Hybridization and population genetics of two macaque species in Sulawesi, Indonesia

This study investigates hybridization and population genetics of two species of macaque monkey in Sulawesi, Indonesia, using molecular markers from mitochondrial, autosomal, and Y-chromosome DNA. Hybridization is the interbreeding of individuals from different parental taxa that are distinguishable by one or more heritable characteristics. Because hybridization can affect population structure of the parental taxa, it is an important consideration for conservation management. On the Indonesian island of Sulawesi an explosive diversification of macaques has occurred; seven of 19 species in the genus Macaca live on this island. The contact zone of the subjects of this study, M. maura and M. tonkeana, is located at the base of the southwestern peninsula of Sulawesi. Land conversion in Sulawesi is occurring at an alarming pace; currently two species of Sulawesi macaque, one of which is M. maura, are classified as endangered species. Results of this study indicate that hybridization among M. maura and M. tonkeana has led to different distributions of molecular variation in mitochondrial DNA and nuclear DNA in the contact zone; mitochondrial DNA shows a sharp transition from M. maura to M. tonkeana haplotypes, but nuclear DNA from the parental taxa is homogenized in a narrow hybrid zone. Similarly, within M. maura divergent mitochondrial DNA haplotypes are geographically structured but population subdivision in the nuclear genome is low or absent. In M. tonkeana, mitochondrial DNA haplotypes are geographically structured and a high level of nuclear DNA population subdivision is present in this species. These results are largely consistent with a macaque behavioral paradigm of female philopatry and obligate male dispersal, suggest that introgression between M. maura and M. tonkeana is restricted to the hybrid zone, and delineate one conservation management unit in M. maura and at least two in M. tonkeana.     

A reassessment of explanations for discordant introgressions of mitochondrial and nuclear genomes

Hybridization is increasingly recognized as a significant evolutionary process, in particular because it can lead to introgression of genes from one species to another. A striking pattern of discordance in the amount of introgression between mitochondrial and nuclear markers exists such that substantial mitochondrial introgression is often found in combination with no or little nuclear introgression. Multiple mechanisms have been proposed to explain this discordance, including positive selection for introgressing mitochondrial variants, several types of sex‐biases, drift, negative selection against introgression in the nuclear genome, and spatial expansion. Most of these hypotheses are verbal, and have not been quantitatively evaluated so far. We use individual‐based, multilocus, computer simulations of secondary contact under a wide range of demographic and genetic scenarios to evaluate the ability of the different mechanisms to produce discordant introgression. Sex‐biases and spatial expansions fail to produce substantial mito‐nuclear discordance. Drift and nuclear selection can produce strong discordance, but only under a limited range of conditions. In contrast, selection on the mitochondrial genome produces strong discordance, particularly when dispersal rates are low. However, commonly used statistical tests have little power to detect this selection. Altogether, these results dismiss several popular hypotheses, and provide support for adaptive mitochondrial introgression.     

History repeated: recent and historical mitochondrial introgression between the current darter Etheostoma uniporum and rainbow darter Etheostoma caeruleum (Teleostei: Percidae)

Incongruence between recognized taxonomy and phylogenetic relationships between two species from a diverse clade (Percidae: Etheostomatinae) of stream fishes was found in a mitochondrial (mt) DNA gene tree. Two darters in subgenus Oligocephalus , Etheostoma uniporum current darter and Etheostoma caeruleum rainbow darter were sampled throughout their sympatric distribution in the Ozark Highlands of the central United States. Sequences from cytochrome (cyt) b and the first intron of the nuclear marker S7 were analysed separately using maximum parsimony and Bayesian methods. Cyt b recovered both species as polyphyletic; E. uniporum haplotypes were interspersed within E. caeruleum . However, both species were monophyletic and non-sister taxa based on S7. The cyt b gene tree pattern is caused by introgressive hybridization resulting in the mtDNA replacement of E. uniporum haplotypes by those of E. caeruleum . Some E. uniporum haplotypes are shared with geographically proximate E. caeruleum , and this is consistent with recent hybridization, while other E. uniporum haplotypes indicate historical sorting of introgressed lineages. The mechanism of introgression is likely asymmetric sneaking behaviour by male E. uniporum , a mating tactic observed in related species. MtDNA replacement may have occurred in E. uniporum due to drift fixation in a historically small female effective population. Additional evidence for darter hybridization will likely be discovered in future molecular genetic surveys of the nearly 200 species in eastern North America.     

