timidus-like timdius timidus-like europaeus-like europaeus-like timdius europaeus-like europaeus-like

AATGA AACGA GGTAA GGTAA AACGA AATGA GGTAA AACGA GGTAA GGTAA AACGA GGTAG GGTAA

timidus timidus europaeus (new) europaeus (new) timidus timidus (new) europaeus (new) timidus europaeus (new) europaeus (new) timidus europaeus europaeus (new)

Consensus species status L. L. L. L. L. L. L. L. L. L. L. L. L.

timidus timidus timidus */hybrid? timidus */hybrid? timidus timidus timidus * timidus europaeus europaeus timidus europaeus europaeus


L. timidus (R4) L. timidus (R5) L. europaeus (R15) L. europaeus (R16) L. timidus (R19) L. timidus (R24) L. timidus (R37) L. timidus (R39) L. europaeus (R41) L. europaeus (R52) Putative hybrid (R55) L. europaeus (R57) L. europaeus (R63)

sample region

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‘‘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 TEGELSTRO¨M 2002; ALVES et al. 2003; MELOFERREIRA 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 bidirectional 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 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.

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