The strength of reproductive isolation in two hybridizing food-deceptive orchid species

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papilionacea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid individuals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F1 hybrids, and that no introgression occurred between parental species. The maternally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification.     

Evidence for strong assortative mating,limited gene flow,and strong differentiation across the blue‐footed/Peruvian booby hybrid zone in northern Peru

Hybrid zones represent natural laboratories in which the processes of divergence and genetic isolation can be examined. The generation and maintenance of a hybrid zone requires mispairing and successful reproduction between organisms that differ in one or more heritable traits. Understanding the dynamics of hybridization between two species requires an understanding of the extent to which they have diverged genetically, the frequency of misparing and hybrid production, and the extent of introgression. Three hundred and twenty one blue‐footed Sula nebouxii and Peruvian S. variegata boobies from the eastern tropical Pacific Ocean were analyzed using 19 putatively neutral genetic markers to evaluate interspecific differentiation, to classify morphological hybrids using Bayesian assignments, and to characterize hybridization using cline theory and Bayesian assignments. The species were well differentiated at mitochondrial and nuclear microsatellites, the hybrid zone was bimodal (contained a high frequency of each parental species but a low frequency of hybrids), and morphologically intermediate individuals were most likely F1 hybrids resulting from mating between female Peruvian boobies and male blue‐footed boobies. Clines in allele frequency could be constrained to share a common geographic centre but could not be constrained to share a common width. Peruvian and blue‐footed boobies hybridize infrequently, potentially due to strong premating reproductive isolation; however, backcrossing appears to facilitate introgression from blue‐footed to Peruvian boobies in this hybrid system.     

Characterization of a contemporaneous hybrid zone between two darter species (Etheostoma bison and E. caeruleum) in the Buffalo River System

Hybrid zones have long intrigued evolutionary biologists and provide a natural laboratory to explore the evolution of reproductive isolation (speciation). Molecular characterization of hybrid zone dynamics can provide insight into the strength of reproductive isolation as well as the underlying evolutionary processes shaping gene flow. Approximately one-third of darter species naturally hybridize making this species-rich North American freshwater teleost fish clade an ideal system to investigate the extent and direction of hybridization. The objective of this study was to use diagnostic microsatellite markers to calculate genetic hybrid index scores of two syntopic, but distantly related darter species, Etheostoma bison and Etheostoma caeruleum. A combination of hybrid index scores, assignment tests, and mitochondrial haplotype profiles uncovered mixed ancestry in approximately 6 % of sampled adult individuals, supporting contemporaneous hybridization that was previously undocumented in E. bison. Moreover, hybrids were not limited to the F₁ generation, but encompassed the entire suite of hybrid categories (F₁, F₂ and backcross hybrids). The low number of hybrids assigned to each hybrid category represents a bimodal hybrid zone, suggesting reproductive isolation is strong (but incomplete) and also advocates for the ability of hybrids to produce second-generation hybrids and backcross into both parental species, mediating introgression across species boundaries. To this end, cytonuclear profiles of the sampled parental species and hybrids were consistent with bidirectional gene flow, although there was an overall trend of asymmetric hybridization between E. caeruleum females and E. bison males. The spatiotemporal variation in hybridization rates and resulting cytonuclear patterns expanded on in this study provide a comparative genetic framework on which future studies can begin to elucidate the underlying processes that not only generate a mosaic hybrid zone, but maintain the distinctness of species in the face of gene flow.     

Hybrid male sterility between the fresh- and brackish-water types of ninespine stickleback Pungitius pungitius (Pisces, Gasterosteidae)

Two ecologically distinct forms, fresh- and brackish-water types, of ninespine stickleback co-exist in several freshwater systems on the coast of eastern Hokkaido. Recent genetic analyses of 13 allozyme loci revealed genetic separation between the two types even though their spawning grounds were in close proximity. On the other hand, there is only a small difference in mitochondrial DNA (mtDNA) sequence between the two types suggesting that they diverged quite recently or that mtDNA introgression occurred between them. To test for postzygotic reproductive isolating mechanisms and hybrid mediated gene flow, we examined the viability and reproductive performance of reciprocal F1 hybrids. The hybrids grew to the adult size normally and both sexes expressed secondary sexual characters in the reciprocal crosses. The female hybrids were reciprocally fertile, while the male hybrids were reciprocally sterile. Histological and flow-cytometric analyses of the hybrid testis revealed that the sterility pattern was classified as 'gametic sterility,' with gonads of normal size but abnormal spermatogenesis. To our knowledge, the present finding is a novel example of one sex hybrid sterility in the stickleback family (Gasterosteidae).     