Cytonuclear discordance across a leopard frog contact zone

We conducted a genetic analysis of the extent of hybridization within and outside a contact zone between two North American leopard frogs, Rana blairi and Rana pipiens by comparing distribution patterns of mitochondrial and nuclear haplotypes. The contact zone, located between South Dakota, Nebraska, and Iowa, USA, was previously examined using morphological data in the 1970s, leading to the conclusion that hybridization was rare between R. pipiens and R. blairi. Our genetic analysis of 51 populations (611 samples) shows strong cytonuclear discordance. Mitochondrial-haplotype distribution matches the same pattern as the documented species spatial distributions based on morphology. However, the geographic distribution of the nuclear haplotypes reveals asymmetrical swamping of the R. pipiens nuclear haplotypes by R. blairi haplotypes. Phylogenetic analyses of both mitochondrial and nuclear markers provide strong evidence for the presence of R. blairi-R. pipiens introgression for the nuclear marker. A pattern of mitochondrial isolation with nuclear introgression is extremely unusual, and predicted to occur much less often than the reverse.     

The biogeography of mitochondrial and nuclear discordance in animals

Combining nuclear (nuDNA) and mitochondrial DNA (mtDNA) markers has improved the power of molecular data to test phylogenetic and phylogeographic hypotheses and has highlighted the limitations of studies using only mtDNA markers. In fact, in the past decade, many conflicting geographic patterns between mitochondrial and nuclear genetic markers have been identified (i.e. mito-nuclear discordance). Our goals in this synthesis are to: (i) review known cases of mito-nuclear discordance in animal systems, (ii) to summarize the biogeographic patterns in each instance and (iii) to identify common drivers of discordance in various groups. In total, we identified 126 cases in animal systems with strong evidence of discordance between the biogeographic patterns obtained from mitochondrial DNA and those observed in the nuclear genome. In most cases, these patterns are attributed to adaptive introgression of mtDNA, demographic disparities and sex-biased asymmetries, with some studies also implicating hybrid zone movement, human introductions and Wolbachia infection in insects. We also discuss situations where divergent mtDNA clades seem to have arisen in the absence of geographic isolation. For those cases where foreign mtDNA haplotypes are found deep within the range of a second taxon, data suggest that those mtDNA haplotypes are more likely to be at a high frequency and are commonly driven by sex-biased asymmetries and/or adaptive introgression. In addition, we discuss the problems with inferring the processes causing discordance from biogeographic patterns that are common in many studies. In many cases, authors presented more than one explanation for discordant patterns in a given system, which indicates that likely more data are required. Ideally, to resolve this issue, we see important future work shifting focus from documenting the prevalence of mito-nuclear discordance towards testing hypotheses regarding the drivers of discordance. Indeed, there is great potential for certain cases of mitochondrial introgression to become important natural systems within which to test the effect of different mitochondrial genotypes on whole-animal phenotypes.     

Recurrent replacement of mtDNA and cryptic hybridization between two sibling bat species Myotis myotis and Myotis blythii

