Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Widespread introgression of mountain hare genes into Fennoscandian brown hare populations

  • Riikka Levänen,

    Roles Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland

    ⨯
  • Carl-Gustaf Thulin,

    Roles Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

    Affiliations Molecular Ecology Group, Department of Wildlife, Fish, and Environmental Studies, Swedish University of Agricultural Sciences, Umeå, Sweden, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden

    ⨯
  • Göran Spong,

    Roles Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing

    Affiliations Molecular Ecology Group, Department of Wildlife, Fish, and Environmental Studies, Swedish University of Agricultural Sciences, Umeå, Sweden, Forestry and Environmental Resources, College of Natural Resources, North Carolina State University, Raleigh, North Carolina, United States of America

    ⨯
  • Jaakko L. O. Pohjoismäki

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Jaakko.Pohjoismaki@uef.fi

    Affiliation Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland

    ⨯

Abstract

In Fennoscandia, mountain hare (Lepus timidus) and brown hare (Lepus europaeus) hybridize and produce fertile offspring, resulting in gene flow across the species barrier. Analyses of maternally inherited mitochondrial DNA (mtDNA) show that introgression occur frequently, but unavailability of appropriate nuclear DNA markers has made it difficult to evaluate the scale- and significance for the species. The extent of introgression has become important as the brown hare is continuously expanding its range northward, at the apparent expense of the mountain hare, raising concerns about possible competition. We report here, based on analysis of 6833 SNP markers, that the introgression is highly asymmetrical in the direction of gene flow from mountain hare to brown hare, and that the levels of nuclear gene introgression are independent of mtDNA introgression. While it is possible that brown hares obtain locally adapted alleles from the resident mountain hares, the low levels of mountain hare alleles among allopatric brown hares suggest that hybridization is driven by stochastic processes. Interspecific geneflow with the brown hare is unlikely to have major impacts on mountain hare in Fennoscandia, but direct competition may.

Citation: Levänen R, Thulin C-G, Spong G, Pohjoismäki JLO (2018) Widespread introgression of mountain hare genes into Fennoscandian brown hare populations. PLoS ONE 13(1): e0191790. https://doi.org/10.1371/journal.pone.0191790

Editor: Riccardo Castiglia, Universita degli Studi di Roma La Sapienza, ITALY

Received: September 13, 2017; Accepted: January 11, 2018; Published: January 25, 2018

Copyright: © 2018 Levänen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files, except for the original SNP genotyping data, which are available from the Dryad database at the following DOI: 10.5061/dryad.n70q6.

Funding: The Swedish Association of Hunting and Management, the Swedish Environmental Protection Agency, the Faculty of Forest Sciences at SLU, Betty Väänänen Foundation (https://www.sll.fi/pohjois-savo/klyy/apurahat), Finnish Game Foundation (http://www.riistasaatio.fi/), Raija and Ossi Tuuliainen Foundation (http://www.tuuliaisensaatio.fi/), Societas pro Fauna et Flora Fennica (http://www.societasfff.fi/), Saastamoinen Foundation (http://saastamoinenfoundation.fi/) and the University of Eastern Finland doctoral school are kindly thanked for funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Species boundaries are frequently challenged by lineage divergence and hybridization [1]. Diverged lineages (i.e. species) are maintained by barriers to gene flow that vary in strength over time, space, or the genome [2]. For closely related species, the barrier may be permeable [3–5]. Changes in ecology, behavior, population dynamics and distribution may all result in increased levels of spatial and temporal sympatry between closely related species, leading to an increased frequency of hybridization events. These often have profound effects on a wide range of individual- and population level processes. At the individual level, hybridization may affect fitness by creating novel combinations of traits adapted to different environments [6–8]. At the population and species level, hybridization may lead to the introgression of new genetic variation, affecting the diversity within and between species [9].

As the genetic variation provided by hybridization is exposed to selection, hybrids experience an increased, decreased or neutral selection effect, and the effect of this selection may vary in direction and strength across the genome and context [1,2,10]. The signs of introgression are therefore unevenly distributed throughout the genome, with some regions showing selection for, and other against, the introduced genetic material [11,12]. At the population level, introgression typically occurs in hybridization zones at species boundaries, resulting in genetic gradients across the population. At the species level, patterns of introgression are also often highly variable and asymmetric, and may have a strong directional bias towards one of the species in the pair [13]. The outcomes and signatures of hybridization are thus highly contingent on temporal and spatial scales, phylogenetic and demographic relationships and the particulars of the hybridization events. For example, introgression could in theory erase a species boundary, thereby redistributing genetic variation, but without affecting the total amount of standing genetic variation. However, introgression might also accelerate speciation or colonization processes by providing adaptive alleles [14].