Contrasting genetic structures across two hybrid zones of a tropical reef fish, Acanthochromis polyacanthus (Bleeker 1855)

Hybrid zones are natural laboratories offering insights into speciation processes. Narrow hybrid zones are less common in the sea than on land consistent with higher dispersal among marine populations. Acanthochromis polyacanthus is an unusual bony marine fish with philopatric dispersal that exists as allopatric stocks of white, bicoloured and black fish on the Great Barrier Reef (GBR). At two latitudes, different morphs coexist and hybridize at narrow contact zones. Sequence data from mitochondrial Hypervariable Region 1 revealed contrasting patterns of introgression across these zones. At the northern hybrid zone, a single clade of mitochondrial haplotypes was found in all white fish, hybrids and tens of kilometres into pure bicoloured stock. At the southern hybrid zone, there was no introgression of mitochondrial genes into black fish and hybrids shared the bicoloured haplotypes. Based on this asymmetry, we postulate that black fish from the southern GBR have experienced a selective sweep of their mitochondrial genome, which has resulted in almost total reproductive isolation.     

Weak premating isolation between Clitarchus stick insect species despite divergent male and female genital morphology

Documenting natural hybrid systems builds our understanding of mate choice, reproductive isolation and speciation. The stick insect species Clitarchus hookeri and C. tepaki differ in their genital morphology and hybridize along a narrow peninsula in northern New Zealand. We utilize three lines of evidence to understand the role of premating isolation and species boundaries: (a) genetic differentiation using microsatellites and mitochondrial DNA; (b) variation in 3D surface topology of male claspers and 2D morphometrics of female opercular organs; and (c) behavioural reproductive isolation among parental and hybrid populations through mating crosses. The genetic data show introgression between the parental species and formation of a genetically variable hybrid swarm. Similarly, the male and female morphometric data show genital divergence between the parental species as well as increased variation within the hybrid populations. This genital divergence has not resulted in reproductive isolation between species, instead weak perimating isolation has enabled the formation of a hybrid swarm. Behavioural analysis demonstrates that the entire mating process influences the degree of reproductive isolation between species undergoing secondary contact. Mechanical isolation may appear strong, whereas perimating isolation is weak.     

Incomplete reproductive isolation between Rhododendron taxa enables hybrid formation and persistence

The evolutionary consequences of hybridization ultimately depend on the magnitude of reproductive isolation between hybrids and their parents. We evaluated the relative contributions of pre-and post-zygotic barriers to reproduction for hybrid formation, hybrid persistence and potential for reproductive isolation of hybrids formed between two Rhododendron species,R. spiciferum and R. spinuliferum. Our study established that incomplete reproductive isolation promotes hybrid formation and persistence and delays hybrid speciation.All pre-zygotic barriers to reproduction leading to hybrid formation are incomplete: parental species have overlapping flowering; they share the same pollinators;reciprocal assessments of pollen tube germination and growth do not differ among parents. The absence of post-zygotic barriers between parental taxa indicates that the persistence of hybrids is likely. Reproductive isolation was incomplete between hybrids and parents in all cases studied, although asymmetric differences in reproductive fitness were prevalent and possibly explain the genetic structure of natural hybrid swarms where hybridization is known to be bidirectional but asymmetric. Introgression, rather than speciation, is a probable evolutionary outcome of hybridization between the two Rhododendron taxa. Our study provides insights into understanding the evolutionary implications of natural hybridization in woody plants.     