The two sibling bat species Myotis myotis and Myotis blythii occur in sympatry over wide areas of Southern and Central Europe. Morphological, ecological and previous genetic evidence supported the view that the two species constitute two well-differentiated groups, but recent phylogenetic analyses have shown that the two species share some mtDNA haplotypes when they occur in sympatry. In order to see whether some genetic exchange has occurred between the two species, we sequenced a highly variable segment of the mitochondrial control region in both species living in sympatry and in allopatry. We also analysed the nuclear diversity of 160 individuals of both species found in two mixed nursery colonies located north and south of the Alps. MtDNA analysis confirmed that European M. blythii share multiple, identical or very similar haplotypes with M. myotis. Since allopatric Asian M. blythii presents mtDNA sequences that are very divergent from those of the two species found in Europe, we postulate that the mitochondrial genome of the European M. blythii has been replaced by that of M. myotis. The analysis of nuclear diversity shows a strikingly different pattern, as both species are well differentiated within mixed nursery colonies (F(ST) = 0.18). However, a Bayesian analysis of admixture reveals that the hybrids can be frequently observed, as about 25% of sampled M. blythii show introgressed genes of M. myotis origin. In contrast, less than 4% of the M. myotis analysed were classified as non-parental genotypes, revealing an asymmetry in the pattern of hybridization between the two species. These results show that the two species can interbreed and that the hybridization is still ongoing in the areas of sympatry. The persistence of well-differentiated nuclear gene pools, in spite of an apparent replacement of mitochondrial genome in European M. blythii by that of M. myotis, is best explained by a series of introgression events having occurred repeatedly during the recent colonization of Europe by M. blythii from Asia. The sharp contrast obtained from the analysis of mitochondrial and nuclear markers further points to the need to cautiously interpret results based on a single class of genetic markers.     

Extensive trans-species mitochondrial polymorphisms in the carabid beetles Carabus subgenus Ohomopterus caused by repeated introgressive hybridization

To study the potential importance of introgressive hybridization to the evolutionary diversification of a carabid beetle lineage, we studied intraspecific and trans-species polymorphisms in the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequence (1083 bp) in four species of the subgenus Ohomopterus (genus Carabus) in central and eastern Honshu, Japan. Of the four species, C. insulicola is parapatric with the other three, and can hybridize naturally with at least two. This species possesses two haplotypes of remote lineages. We classified ND5 haplotypes using polymerase chain reaction-restriction fragment length polymorphism with TaqI endonuclease for 524 specimens, and sequenced 143 samples. Analysis revealed that each species was polyphyletic in its mitochondrial DNA phylogeny, representing a marked case of trans-species polymorphism. Recent one-way introgression of mitochondria from C. arrowianus nakamurai to C. insulicola, and from C. insulicola to C. esakii, was inferred from the frequency of identical sequences between these species and from direct evidence of hybridization in their contact zones. Other intraspecific polymorphisms in the four species may be due to undetected introgressive hybridization (e.g. C. insulicola to C. maiyasanus) or from stochastic lineage sorting of ancestral polymorphisms. This beetle group has a genital lock-and-key system, with species-specific or subspecies-specific genital morphology that may act as a barrier to hybridization. However, our results demonstrate that introgressive hybridization has occurred multiple times, at least for mitochondria, despite differences among, and stability within, morphological characters that distinguish local populations. Thus, hybridization and introgression could have been key processes in the evolutionary diversification of Ohomopterus.     

Sequential adaptive introgression of the mitochondrial genome in Drosophila yakuba and Drosophila santomea

Interspecific hybridization provides the unique opportunity for species to tap into genetic variation present in a closely related species and potentially take advantage of beneficial alleles. It has become increasingly clear that when hybridization occurs, mitochondrial DNA (mtDNA) often crosses species boundaries, raising the possibility that it could serve as a recurrent target of natural selection and source of species' adaptations. Here we report the sequences of 46 complete mitochondrial genomes of Drosophila yakuba and Drosophila santomea, two sister species known to produce hybrids in nature (~3%). At least two independent events of mtDNA introgression are uncovered in this study, including an early invasion of the D. yakuba mitochondrial genome that fully replaced the D. santomea mtDNA native haplotypes and a more recent, ongoing event centred in the hybrid zone. Interestingly, this recent introgression event bears the signature of Darwinian natural selection, and the selective haplotype can be found at low frequency in Africa mainland populations of D. yakuba. We put forward the possibility that, because the effective population size of D. santomea is smaller than that of D. yakuba, the faster accumulation of mildly deleterious mutations associated with Muller's ratchet in the former species may have facilitated the replacement of the mutationally loaded mitochondrial genome of D. santomea by that of D. yakuba.     