From a conservation perspective, introgression create challenges for management efforts, typically by blurring the species barrier [15], affecting the distribution and amount genetic variation, and obfuscating the delineation of appropriate management units [16,17]. Moreover, hybridization can lead to genetic swamping, where hybrids overwhelm or outcompete the rarer species, or to demographic swamping where inferior hybrids lead to a lower population growth rate [18]. In both cases, the loss of species or locally adapted populations is at risk. A contrasting view is that hybridization offers genetic rescue to genetically impoverished populations in risk of genetic meltdown or as a means for species to more rapidly adapt to ecological change [12,14]. Whether viewed as a threat or opportunity, hybridization clearly present challenges for conservation. For example how prevalent it is, which genetic and demographic effects it has, and whether can it be considered independent of or caused by anthropogenic factors. Improved genetic methods allow for more comprehensive understanding of the genomics of introgression and its effect on all levels of biological diversity.

In Europe, northern Fennoscandia represents the northernmost contact zone between brown hare (Lepus europaeus Pallas) and mountain hare (Lepus timidus L.). Benefitted by translocations and, potentially, climate change, the brown hare has been extending its distribution northwards since the 19th century. In contrast, mountain hare populations have been in decline in Finland and southern Sweden [19,20]. Currently, brown hares are abundant in southern and central Sweden and Finland where they mainly exist in sympatry with the mountain hare. Previous research have shown transmission of mitochondrial DNA (mtDNA) from mountain hares into brown hare populations [21,22], where the genetic material is maintained for generations even in geographical areas where mountain hares have become extinct [21,23,24]. Because of the preservation of mtDNA linages the question has been raised whether this introgression has adaptive value or maintained simply due to stochastic demographic processes [13,21,25,26]. Increased frequency of introgressed mtDNA could happen through genetic drift in the extremity of the species distribution, where population density is low and population growth is rapid [27]. Once the population increases and expands its range, for example due to climate change, the introgressed mtDNA could also propagate and imprint into the local resident population, in particular if certain mtDNA lineages generate adaptive advantages. This is suggested to have happened in the Iberian peninsula, where mountain hare mtDNA has been introduced to brown hare population through repeated introgression along the expansion front of the brown hare after last glacial maximum [28]. Recent work done on Iberian hares, involving nuclear DNA markers, suggests that mtDNA introgression among Lepus is driven by demographic processes [29], facilitated by continuous changes of species distribution in the wake of quaternary climate oscillations [30], which could be generalized to explain the common nature of the phenomenon.

The overlap in distribution of mountain hares and brown hares in Fennoscandia and their concurrent population expansion and contraction, presents an outstanding opportunity to study ongoing genetic interactions between two closely related species adapted to different ecological conditions that are now changing rapidly.

Here we analyze 6833 SNP markers across the genome to quantify introgression among Finnish- and Swedish mountain hares and brown hares. We show introgression of mountain hare markers into brown hare, but not vice versa, to be frequent in areas of sympatry. The introgression was independent on levels of mtDNA introgression, confirming the highly asymmetric nature of the genetic interaction between the two species. Genotyping also revealed differences in the genetic diversity of brown hares from the two countries, as expected from their different colonization histories. Besides identifying new genetic markers for Lepus, our study provides evidence of genetic swamping by an invasive species (brown hare) at the expense of a resident species (mountain hare). As ecological change forces species to adapt by changes in distribution or behavior, the demographic and genetic consequences of increased levels of sympatry and hybridization are important to consider for management.

