Evaluating the role of ecological isolation in maintaining the species boundary between <Emphasis Type="Italic">Silene dioica</Emphasis> and <Emphasis Type="Italic">S. latifolia</Emphasis>

The relative importance of floral versus ecological isolation in preventing introgression remains unclear. This study examines whether ecological isolation can explain the continuing integrity of Silene dioica and S. latifolia where floral isolation is weak and hybrids are fully viable. Eighteen small replicate founder populations of 6 individuals (3 males and 3 females) of either S. latifolia, S. dioica or hybrids were created in woodland and in open sites in southern UK. Survival, reproduction and introgression of these populations were examined over 9 years. S. latifolia and hybrid plants suffered higher mortality than S. dioica in woodland. In open sites, there was extensive introgression, with few or no pure S. latifolia or S. dioica surviving by the end of the experiment. The experiment suggests that the integrity of S. dioica is maintained by its ability to survive in shaded habitats where S. latifolia and hybrids cannot persist. However, how S. latifolia survives as a distinct species in the study area remains a puzzle. Immigration from regions where S. latifolia occurs in isolation (i.e. large-scale ecological isolation) may balance introgression in the study area.     

Searching for the genetic footprint of ancient and recent hybridization

Determining the long‐term consequences of hybridization remains a central quest for evolutionary biologists. A particular challenge is to establish whether and to what extent widespread hybridization results in gene flow (introgression) between parental taxa. In this issue of Molecular Ecology, Jordan et al. ( 2018 ) search for evidence of gene flow between two closely related species of Geum (Rosaceae), which hybridize readily in contemporary populations and where hybrid swarms have been recorded for at least 200 years (Ruhsam, Hollingsworth, & Ennos, 2013 ). The authors find mixed evidence of ancient introgression when analysing allopatric populations. Intriguingly, when analysing populations of a region where the two species occur either mixed in the same population or in close proximity, and where hybrids are presently common, Jordan and colleagues find that the majority of randomly sampled individuals analysed (92/96) show no evidence of introgression (defined as individuals with admixture coefficients of <1%). The few individuals identified as hybrids are shown to likely be F1 or early‐generation backcrosses, indicating that even in sympatric regions, hybridization does not penetrate beyond a few generations. Based on their findings, Geum seems to be an example of little to no introgression despite contemporary hybridization.     

Persistence of sunflower crop traits and fitness in Helianthus petiolaris populations

Transgenic plants have increased interest in the study of crop gene introgression in wild populations. Genes (or transgenes) conferring adaptive advantages persist in introgressed populations, enhancing competitiveness of wild or weedy plants. This represents an ecological risk that could increase problems of weed control. Introgression of cultivar alleles into wild plant populations via crop–wild hybridisations is primarily governed by their fitness effect. To evaluate this, we studied the second generation of seven wild–crop interspecific hybrids between weedy Helianthus petiolaris and cultivated sunflower, H. annuus var. macrocarpus. The second generation comprised open‐pollinated progeny and backcrosses to the wild parent, mimicking crosses that occur in natural situations. We compared a number of morphological, life history and fitness traits. Multivariate analysis showed that the parental species H. annuus and H. petiolaris differed in a number of morphological traits, while the second hybrid generation between them was intermediate. Sunflower crop introgression lowered fitness of interspecific hybrids, but fitness parameters tended to recover in the following generation. Relative frequency of wild/weedy and introgressed plants was estimated through four generations, based on male and female parent fitness. In spite of several negative selection coefficients observed in the second generation, introgressed plants could be detected in stands of <100 weedy H. petiolaris populations. The rapid recovery of fecundity parameters leads to prediction that any trait conferring an ecological advantage will diffuse into the wild or weedy population, even if F1 hybrids have low fitness.     