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold‐resistant leaf beetles, Gonioctena quinquepunctata and G. intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD‐seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species‐specific SNPs (displaying a fixed difference between species) a priori and second by using model‐based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.     

Geographical extent of Arctic char (Salvelinus alpinus) mtDNA introgression in brook char populations (S. fontinalis) from eastern Québec, Canada

The geographical extent of Arctic char ( Salvelinus alpinus ) mitochondrial DNA introgression into brook char ( Salvelinus fontinalis ) populations found in eastern Québec was determined by analysing a total of 598 fish from 29 lakes. The nuclear genome was analysed by protein electrophoresis, whereas the ND-5,6 portion of the mitochondrial genome was analysed by restriction fragment length polymorphism. This survey revealed that introgressed S. fontinalis populations are restricted to only one river subdrainage of the Portneuf basin, where Arctic char is completely absent. Elsewhere, nonintrogressed pure S. fontinalis populations populate the lakes. These findings suggest that the initial hybridization event between the species is ancient and probably occurred shortly after recolonization of the area. At that time, the species would have been in contact and the chances of reproductive isolation mechanisms breaking down would have been high. We discuss the possibility that a combination of biogeographical conditions coupled with positive selection for mtDNA introgression led to the present-day distribution of introgressed S. fontinalis in northeastern North America.     

Repeated unidirectional introgression of nuclear and mitochondrial DNA between four congeneric Tanganyikan cichlids

With an increasing number of reported cases of hybridization and introgression, interspecific gene flow between animals has recently become a widely accepted and broadly studied phenomenon. In this study, we examine patterns of hybridization and introgression in Ophthalmotilapia spp., a genus of cichlid fish from Lake Tanganyika, using mitochondrial and nuclear DNA from all four species in the genus and including specimens from over 800 km of shoreline. These four species have very different, partially overlapping distribution ranges, thus allowing us to study in detail patterns of gene flow between sympatric and allopatric populations of the different species. We show that a significant proportion of individuals of the lake-wide distributed O. nasuta carry mitochondrial and/or nuclear DNA typical of other Ophthalmotilapia species. Strikingly, all such individuals were found in populations living in sympatry with each of the other Ophthalmotilapia species, strongly suggesting that this pattern originated by repeated and independent episodes of genetic exchange in different parts of the lake, with unidirectional introgression occurring into O. nasuta. Our analysis rejects the hypotheses that unidirectional introgression is caused by natural selection favoring heterospecific DNA, by skewed abundances of Ophthalmotilapia species or by hybridization events occurring during a putative spatial expansion in O. nasuta. Instead, cytonuclear incompatibilities or asymmetric behavioral reproductive isolation seem to have driven repeated, unidirectional introgression of nuclear and mitochondrial DNA into O. nasuta in different parts of the lake.     

Introgressive hybridization in fishes: the biochemical evidence

Biochemical methods can detect variation at individual genetic loci, making possible the direct assessment of natural hybridization and introgression between fish populations. Protein electro-phoresis has been used to confirm and extend knowledge of many situations where species hybrids have been detected by morphological analyses. New cases of natural hybridization, including some at the subspecies level, have also been identified. Biochemical studies have provided the first conclusive evidence of natural post F₁ hybrids and of introgression between fish taxa. The strongest cases for introgression have used a combined analysis of nuclear protein genes and taxaspecific maternally inherited mitochondrial DNA variation. Information on the significance of introgression as a source of gene flow between taxa, particularly below the species level where sympatric subspecies and sibling species are involved, should expand in the future as the numbers and types of nuclear and mitochondrial DNA loci which can be assayed for variation increase. The full importance of introgressive hybridization in speciation may then be understood.     