Materials and methods

Tissue samples and DNA isolation

For the genome-wide SNP analysis, DNA was isolated from 22 mountain hares and 27 brown hares (muscle from the base of the ear), including specimens with introgressed mtDNA and representing allopatric as well as sympatric populations almost throughout the full range of the species’ distribution in Sweden and Finland (Table 1, Fig 1). Hunters collected the specimens during normal hunting season, following the regional hunting seasons and legislation, using 12 to 20 gauge shotguns with 3–4 mm shot size. No animal was killed for research purposes only. All specimens were initially identified using morphological characters. The samples included in this study belong to a larger DNA collection of 904 Finnish and 1270 Swedish hares, sorted according to the species, country of origin, mtDNA haplotype as well as allopatric/sympatric occurrence. Populations at the extreme ends of the range, with high certainty of no contemporary contact with the other species, were assigned as allopatric populations (Fig 1). While specimens with conspecific mtDNA haplotype were selected randomly, specimens with introgressed mtDNA were intentionally included in to the study to test the degree of nuclear DNA admixture as well as to monitor for the accuracy of morphological species determination. As pointed out earlier, mtDNA introgression in hares is highly asymmetric, from mountain hare to brown hare and the transfer of mtDNA from brown hares to mountain hares have been considered as rare events and generally of less ecological or genetic significance [31]. DNA was isolated using Chelex® 100 (Bio-Rad) method [32], following manufacturers’ recommendations.

thumbnail
Download:
Table 1. DNA samples used in the study by country, population and mtDNA genotype.

https://doi.org/10.1371/journal.pone.0191790.t001

thumbnail
Download:
Fig 1. Sample distribution across Sweden and Finland.

https://doi.org/10.1371/journal.pone.0191790.g001

mtDNA genotyping

Mitochondrial DNA was genotyped from all samples by amplifying a 669 bp cytochrome b (Cytb) fragment using polymerase chain reaction (PCR) and LCYTBF and LCYTBR primers [33]. The identity of PCR for the species-specific reference products was confirmed by Sanger sequencing (GATC Biotech AG, Germany). The PCR products were digested with AluI restriction enzyme and run over a 3% high resolution agarose gel electrophoresis to reveal restriction fragment lengths and the subsequent genotype [23,33].

SNP genotyping and analysis

199,693 SNPs were analyzed using the commercially available GeneChip™ Rabbit Gene 1.0 ST Array genotyping DNA-chip (Thermo Fischer Scientific #902238). Analyses of population structure and -admixture were performed using 6833 polymorphic loci informative for both hare species (S1 File). The genotype data together with the detailed specimen information can be accessed at the Dryad Data repository (doi:10.5061/dryad.n70q6). Basic population genetic analyses, such as computing allele frequencies and testing for Hardy-Weinberg equilibrium, were performed using Arlequin 3.5. [34]. The PCA analyses to cluster specimens based on the SNP genotypes, was performed in R using the package SNPRelate found at https://bioconductor.org/biocLite.R. Genetic diversity in the hare species was assessed using STRUCTURE 2.3.4 [35,36], using the admixture model, 2–10 populations (K), three iterations with 1 million MCMC repetitions for burnin period of 500,000 [29]. Sampling locations were used as prior information (LOC prior) to expose any shallow population structures. Iterations were matched using CLUMPP v1.1.2 [37]. K or the best number of populations was obtained using STRUCTURE HARVESTER [38]. The detected 192 species-specific SNPs (see Results and Discussion) were used to estimate the degree and direction of hybridization using NEWHYBRIDS software [39], implementing a Gibbs sampler to estimate the posterior probability for individuals falling into defined hybrid categories. The default Prior and Theta settings were used with 100,000 sweeps before and after burnin. Mitochondrial DNA haplotype was also included in the NEWHYBRID analysis as a haploid (“dominant”) marker.

Results and discussion

The genotyping data enabled us to detect highly asymmetrical introgression of nuclear genes from L. timidus to L. europaeus at average level of 2% in regions of sympatry but being almost absent in allopatric populations. In total, 62,730 SNP genotypes had 100% call rate for the 49 samples and 6833 of these were polymorphic (Table 2, S1 and S2 Files). Although no phylogenetic dating of Lepus and Oryctolagus divergence has been made, comparative evidence from Ig gene sequences suggests that the two genera could be as distant as mice and rats [40], which split almost 12 Ma ago [41]. Although SNPs that have remained variable between the species during long evolutionary histories, could be enriched in loci under balancing selection, only 277 loci in brown hare and 109 in mountain hare were not in Hardy-Weinberg equilibrium (S3 File). It may be that the number of analyzed loci has been effective in picking up rare neutral loci that have retained variation across genera. It should be noted that the shared polymorphisms are rare, resulting in low minor allele frequencies in the two species (Table 2). Interestingly, none of the alleles was monomorphic in one species, but typically present at low frequency also in the other one. This might not be surprising, as drift alone would require 9–12 Ne (= size of the effective population) generations to make the diverging species monophyletic at more than 95% of the loci during allopatric speciation [42]. We identified only 192 SNPs having allele frequencies of over 80% in one species but not in the other, which could be useful in species identification (S2 File).