Heterogeneity and concordance in locus‐specific differentiation and introgression between species of towhees

The maintenance or breakdown of reproductive isolation is an observable outcome of secondary contact between species. In cases where hybrids beyond the F1 are formed, the representation of each species' ancestry can vary dramatically among genomic regions. This genomic heterogeneity in ancestry and introgression can offer insight into evolutionary processes, particularly if introgression is compared in multiple hybrid zones. Similarly, considerable heterogeneity exists across the genome in the extent to which populations and species have diverged, reflecting the combined effects of different evolutionary processes on genetic variation. We studied hybridization across two hybrid zones of two phenotypically well‐differentiated bird species in Mexico (Pipilo maculatus and P. ocai), to investigate genomic heterogeneity in differentiation and introgression. Using genotyping‐by‐sequencing (GBS) and hierarchical Bayesian models, we genotyped 460 birds at over 41 000 single nucleotide polymorphism (SNP) loci. We identified loci exhibiting extreme introgression relative to the genome‐wide expectation using a Bayesian genomic cline model. We also estimated locus‐specific F_ST and identified loci with exceptionally high genetic divergence between the parental species. We found some concordance of locus‐specific introgression in the two independent hybrid zones (6–20% of extreme loci shared across zones), reflecting areas of the genome that experience similar gene flow when the species interact. Additionally, heterogeneity in introgression and divergence across the genome revealed another subset of loci under the influence of locally specific factors. These results are consistent with a history in which reproductive isolation has been influenced by a common set of loci in both hybrid zones, but where local environmental and stochastic factors also lead to genomic differentiation.     

Morphological distinctiveness of Ligularia tongolensis and L. cymbulifera is maintained between habitats despite bidirectional and asymmetrical introgression in multiple hybrid zones

Natural hybridization is a crucial evolutionary process and a long-standing topic of study in evolutionary biology. Hybrid zones, where two congeneric species interact, can provide insight into the process of natural hybridization, especially with respect to how taxon diversity is maintained. In this study, we used double digest restriction-site associated DNA sequencing technology (ddRAD-seq) to examine genetic structure and estimate introgression in four hybrid zones of Ligularia tongolensis and Ligularia cymbulifera. Our analysis demonstrated that parental species were highly differentiated, whereas pairwise F_ST between parents and their hybrids was low, indicating that sympatric sites can form hybrid zones. As most F₁ hybrid individuals were observed within these zones, our finding also implied the presence of substantial barriers to interbreeding. Furthermore, some individuals that possessed the typical morphology of the parental species belonged to the F₁ generation. Genomic clines analysis revealed that a large fraction of single nucleotide polymorphisms (SNPs) deviated from a model of neutral introgression in the four hybrid zones, and most SNPs exhibited selection favoring the L. cymbulifera genotype. Bidirectional but asymmetric introgression was revealed as evident in the four hybrid zones. Habitat differences between the four hybrid zones may affect isolation barriers between both species. Taken together, these findings suggest that where incomplete reproductive barriers allow natural hybridization, the introgression between species generates rich genetic recombination that contributes to the fast adaptation and diversification of the widespread Ligularia in the Hengduan Mountains Region (HMR).     

Variation in contact zone dynamics between two species of topminnows, Fundulus notatus and F. olivaceus, across isolated drainage systems

Spatially variable selection pressure within heterogeneous environments can result in the evolution of specialist phenotypes that facilitate co-occurrence of closely related species and limit genetic exchange. If divergent selection pressures maintain reproductive isolation, hybridization is expected to correlate with the strength of underlying ecological gradients and the traits shaped by adaptive processes. We sampled ten replicate topminnow (Fundulus olivaceus and Fundulus notatus) hybrid zones in isolated drainages throughout central and southern North America. In all drainages, species were distributed in an upstream–downstream manner with contact zones localized at confluences featuring abrupt shifts from tributary to river habitat. In two drainages, the typical up and downstream positions of species were reversed. Phenotype differences between the species reflect predicted selection differences along stream gradients. Downstream populations (lower food availability and greater predator pressure) generally showed larger investment in reproduction (higher gonadal somatic index), smaller body size and lower somatic condition compared to upstream populations. Phenotypic differences between the species in the two reversed drainages were consistent with convergence of life history traits in the respective habitats. Phenotypes of individuals of hybrid origin (F₁ hybrids or backcrosses) were not significantly different from the average of the two parental forms, though there were trends towards reduced fitness. The prevalence of hybridization among drainages ranged from no hybrids in two drainages to near random mating. The strongest correlates of hybridization rate among replicate hybrid zones were similarity in body shape and the homogeneity of habitat through tributary-river confluences. The two reversed orientation hybrid zones also exhibited high prevalence of hybrids suggesting that phenotypic convergence could lead to increased hybridization.     