Perpendicular axes of differentiation generated by mitochondrial introgression

Differential introgression of mitochondrial vs. nuclear DNA generates discordant patterns of geographic variation and can promote population divergence and speciation. We examined a potential case of mitochondrial introgression leading to two perpendicular axes of differentiation. The Eastern Yellow Robin Eopsaltria australis, a widespread Australian bird, shows a deep mitochondrial split that is perpendicular to north–south nuclear DNA and plumage colour differentiation. We propose a scenario to explain this pattern: (i) first, both nuclear and mitochondrial genomes differentiated in concert during north–south population divergence; (ii) later, their histories disconnected after two mitochondrial introgression events resulting in a deep mitochondrial split perpendicular to the nuclear DNA structure. We explored this scenario by coalescent modelling of ten mitochondrial genes and 400 nuclear DNA loci. Initial mitochondrial and nuclear genome divergences were estimated to have occurred in the early Pleistocene, consistent with the proposed scenario. Subsequent climatic transitions may have driven later mitochondrial introgression. We consider neutral introgression unlikely and instead propose that the evidence is more consistent with adaptive mitochondrial introgression and selection against incompatible mitochondrial‐nuclear combinations. This likely generated an axis of coastal‐inland mitochondrial differentiation in the face of nuclear gene flow, perpendicular to the initial north–south axis of differentiation (reflected in genomewide nuclear DNA and colour variation).     

Chlorophyll-binding proteins revisited - a multigenic family of light-harvesting and stress proteins from a brown algal perspective

Background

Mitochondrial introgression may result in the mitochondrial genome of one species being replaced by that of another species without leaving any trace of past hybridization in its nuclear genome. Such introgression can confuse the species genealogy estimates and lead to absurd inferences of species history. We used a phylogenetic approach to explore the potential mitochondrial genome introgression event(s) between two closely related green pond frog species, Pelophylax nigromaculatus and P. plancyi.

Results

DNA sequence data of one mitochondrial and two nuclear genes from an extensive sampling of the two species were collected, and the genealogies of the three genes were constructed and compared. While the two nuclear genes congruently showed mutual reciprocal monophyly of both species, the mitochondrial phylogeny separated a Korean P. nigromaculatus clade, a paraphyletic central China P. plancyi assemblage, and a large well-supported introgression clade. Within the introgression clade, the mitochondrial haplotypes of the two species were mixed together. This reticulated pattern can be most parsimoniously explained by an ancient mitochondrial introgression event from P. plancyi to P. nigromaculatus that occurred at least 1.36 MYA, followed by multiple recent introgression events from P. nigromaculatus back to P. plancyi within the last 0.63 MY. The re-constitution of previously co-adapted genomes in P. plancyi may be responsible for the recent rampant introgression events. The Korean P. nigromaculatus clade likely represents the only surviving "true" mitochondrial lineage of P. nigromaculatus, and the central China P. plancyi assemblage likely represents the "original" P. plancyi mitochondrial lineage. Refugia in the Korean Peninsula and central China may have played a significant role in preserving these ancient lineages.

Conclusions

The majority of individuals in the two species have either introgressed (P. nigromaculatus) or reclaimed (P. plancyi) mitochondrial genomes while no trace of past hybridization in their nuclear genomes was detected. Asymmetrical reproductive ability of hybrids and continuous backcrossing are likely responsible for the observed mitochondrial introgression. This case is unique in that it includes an ancient "forward" introgression and many recent "backward" introgressions, which re-constitutes the original nuclear and mitochondrial genomes of P. plancyi. This hybrid system provides an excellent opportunity to study cyto-nuclear interaction and co-adaptation.     