thumbnail
Download:
Table 2. SNP heterozygosity levels and minor allele frequencies for the 6833 polymorphic SNPs by hare species and country of origin.

https://doi.org/10.1371/journal.pone.0191790.t002

Genetic diversity in Fennoscandian hare species

Although the small number of polymorphic loci, ambiguity of chromosomal locations and relatively low levels of minor alleles as well as mean heterozygosity, resulting from using across species SNP array, disallowe more detailed population genetic analyses, the 6833 loci do pick up differences between the two hare species and work as a proxy to compare genetic diversity in Sweden and Finland. Interestingly, the Swedish brown hares show higher heterozygosity levels and minor allele frequencies than their Finnish counterparts, whereas the mountain hares from the two countries do not differ (Table 2). The high heterozygosity in Swedish brown hares correlate also with the genetic diversity as revealed by grouping the specimens by genetic similarity using PCA, where the Swedish brown hare genotypes are more scattered compared to the Finnish specimens or mountain hares (Fig 2A).

thumbnail
Download:
Fig 2. Genetic similarities and population structures among Fennoscandian hares.

(A) Clustering of the 48 genotyped specimens using principal component analysis (PCA) of the SNP data. While the Swedish and Finnish brown hare populations are genetically distinct, the mountain hares from the two countries clearly belong to the same Fennoscandian population. Orange fill: Brown hare, Light blue fill: Finnish mountain hare specimens, Dark blue fill: Swedish mountain hares. Color of the outer ring marks mtDNA genotype whereas allopatric brown hares are marked with black outer ring. Note how the individual hybrid specimen is with brown hare mtDNA is located midway of the two species. Specimens with introgressed mtDNA are otherwise embedded among conspecific samples. (B) Population structure among all 48 samples. A high degree of differentiation between the two species apart for the one hybrid (*). The yellow color, representing typical mountain hare allele combinations, trails into the brown hare clusters. Two ancestral populations (K = 2) was chosen to illustrate hybridization between the two species.

https://doi.org/10.1371/journal.pone.0191790.g002

It is likely that the overall higher heterozygosity in the brown hares compared to mountain hares does not only result from the biased gene flow from mountain hares to brown hares. For example, the country-specific differences in the genetic diversity among brown hares can be explained by the differential colonization histories in the two countries. Brown hares were first introduced to Sweden in 1858 to the island of Ven in the Öresund straight between Sweden and Denmark [43]. After translocations to the Swedish mainland, the species was well established in the south in the late 19th century and subsequently expanded to southern and central Sweden [19]. The brown hare is still expanding northwards in Sweden [44].

Swedish brown hares show high mtDNA haplotype diversity, likely due to admixture due to repeated introductions of brown hares from several geographical areas of the native (continental) range of brown hares [45]. This high mtDNA diversity has likely been accompanied by a diversity of nuclear DNA, explaining the differences in heterozygosity (Table 2), as well as in genetic distance (Fig 2A) between Swedish and Finnish brown hares. In addition, the continuous introgression of mountain hare mtDNA to brown hares in Sweden [21] has led to transfer of nuclear DNA over the species barrier [46], further adding to the genetic composition of brown hares in Sweden. In contrast, the brown hare has established in Finland through spontaneous immigration, mainly during the 20th century in association with a general, northeastward expansion of brown hares noted by Thenius [47]. In Finland, and elsewhere, this expansion has likely been assisted by translocation and supplementary releases, although this has not been properly documented.

Thus, the genotypic differences in SNPs observed between Swedish and Finnish brown hares in the current study, may reflect the differences in the population history of the recurrent populations. The composition of the Swedish brown hare gene pool is a result of admixture of hares from different areas in continental Europe [39] along with introgression from mountain hares [21,51], while Finnish brown hares likely reflect the stepwise and/or gradual expansion pattern observed at the edge of a species distribution [31,48], in association with introgression from mountain hares [49].

Admixture between mountain hare and brown hare

Population structure analysis enabled us to identify species-specific genetic markers and confirm introgression between mountain hares and brown hares. Based on the NEWHYBRID analysis of the species-specific markers, introgression was highly asymmetric, with brown hares being classed as backcrosses whereas all mountain hares included in the study were ranked as purebred (S4 File). The average degree of backcrossing to L. europaeus background cannot be reliably estimated as retention of ancestral polymorphism because of incomplete lineage sorting (ILS) and secondary gene flow through introgression, produce very similar patterns of shared genetic diversity between two species [50,51]. For example, after five generations of backcrosses, only 1.6% of introgressed alleles would be retained, low enough number to be sampled by chance in case of 192 markers. Interestingly, individuals with introgressed mtDNA did not differ significantly from the other sympatric specimens. This is likely to reflect a situation, where hybridization in the areas of sympatry is frequent, but that the most of the mountain hare genotypes are not selectively maintained in the brown hare population but are diluted out in the regions were mountain hares are not present [21,26,28,29,33,52].