Genetic Evidence of Hybridization between the Endangered Native Species Iguana delicatissima and the Invasive Iguana iguana (Reptilia,Iguanidae) in the Lesser Antilles: Management Implications

The worldwide increase of hybridization in different groups is thought to have become more important with the loss of isolating barriers and the introduction of invasive species. This phenomenon could result in the extinction of endemic species. This study aims at investigating the hybridization dynamics between the endemic and threatened Lesser Antillean iguana (Iguana delicatissima) and the invasive common green iguana (Iguana iguana) in the Lesser Antilles, as well as assessing the impact of interspecific hybridization on the decline of I. delicatissima. 59 I. delicatissima (5 localities), 47 I. iguana (12 localities) and 27 hybrids (5 localities), who were all identified based on morphological characters, have been genotyped at 15 microsatellites markers. We also sequenced hybrids using ND4 mitochondrial loci to further investigate mitochondrial introgression. The genetic clustering of species and hybrid genetic assignment were performed using a comparative approach, through the implementation of a Discriminant Analysis of Principal Component (DAPC) based on statistics, as well as genetic clustering approaches based on the genetic models of several populations (Structure, NewHybrids and HIest), in order to get full characterization of hybridization patterns and introgression dynamics across the islands. The iguanas identified as hybrids in the wild, thanks to morphological analysis, were all genetically F1, F2, or backcrosses. A high proportion of individuals were also the result of a longer-term admixture. The absence of reproductive barriers between species leads to hybridization when species are in contact. Yet morphological and behavioral differences between species could explain why males I. iguana may dominate I. delicatissima, thus resulting in short-term species displacement and extinction by hybridization and recurrent introgression from I. iguana toward I. delicatissima. As a consequence, I. delicatissima gets eliminated through introgression, as observed in recent population history over several islands. These results have profound implications for species management of the endangered I. delicatissima and practical conservation recommendations are being discussed in the light of these findings.     

Breakdown of species boundaries in Mandevilla: floral morphological intermediacy,novel fragrances and asymmetric pollen flow

• Phenotypic intermediacy is an indicator of putative hybrid origin and has provided the main clues to discovering hybrid plants in nature. Mandevilla pentlandiana and M. laxa (Apocynaceae) are sister species with clear differences in floral phenotype and associated pollinator guilds: diurnal Hymenoptera and nocturnal hawkmoths, respectively. The presence of individuals with intermediate phenotypes in a wild population raises questions about the roles of visual and olfactory signals (i.e. corolla morphology and floral fragrances) as barriers to interbreeding, and how the breakdown of floral isolation occurs.
• We examined phenotypic variation in a mixed Mandevilla population, analysing the chemical composition of floral fragrances, characterising floral shape through geometric morphometrics and assessing individual grouping through taxonomically relevant traits and an unsupervised learning algorithm. We quantified the visitation frequencies of floral visitors and tracked their foraging movements using pollen analogues.
• The presence of morphologically intermediate individuals and pollen analogue movement suggested extensive hybridisation between M. laxa and M. pentlandiana, along with asymmetrical rates of backcrossing between these putative hybrids and M. laxa. Floral volatiles from putative hybrid individuals showed a transgressive phenotype, with additional compounds not emitted by either parental species.
• Our results suggest the presence of a hybrid swarm between sympatric M. pentlandiana and M. laxa and indicate that initial hybridisation events between these parental species are rare, but once they occur, visits between putative hybrids and M. laxa are common and facilitate continued introgression.

     

Bayesian estimation of genomic clines

We developed a Bayesian genomic cline model to study the genetic architecture of adaptive divergence and reproductive isolation between hybridizing lineages. This model quantifies locus‐specific patterns of introgression with two cline parameters that describe the probability of locus‐specific ancestry as a function of genome‐wide admixture. ‘Outlier’ loci with extreme patterns of introgression relative to most of the genome can be identified. These loci are potentially associated with adaptive divergence or reproductive isolation. We simulated genetic data for admixed populations that included neutral introgression, as well as loci that were subject to directional, epistatic or underdominant selection, and analysed these data using the Bayesian genomic cline model. Under many demographic conditions, underdominance or directional selection had detectable and predictable effects on cline parameters, and ‘outlier’ loci were greatly enriched for genetic regions affected by selection. We also analysed previously published genetic data from two transects through a hybrid zone between Mus domesticus and M. musculus. We found considerable variation in rates of introgression across the genome and particularly low rates of introgression for two X‐linked markers. There were similarities and differences in patterns of introgression between the two transects, which likely reflects a combination of stochastic variability because of genetic drift and geographic variation in the genetic architecture of reproductive isolation. By providing a robust framework to quantify and compare patterns of introgression among genetic regions and populations, the Bayesian genomic cline model will advance our understanding of the genetics of reproductive isolation and the speciation process.     