Current and historical hybridization with differential introgression among three species of cyprinid fishes (genus <Emphasis Type="Italic">Cyprinella</Emphasis>)

Hybridization is common among freshwater fishes, particular among the Cyprinidae. We used two mitochondrial genes and one nuclear gene to characterize hybridization among two species pairs of Cyprinella in southwestern North America. Genalogical patterns revealed that C. lutrensis and C. venusta are currently hybridizing in several localities producing apparent F₁, F₂ and backcross generations, yet there was no evidence for introgression outside of local hybrid zones. Alternatively, mitochondrial haplotypes from C. lutrensis appear to have introgressed into a C. lepida population in the Nueces River completely replacing the native C. lepida haplotype. There was no evidence of introgression of nuclear DNA and there does not appear to be ongoing hybridization. The population of C. lepida from the nearby Frio River exhibits no evidence of hybridization with C. lutrensis. Thus, contact between C. lutrensis and C. venusta results in the formation of localized hybrid swarms, while contact between C. lutrensis and C. lepida has resulted in complete mitochondrial introgression in the Nueces River or no apparent hybridization in the Frio River. The three different outcomes of contact between these species illustrate the variable nature of interspecific reproductive interactions and provide an excellent system in which to better understand the factors influencing hybridization among freshwater fishes.     

The on‐again,off‐again relationship between mitochondrial genomes and species boundaries

The study of reproductive isolation and species barriers frequently focuses on mitochondrial genomes and has produced two alternative and almost diametrically opposed narratives. On one hand, mtDNA may be at the forefront of speciation events, with co‐evolved mitonuclear interactions responsible for some of the earliest genetic incompatibilities arising among isolated populations. On the other hand, there are numerous cases of introgression of mtDNA across species boundaries even when nuclear gene flow is restricted. We argue that these seemingly contradictory patterns can result from a single underlying cause. Specifically, the accumulation of deleterious mutations in mtDNA creates a problem with two alternative evolutionary solutions. In some cases, compensatory or epistatic changes in the nuclear genome may ameliorate the effects of mitochondrial mutations, thereby establishing coadapted mitonuclear genotypes within populations and forming the basis of reproductive incompatibilities between populations. Alternatively, populations with high mitochondrial mutation loads may be rescued by replacement with a more fit, foreign mitochondrial haplotype. Coupled with many nonadaptive mechanisms of introgression that can preferentially affect cytoplasmic genomes, this form of adaptive introgression may contribute to the widespread discordance between mitochondrial and nuclear genealogies. Here, we review recent advances related to mitochondrial introgression and mitonuclear incompatibilities, including the potential for cointrogression of mtDNA and interacting nuclear genes. We also address an emerging controversy over the classic assumption that selection on mitochondrial genomes is inefficient and discuss the mechanisms that lead lineages down alternative evolutionary paths in response to mitochondrial mutation accumulation.     

Genetic structure and distinctness of Apis mellifera L. populations from the Canary Islands

The genetic structure of Apis mellifera populations from the Canary Islands has been assessed by mitochondrial (restriction fragment length polymorphisms of the intergenic transfer RNAleu-COII region) and nuclear (microsatellites) studies. These populations show a low level of genetic variation in terms of average number of alleles and degree of heterozygosity. Significant differences in the distribution of alleles were found in both data sets, confirming the genetic differentiation among some of the islands but not within them. Two mitochondrial haplotypes characteristic of the Canary Islands are found at high frequencies, although populations are introgressed by imported honeybees of eastern European C lineage. This introgression is rather high on Tenerife and El Hierro and low on Gran Canaria and La Gomera, whereas on La Palma it has not been recorded. The finding of microsatellite alleles characteristic of the eastern European lineage corroborates the genetic introgression. Phylogenetic analyses indicate that the Canarian honeybees are differentiated from other lineages and provide genetic evidence of their African origin.     

Introgression of mitochondrial DNA among lineages in a hybridogenetic ant

We report a remarkable pattern of incongruence between nuclear and mitochondrial variations in a social insect, the desert ant Cataglyphis hispanica. This species reproduces by social hybridogenesis. In all populations, two distinct genetic lineages coexist; non-reproductive workers develop from hybrid crosses between the lineages, whereas reproductive offspring (males and new queens) are typically produced asexually by parthenogenesis. Genetic analyses based on nuclear markers revealed that the two lineages remain highly differentiated despite constant hybridization for worker production. Here, we show that, in contrast with nuclear DNA, mitochondrial DNA (mtDNA) does not recover the two lineages as monophyletic. Rather, mitochondrial haplotypes cluster according to their geographical origin. We argue that this cytonuclear incongruence stems from introgression of mtDNA among lineages, and review the mechanisms likely to explain this pattern under social hybridogenesis.     