As a curiosity, the mountain hare like hybrid (Table 1 and S4 File) having L. europaeus mtDNA represented 0.56 timidus and 0.44 europaeus share of ancestral populations in STRUCTURE analysis (Fig 2). Although it had only 12% total marker heterozygosity, representing a midway between the mean heterozygosities among the species (Table 2). 86% of the 192 species-specific loci (S2 File) were heterozygous (S5 File). When also the NEWHYBRID analysis gave posterior probability value of 1.00 for F1 hybrid assignment in (S4 File)., we suggest that this individual is a genuine first generation hybrid between a female brown hare (mtDNA donor) and male mountain hare. We were also able to confirm that the few other examples of brown hare mtDNA being introgressed into mountain hare were genuine observations and not misidentifications by hunters (Fig 2, S4 File). Interestingly, these specimens had only traces of brown hare specific nuclear markers in contrast to the patterns of genetic imprint among brown hares with mountain hare mtDNA (S4 File).

Concluding remarks

Our study represents the first attempt to assess nuclear gene introgression in hares using a SNP-chip designed for a related species (the rabbit) and, more specifically, it is the first such study focused on mountain hares and brown hares in Fennoscandia. While it is clear that the next generation methods, such as Double Digest Restriction Associated DNA (ddRAD) sequencing [53], provide more powerful alternatives for genotyping species without prior knowledge of their genomes, exploitation of existing and standardized genome-wide genotyping methods can sometimes be cost-efficient and fast, providing useful data for population genetic analysis. The low levels of minor allele frequencies and heterozygosity among the 6833 polymorphic markers indicate that the detected SNPs are not optimal for detailed population analysis. For example, a commercial dog SNP panels typically give 0.30–0.40 heterozygosity rates within breed [54]. However, SNPs that show relatively little variation could constitute highly species-specific markers. Such conservative polymorphisms could be more useful in comparative studies than common polymorphisms used to differentiate individuals. Therefore, we believe that the discovered SNPs in this study prove to be useful as a future resource for hare population genetics.

Our results provide support for the preferential introgression of nuclear genes from L. timidus to L. europaeus in Fennoscandia, as previously reported for mtDNA [21,22,49]. Based on the mtDNA evidence, introgression is most frequent at the leading edge of brown hare range expansion [21,49]. Contrary to the comprehensive study using 100 SNPs obtained from RNA-seq transcriptome data from 314 L. granatensis specimens, assessing the patterns of the historical introgression events [29], our study aimed to validate the degree of nuclear marker admixture during ongoing contact between the two species in Fennoscandia. While it is likely that mtDNA introgression and preservation can be explained by demography [29], it is plausible that brown hare could also obtain locally adapted alleles from the resident mountain hares, which are expected to represent only a tiny fraction of the total genome. A limitation of transcriptome studies is that they provide expression data and transcript genotypes only from the sampled tissue, such as kidney or liver, and might not be able to capture genes influencing traits such as coat color variation [55,56], muscle metabolism [57], diet specialization [58] and immunity [59]. At present, comprehensive surveys of this type of adaptive variation in species with unknown genomes are generally not possible. However, already the current next generation sequencing methods enable the simultaneous genotyping of dozens of candidate gene loci, enabling the correlation of genotypes with phenotypes and the detection of single adaptive alleles. As the costs of whole genome sequencing are constantly dropping, it is likely that ad hoc comparisons of genomes become the standard practice in population genetics in the near future.

Although brown hare may outcompete mountain hare under certain conditions [19,60], the two species has coexisted in sympatry in most of Fennoscandia for decades. While hybridization between sympatric species can be a threat to endangered species, theoretically in less than five generations [61], genetic introgression in our hare-model system has significance mainly to the receiving brown hare population. Shortened snow-covered season, resulting in a dramatic mismatch with the protective white, may pose an additional threat to mountain hares regardless of brown hare’s range expansion. Although the mountain hare populations might be contracting at the southern edges of the species’ distribution [20], for a cold-adapted species, mountain hare has shown a remarkable resilience in the past [62] and is likely survive in Fennoscandia in the future.