Genomic Characterization of Interspecific Hybrids between the Scallops Argopecten purpuratus and A. irradians irradians

The Peruvian scallop (Argopecten purpuratus) has been introduced to China and has successfully been hybridized with the bay scallop (A. irradians irradians). The F1 hybrids of these two scallops exhibited a large increase in production traits and some other interesting new characteristics. To understand the genetic basis of this heterosis, nuclear gene and partial mtDNA sequences, and genomic in situ hybridization (GISH) were employed to analyze the genomic organization of the hybrids. Amplification of the ribosomal DNA internal transcribed spacer (ITS) showed that the parental ITS sequences were present in all the hybrid individuals, illustrating that the hybrid offspring inherited nuclear DNA from both parents. Sequence analyses of the ITS region further confirmed that the hybrids harbored alleles from their parents; some recombinant variants were also detected, which revealed some alterations in the nuclear genetic material of the hybrids. The analysis of mitochondrial 16S rDNA showed that the hybrids possessed sequences that were identical to the 16S rDNA of the female parents, proving a matrilineal inheritance of mitochondrial genes in scallops. In addition, GISH clearly discriminated between the parental chromosomes and indicated a combination of haploid genomes of duplex parents in the hybrids. The genetic analyses in our study illustrated that the F1 hybrids inherited nuclear material from both parents and cytoplasmic genetic material maternally, and some variations occurred in the genome, which might contribute to a further understanding of crossbreeding and heterosis in scallop species.     

Unexpected ancestry of Populus seedlings from a hybrid zone implies a large role for postzygotic selection in the maintenance of species

In the context of potential interspecific gene flow, the integrity of species will be maintained by reproductive barriers that reduce genetic exchange, including traits associated with prezygotic isolation or poor performance of hybrids. Hybrid zones can be used to study the importance of different reproductive barriers, particularly when both parental species and hybrids occur in close spatial proximity. We investigated the importance of barriers to gene flow that act early vs. late in the life cycle of European Populus by quantifying the prevalence of homospecific and hybrid matings within a mosaic hybrid zone. We obtained genotypic data for 11 976 loci from progeny and their maternal parents and constructed a Bayesian model to estimate individual admixture proportions and hybrid classes for sampled trees and for the unsampled pollen parent. Matings that included one or two hybrid parents were common, resulting in admixture proportions of progeny that spanned the whole range of potential ancestries between the two parental species. This result contrasts strongly with the distribution of admixture proportions in adult trees, where intermediate hybrids and each of the parental species are separated into three discrete ancestry clusters. The existence of the full range of hybrids in seedlings is consistent with weak reproductive isolation early in the life cycle of Populus. Instead, a considerable amount of selection must take place between the seedling stage and maturity to remove many hybrid seedlings. Our results highlight that high hybridization rates and appreciable hybrid fitness do not necessarily conflict with the maintenance of species integrity.     