The rise and fall of the mountain hare (Lepus timidus) during Pleistocene glaciations: expansion and retreat with hybridization in the Iberian Peninsula

The climatic fluctuations during glaciations have affected differently arctic and temperate species. In the northern hemisphere, cooling periods induced the expansion of many arctic species to the south, while temperate species were forced to retract in southern refugia. Consequently, in some areas the alternation of these species set the conditions for competition and eventually hybridization. Hares in the Iberian Peninsula appear to illustrate this phenomenon. Populations of Iberian hare (Lepus granatensis), brown hare (Lepus europaeus) and broom hare (Lepus castroviejoi) in Northern Iberia harbour mitochondrial haplotypes from the mountain hare (Lepus timidus), a mainly boreal and arctic species presently absent from the peninsula. To understand the history of this past introgression we analysed sequence variation and geographical distribution of mitochondrial control region and cytochrome b haplotypes of L. timidus origin found in 378 specimens of these four species. Among 124 L. timidus from the Northern Palaearctic and the Alps we found substantial nucleotide diversity (2.3%) but little differentiation between populations. Based on the mismatch distribution of the L. timidus sequences, this could result from an expansion at a time of temperature decrease favourable to this arctic species. The nucleotide diversity of L. timidus mtDNA found in Iberian L. granatensis, L. europaeus and L. castroviejoi (183, 70 and 1 specimens, respectively) was of the same order as that in L. timidus over its range (1.9%), suggesting repeated introgression of multiple lineages. The structure of the coalescent of L. granatensis sequences indicates that hybridization with L. timidus was followed by expansion of the introgressed haplotypes, as expected during a replacement with competition, and occurred when temperatures started to rise, favouring the temperate species. Whether a similar scenario explains the introgression into Iberian L. europaeus remains unclear but it is possible that it hybridized with already introgressed L. granatensis.     

Postglacial species displacement in Triturus newts deduced from asymmetrically introgressed mitochondrial DNA and ecological niche models

ABSTRACT: BACKGROUND: If the geographical displacement of one species by another is accompanied by hybridization, mitochondrial DNA can introgress asymmetrically, from the outcompeted species into the invading species, over a large area. We explore this phenomenon using the two parapatric crested newt species, Triturus macedonicus and T. karelinii, distributed on the Balkan Peninsula in south-eastern Europe, as a model. RESULTS: We first delimit a ca. 54,000 km2 area in which T. macedonicus contains T. karelinii mitochondrial DNA. This introgression zone bisects the range of T. karelinii, cutting off a T. karelinii enclave. The high similarity of introgressed mitochondrial DNA haplotypes with those found in T. karelinii suggests a recent transfer across the species boundary. We then use ecological niche modeling to explore habitat suitability of the location of the present day introgression zone under current, mid-Holocene and Last Glacial Maximum conditions. This area was inhospitable during the Last Glacial Maximum for both species, but would have been habitable at the mid-Holocene. Since the mid-Holocene, habitat suitability generally increased for T. macedonicus, whereas it decreased for T. karelinii. CONCLUSION: The presence of a T. karelinii enclave suggests that T. karelinii was the first to colonize the area where the present day introgression zone is positioned after the Last Glacial Maximum. Subsequently, we propose T. karelinii was outcompeted by T. macedonicus, which captured T. karelinii mitochondrial DNA via introgressive hybridization in the process. Ecological niche modeling suggests that this replacement was likely facilitated by a shift in climate since the mid-Holocene. We suggest that the northwestern part of the current introgression zone was probably never inhabited by T. karelinii itself, and that T. karelinii mitochondrial DNA spread there through T. macedonicus exclusively. Considering the spatial distribution of the introgressed mitochondrial DNA and the signal derived from ecological niche modeling, we do not favor the hypothesis that foreign mitochondrial DNA was pulled into the T. macedonicus range by natural selection.