Supporting information

S1 File. Probe sequences for the SNP markers used in the study.

https://doi.org/10.1371/journal.pone.0191790.s001

(XLSX)

S2 File. Allele frequencies for all analyzed loci used and a list species diagnostic SNP markers.

https://doi.org/10.1371/journal.pone.0191790.s002

(XLSX)

S3 File. List of loci not in Hardy-Weinberg equilibrium in brown hare and mountain hare.

Species on separate spreadsheets.

https://doi.org/10.1371/journal.pone.0191790.s003

(XLSX)

S4 File. Posterior probabilities for different hybrid classes based on NEWHYBRID analysis.

https://doi.org/10.1371/journal.pone.0191790.s004

(XLSX)

S5 File. F1 hybrid genotyping using the species-specific loci listed in S2 File.

https://doi.org/10.1371/journal.pone.0191790.s005

(XLSX)

Acknowledgments

RL, JP and CGT thank all hunters and Finnish food safety authority for helping with sample collections, and A. Pynttäri, I. Assimakopoulou, J. Gnjatović, L. Barbier and P. Mutka for laboratory assistance. The Swedish Association of Hunting and Management, the Swedish Environmental Protection Agency, the Faculty of Forest Sciences at SLU, Betty Väänänen Foundation, Finnish Game Foundation, Raija and Ossi Tuuliainen Foundation, Societas pro Fauna et Flora Fennica, Saastamoinen Foundation and the University of Eastern Finland doctoral school are kindly thanked for funding.