Long Distance Linkage Disequilibrium and Limited Hybridization Suggest Cryptic Speciation in Atlantic Cod

Hybrid zones provide unprecedented opportunity for the study of the evolution of reproductive isolation, and the extent of hybridization across individuals and genomes can illuminate the degree of isolation. We examine patterns of interchromosomal linkage disequilibrium (ILD) and the presence of hybridization in Atlantic cod, Gadus morhua, in previously identified hybrid zones in the North Atlantic. Here, previously identified clinal loci were mapped to the cod genome with most (∼70%) occurring in or associated with (<5 kb) coding regions representing a diverse array of possible functions and pathways. Despite the observation that clinal loci were distributed across three linkage groups, elevated ILD was observed among all groups of clinal loci and strongest in comparisons involving a region of low recombination along linkage group 7. Evidence of ILD supports a hypothesis of divergence hitchhiking transitioning to genome hitchhiking consistent with reproductive isolation. This hypothesis is supported by Bayesian characterization of hybrid classes present and we find evidence of common F1 hybrids in several regions consistent with frequent interbreeding, yet little evidence of F2 or backcrossed individuals. This work suggests that significant barriers to hybridization and introgression exist among these co-occurring groups of cod either through strong selection against hybrid individuals, or genetic incompatibility and intrinsic barriers to hybridization. In either case, the presence of strong clinal trends, and little gene flow despite extensive hybridization supports a hypothesis of reproductive isolation and cryptic speciation in Atlantic cod. Further work is required to test the degree and nature of reproductive isolation in this species.     

Reproductive Isolation of Hybrid Populations Driven by Genetic Incompatibilities

Despite its role in homogenizing populations, hybridization has also been proposed as a means to generate new species. The conceptual basis for this idea is that hybridization can result in novel phenotypes through recombination between the parental genomes, allowing a hybrid population to occupy ecological niches unavailable to parental species. Here we present an alternative model of the evolution of reproductive isolation in hybrid populations that occurs as a simple consequence of selection against genetic incompatibilities. Unlike previous models of hybrid speciation, our model does not incorporate inbreeding, or assume that hybrids have an ecological or reproductive fitness advantage relative to parental populations. We show that reproductive isolation between hybrids and parental species can evolve frequently and rapidly under this model, even in the presence of substantial ongoing immigration from parental species and strong selection against hybrids. An interesting prediction of our model is that replicate hybrid populations formed from the same pair of parental species can evolve reproductive isolation from each other. This non-adaptive process can therefore generate patterns of species diversity and relatedness that resemble an adaptive radiation. Intriguingly, several known hybrid species exhibit patterns of reproductive isolation consistent with the predictions of our model.     

Asymmetrical crossing barriers in angiosperms

Patterns of reproductive isolation between species may provide insight into the mechanisms and evolution of barriers to interspecific gene exchange. We used data from published interspecific hybridization experiments from 14 genera of angiosperms in order to test for the presence of asymmetrical barriers to gene exchange. Reproductive isolation was examined at three life-history stages: the ability of interspecific crosses to produce seeds, the viability of F1 hybrids, and the fertility of F1 hybrids. Statistically significant asymmetries in the strength of reproductive isolation between species were detected in all genera and at each of the three life-history stages. Asymmetries in seed production may be caused by a variety of mechanisms including differences in stigma/style lengths, self compatibility and differential fruit abortion. Asymmetries in post-zygotic isolation are probably caused by nuclear-cytoplasmic interactions. Asymmetrical reproductive isolation between plant taxa may have important implications for the dynamics of hybrid zones, the direction of genetic introgression and the probability of reinforcement.     

Reproductive isolation due to the genetic incompatibilities between Thrichomys pachyurus and two subspecies of Thrichomys apereoides (Rodentia, Echimyidae).

We tested intrinsic reproductive isolation between 3 taxa of the South American caviomorph rodent Thrichomys (Rodentia, Echimyidae): T. pachyurus, T. apereoides subsp. apereoides and T. apereoides subsp. laurentius. They were mated in captivity and produced viable progeny. Some F1 hybrid females were fertile, whereas all F1 males were sterile. Histological examination revealed meiotic arrest at the primary spermatocyte stage. No sperm was detected in testes or epididymes. Electron microscopic analysis of surface spread synaptonemal complexes revealed a complete failure of chromosome pairing in F1 hybrids of T. pachyurus with T. apereoides subsp. laurentius and T. apereoides subsp. apereoides. In the male hybrids between T. apereoides subsp. apereoides and T. apereoides subsp. laurentius, meiosis did not proceed beyond diplotene, although all of the chromosomes, including heteromorphic ones, paired in an orderly fashion. Backcross males with homomorphic karyotypes showed segregation in meiosis progression. This indicates that male hybrid sterility is due to genetic, but not chromosomal, incompatibility of the parental taxa.     