References

  1. 1. Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJ, et al. (2013) Hybridization and speciation. J Evol Biol 26: 229–246. pmid:23323997
  2. 2. Harrison RG, Larson EL (2014) Hybridization, introgression, and the nature of species boundaries. J Hered 105 Suppl 1: 795–809.
  3. 3. Asensio N, Jose-Dominguez JM, Kongrit C, Brockelman WY (2017) The ecology of white-handed and pileated gibbons in a zone of overlap and hybridization in Thailand. Am J Phys Anthropol 163: 716–728. pmid:28726303
  4. 4. Beaumont M, Barratt EM, Gottelli D, Kitchener AC, Daniels MJ, et al. (2001) Genetic diversity and introgression in the Scottish wildcat. Mol Ecol 10: 319–336. pmid:11298948
  5. 5. Veen T, Borge T, Griffith SC, Saetre GP, Bures S, et al. (2001) Hybridization and adaptive mate choice in flycatchers. Nature 411: 45–50. pmid:11333971
  6. 6. Bay RA, Ruegg K (2017) Genomic islands of divergence or opportunities for introgression? Proc Biol Sci 284.
  7. 7. Racimo F, Marnetto D, Huerta-Sanchez E (2017) Signatures of Archaic Adaptive Introgression in Present-Day Human Populations. Mol Biol Evol 34: 296–317. pmid:27756828
  8. 8. Tigano A, Friesen VL (2016) Genomics of local adaptation with gene flow. Mol Ecol 25: 2144–2164. pmid:26946320
  9. 9. Koju NP, He K, Chalise MK, Ray C, Chen Z, et al. (2017) Multilocus approaches reveal underestimated species diversity and inter-specific gene flow in pikas (Ochotona) from southwestern China. Mol Phylogenet Evol 107: 239–245. pmid:27838310
  10. 10. Mallet J, Besansky N, Hahn MW (2016) How reticulated are species? Bioessays 38: 140–149. pmid:26709836
  11. 11. Payseur BA, Rieseberg LH (2016) A genomic perspective on hybridization and speciation. Molecular Ecology 25: 2337–2360. pmid:26836441
  12. 12. Hedrick PW (2013) Adaptive introgression in animals: examples and comparison to new mutation and standing variation as sources of adaptive variation. Molecular Ecology 22: 4606–4618. pmid:23906376
  13. 13. Currat M, Ruedi M, Petit RJ, Excoffier L (2008) The hidden side of invasions: Massive introgression by local genes. Evolution 62: 1908–1920. pmid:18452573
  14. 14. Hamilton JA, Miller JM (2016) Adaptive introgression as a resource for management and genetic conservation in a changing climate. Conservation Biology 30: 33–41. pmid:26096581
  15. 15. Milan-Garcia Y, Ramos-Targarona R, Perez-Fleitas E, Sosa-Rodriguez G, Guerra-Manchena L, et al. (2015) Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situlex situ conservation. Heredity 114: 272–280. pmid:25335559
  16. 16. Daniels MJ, Corbett L (2003) Redefining introgressed protected mammals: when is a wildcat a wild cat and a dingo a wild dog? Wildlife Research 30: 213–218.
  17. 17. Gil-Sanchez JM, Jaramillo J, Barea-Azcon JM (2015) Strong spatial segregation between wildcats and domestic cats may explain low hybridization rates on the Iberian Peninsula. Zoology 118: 377–385. pmid:26358989
  18. 18. Todesco M, Pascual MA, Owens GL, Ostevik KL, Moyers BT, et al. (2016) Hybridization and extinction. Evolutionary Applications 9: 892–908. pmid:27468307
  19. 19. 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 Review 33: 29–42.
  20. 20. Pedersen S, Odden M, Pedersen HC (2017) Climate change induced molting mismatch? Mountain hare abundance reduced by duration of snow cover and predator abundance. Ecosphere 8: e01722.
  21. 21. Thulin C-G, 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? Journal of Zoology 258: 299–306.
  22. 22. Thulin C-G, Jaarola M, Tegelström H (1997) The occurrence of mountain hare mitochondrial DNA in wild brown hares. Mol Ecol 6: 463–467. pmid:9161014
  23. 23. Alves PC, Ferrand N, Suchentrunk F, Harris DJ (2003) Ancient introgression of Lepus timidus mtDNA into L-granatensis and L-europaeus in the Iberian Peninsula. Molecular Phylogenetics and Evolution 27: 70–80. pmid:12679072
  24. 24. Alves PC, Melo-Ferreira J, Freitas H, Boursot P (2008) The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus. Philosophical Transactions of the Royal Society B-Biological Sciences 363: 2831–2839.
  25. 25. Chan KMA, Levin SA (2005) Leaky prezygotic isolation and porous genomes: Rapid introgression of maternally inherited DNA. Evolution 59: 720–729. pmid:15926684
  26. 26. Melo-Ferreira J, Vilela J, Fonseca MM, da Fonseca RR, Boursot P, et al. (2014) The Elusive Nature of Adaptive Mitochondrial DNA Evolution of an Arctic Lineage Prone to Frequent Introgression. Genome Biology and Evolution 6: 886–896. pmid:24696399
  27. 27. Edmonds CA, Lillie AS, Cavalli-Sforza LL (2004) Mutations arising in the wave front of an expanding population. Proceedings of the National Academy of Sciences of the United States of America 101: 975–979. pmid:14732681
  28. 28. Melo-Ferreira J, Farelo L, Freitas H, Suchentrunk F, Boursot P, et al. (2014) Home-loving boreal hare mitochondria survived several invasions in Iberia: the relative roles of recurrent hybridisation and allele surfing. Heredity 112: 265–273. pmid:24149657
  29. 29. Marques JP, Farelo L, Vilela J, Vanderpool D, Alves PC, et al. (2017) Range expansion underlies historical introgressive hybridization in the Iberian hare. Scientific Reports 7.
  30. 30. Hewitt GM (2004) Genetic consequences of climatic oscillations in the Quaternary. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 359: 183–195.
  31. 31. Ibrahim KM, Nichols RA, Hewitt GM (1996) Spatial patterns of genetic variation generated by different forms of dispersal during range expansion. Heredity 77: 282–291.
  32. 32. Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10: 506–513. pmid:1867860
  33. 33. Melo-Ferreira J, Boursot P, Suchentrunk F, Ferrand N, Alves PC (2005) Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia. Molecular Ecology 14: 2459–2464. pmid:15969727
  34. 34. Excoffier L, Lischer HE (2010) Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: 564–567. pmid:21565059
  35. 35. Falush D, Stephens M, Pritchard JK (2007) Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes 7: 574–578. pmid:18784791
  36. 36. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155: 945–959. pmid:10835412
  37. 37. Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics 23: 1801–1806. pmid:17485429
  38. 38. Earl DA, Vonholdt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources 4: 359–361.
  39. 39. Anderson EC, Thompson EA (2002) A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160: 1217–1229. pmid:11901135
  40. 40. Lanning DK, Esteves PJ, Knight KL (2017) The remnant of the European rabbit (Oryctolagus cuniculus) IgD gene. PLoS One 12: e0182029. pmid:28832642
  41. 41. Kimura Y, Hawkins MT, McDonough MM, Jacobs LL, Flynn LJ (2015) Corrected placement of Mus-Rattus fossil calibration forces precision in the molecular tree of rodents. Sci Rep 5: 14444. pmid:26411391
  42. 42. Hudson RR, Coyne JA (2002) Mathematical consequences of the genealogical species concept. Evolution 56: 1557–1565. pmid:12353748
  43. 43. Lönnberg E (1905) On hybrids between Lepus timidus L. and Lepus europeus Pall. from southern Sweden. Proceedings of Zoological Society of London 1: 278–287.
  44. 44. Jansson G, Pehrson A (2007) The recent expansion of the brown hare (Lepus europaeus) in Sweden with possible implications to the mountain hare (L. timidus). European Journal of Wildlife Research 53: 125–130.
  45. 45. Thulin CG, Tegelström H (2001) High mtDNA haplotype diversity among introduced Swedish brown hares Lepus europaeus. Acta Theriologica 46: 375–384.
  46. 46. Thulin C-G, Stone J, Tegelström H, Walker CW (2006) Species assignment and hybrid identification among Scandinavian hares Lepus europaeus and L. timidus. Wildlife Biology 12: 29–38.
  47. 47. Thenius E (1980) Grundzüge der Faunen- und Verbreitungsgesichte der Säugetiere. Stuttgart: Gustav Fisher Verlag.
    • 48. Hewitt GM (1999) Post-glacial re-colonization of European biota. Biological Journal of the Linnean Society 68: 87–112.
    • 49. Levänen R, Kunnasranta M, Pohjoismäki J (2018) Mitochondrial DNA introgression at the northern edge of the brown hare (Lepus europaeus) range. Ann Zool Fennici 55: 15–24.
    • 50. Charlesworth B, Bartolome C, Noel V (2005) The detection of shared and ancestral polymorphisms. Genet Res 86: 149–157. pmid:16207392
    • 51. Sousa V, Hey J (2013) Understanding the origin of species with genome-scale data: modelling gene flow. Nat Rev Genet 14: 404–414. pmid:23657479
    • 52. Fredsted T, Wincentz T, Villesen P (2006) Introgression of mountain hare (Lepus timidus) mitochondrial DNA into wild brown hares (Lepus europaeus) in Denmark. BMC Ecol 6: 17. pmid:17105672
    • 53. Andrews KR, Good JM, Miller MR, Luikart G, Hohenlohe PA (2016) Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet 17: 81–92. pmid:26729255
    • 54. Kumpulainen M, Anderson H, Svevar T, Kangasvuo I, Donner J, et al. (2017) Founder representation and effective population size in old versus young breeds-genetic diversity of Finnish and Nordic Spitz. J Anim Breed Genet.
    • 55. Corso J, Mundy NI, Fagundes NJR, de Freitas TRO (2016) Evolution of dark colour in toucans (Ramphastidae): a case of molecular adaptation? Journal of Evolutionary Biology 29: 2530–2538. pmid:27654325
    • 56. Zimova M, Mills LS, Nowak JJ (2016) High fitness costs of climate change-induced camouflage mismatch. Ecology Letters 19: 299–307. pmid:26799459
    • 57. Cardona A, Pagani L, Antao T, Lawson DJ, Eichstaedt CA, et al. (2014) Genome-Wide Analysis of Cold Adaptation in Indigenous Siberian Populations. Plos One 9.
    • 58. Fumagalli M, Moltke I, Grarup N, Racimo F, Bjerregaard P, et al. (2015) Greenlandic Inuit show genetic signatures of diet and climate adaptation. Science 349: 1343–1347. pmid:26383953
    • 59. Smith S, de Bellocq JG, Suchentrunk F, Schaschl H (2011) Evolutionary genetics of MHC class II beta genes in the brown hare, Lepus europaeus. Immunogenetics 63: 743–751. pmid:21688061
    • 60. Reid N (2011) European hare (Lepus europaeus) invasion ecology: implication for the conservation of the endemic Irish hare (Lepus timidus hibernicus). Biological Invasions 13: 559–569.
    • 61. Wolf DE, Takebayashi N, Rieseberg LH (2001) Predicting the risk of extinction through hybridization. Conservation Biology 15: 1039–1053.
    • 62. Smith S, Sandoval-Castellanos E, Lagerholm VK, Napierala H, Sablin M, et al. (2017) Nonreceding hare lines: genetic continuity since the Late Pleistocene in European mountain hares (Lepus timidus). Biological Journal of the Linnean Society 120: 891–908.