Hybridization dynamics between sympatric species of trout: loss of reproductive isolation

Although reinforcement should enhance reproductive barriers in sympatric species, sympatric trout species do hybridize. Using mitochondrial and nuclear species markers, we investigated hybridization directionality, hybrid mating biases, and selection against hybrids in 13 sympatric cut-throat and rainbow trout populations on Vancouver Island, Canada. Approximately 50% of the genotyped fish were hybrid (F1 or higher-order) and populations ranged from very recent (all F1 hybrids) to extremely advanced higher-order hybridization. Overall, interbreeding was reciprocal, although some populations showed directional hybridization. Pronounced cytonuclear disequilibrium in post-F1 hybrids indicated a remarkable mating bias not previously reported, which is most likely because of behavioural reproductive preferences. Selection against hybrids was observed in only two populations, indicative of extrinsic selection. Two populations were 'hybrid swarms', with a complete loss of reproductive isolation. The complex hybridization dynamics in this system represent a valuable natural experiment of the genetic and evolutionary implications of recent and on-going interspecific hybridization.     

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post‐mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybrid speciation to occur. Here, we investigate the potential of behavioural mate choice to generate assortative mating among hybrids and parental species. We made three‐first‐generation hybrid crosses between different species of African cichlid fish. In three‐way mate‐choice experiments, we allowed hybrid and nonhybrid females to mate with either hybrid or nonhybrid males. We found that hybrids generally mated nonrandomly and that hybridization can lead to the expression of new combinations of traits and preferences that behaviourally isolate hybrids from both parental species. Specifically, we find that the phenotypic distinctiveness of hybrids predicts the symmetry and extent of their reproductive isolation. Our data suggest that behavioural mate choice among hybrids may facilitate the establishment of isolated hybrid populations, even in proximity to one or both parental species.     

Environment-specific heterozygote deficiency and developmental instability in hybrid Mytilus

The multiple discrete hybrid zones that characterize Mytilus blue mussels allow a novel, non-manipulative, examination of the selective pressures that create and maintain species. If endogenous genetic incompatibility is solely responsible for post-zygotic isolation, then individuals of a specified hybrid genotype are expected to show similar average fitness across environments. However, if hybrid fitness differs across environments, then exogenous selection is implicated, either via ecological selection or environment-specific expression of intrinsic genetic incompatibilities. Correspondence between developmental instability of hybrids and heterozygote deficiency, estimated in two M. trossulus×M. galloprovincialis hybrid zones on the coast of North America, indicates that environment-dependent selection against hybrids may contribute to reproductive isolation among Pacific Mytilus species.     

Geographic variation in the structure of oak hybrid zones provides insights into the dynamics of speciation

Studying geographic variation in the rate of hybridization between closely related species could provide a useful window on the evolution of reproductive isolation. Reinforcement theory predicts greater prezygotic isolation in areas of prolonged contact between recently diverged species than in areas of recent contact, which implies that old contact zones would be dominated by parental phenotypes with few hybrids (bimodal hybrid zones), whereas recent contact zones would be characterized by hybrid swarms (unimodal hybrid zones). Here, we investigate how the hybrid zones of two closely related Chinese oaks, Quercus mongolica and Q. liaotungensis, are structured geographically using both nuclear and chloroplast markers. We found that populations of Q. liaotungensis located around the Changbai Mountains in Northeast China, an inferred glacial refugium, were introgressed by genes from Q. mongolica, suggesting historical contact between the two species in this region. However, these introgressed populations form sharp bimodal hybrid zones with Q. mongolica. In contrast, populations of Q. liaotungensis located in North China, which show no sign of ancient introgression with Q. mongolica, form unimodal hybrid zones with Q. mongolica. These results are consistent with the hypothesis that selection against hybrids has had sufficient time to reinforce the reproductive barriers between Q. liaotungensis and Q. mongolica in Northeast China but not in North China.