Detecting introgressive hybridisation in rock partridge populations (<Emphasis Type="Italic">Alectoris graeca</Emphasis>) in Greece through Bayesian admixture analyses of multilocus genotypes

The nominal subspecies of rock partridge (Alectoris graeca graeca) is widely distributed in Greece, where populations are declining due to over-hunting and habitat changes. Captive-reared chukars (A. chukar) have been massively released throughout the country, raising fear that introgressive hybridisation might have disrupted local adaptations leading to further population declines. In this study we used mtDNA control-region sequences and Bayesian admixture analyses of multilocus genotypes determined at eight microsatellite loci, to assess the extent of introgressive hybridisation in 319 wild rock partridges collected in Greece. A neighbour-joining tree split the mtDNA haplotypes into three strongly supported clades, corresponding to rock, red-legged (A.␣rufa) and chukar partridges. We did not detect any case of maternal introgression. In contrast, admixture analyses of microsatellite genotypes identified from four to 28 putative hybrids (according to different assignment criteria), corresponding to 1.2–8.8% of the samples, which were widespread throughout all the country. Power and limits of admixture analyses were assessed using simulated hybrid genotypes, which revealed that a small number of markers can detect all first and second generation hybrids (F ₁ and F ₂), and up to 90% of the first generation backrossess. Thus, the true proportion of recently introgressed rock partridges in Greece might be ca. 20%. These findings indicate that introgressive hybridisation is widespread, suggesting that released captive-bred partridges have reproduced and hybridised in nature polluting the gene pool of wild rock partridge populations in Greece.     

Fine‐scale geographic patterns of gene flow and reproductive character displacement in Drosophila subquinaria and Drosophila recens

When two species are incompletely isolated, strengthening premating isolation barriers in response to the production of low fitness hybrids may complete the speciation process. Here, we use the sister species Drosophila subquinaria and Drosophila recens to study the conditions under which this reinforcement of species boundaries occurs in natural populations. We first extend the region of known sympatry between these species, and then we conduct a fine‐scale geographic survey of mate discrimination coupled with estimates of gene flow within and admixture between species. Within D. subquinaria, reinforcement is extremely effective: we find variation in mate discrimination both against D. recens males and against conspecific allopatric males on the scale of a few kilometres and in the face of gene flow both from conspecific populations and introgression from D. recens. In D. recens, we do not find evidence for increased mate discrimination in sympatry, even where D. recens is rare, consistent with substantial gene flow throughout the species’ range. Finally, we find that introgression between species is asymmetric, with more from D. recens into D. subquinaria than vice versa. Within each species, admixture is highest in the geographic region where it is rare relative to the other species, suggesting that when hybrids are produced they are of low fitness. In sum, reinforcement within D. subquinaria is effective at maintaining species boundaries, but even when reinforcing selection is strong it may not always result in a pattern of strong reproductive character displacement due to variation in the frequency of hybridization and gene flow from neighbouring populations.     

Detecting adaptive trait loci in nonmodel systems: divergence or admixture mapping?

Mapping adaptive trait loci (ATL) underlying ecological divergence is an essential step towards understanding the processes that generate phenotypic diversity. Technological advances have made it possible to sequence exomes in nonmodel systems, providing an efficient means of analysing functional genetic variants. Divergence scans of genetic markers for outlier loci, or ‘divergence mapping’, have been used to map locally adapted genes, but this approach is likely to be underpowered when background divergence is elevated. Genotype–phenotype association tests in admixed populations, or ‘admixture mapping’, may provide a useful approach for mapping locally adapted loci when neutral divergence is high. To determine the power and limits of divergence mapping, we simulated exomes containing a single ATL across two parental populations of varying neutral divergence, estimated divergence and quantified the power to identify the ATL. We found that divergence mapping had very high power when background F_ST is <0.2, but decreased dramatically above this level. To evaluate the utility of admixture mapping, we simulated exomes from admixed populations, then simulated phenotypes, conducted genotype–phenotype association tests and found that even two generations of random mating after admixture could provide high mapping power in scenarios where pure divergence mapping was ineffective (F_ST = 0.35). Moreover, admixture mapping had high power across all levels of divergence after 20 generations since admixture. Together with high‐throughput exome sequencing, admixture mapping could be used to map ATL in systems such as Heliconius butterflies or Gryllus crickets when experimental design and analytical approach are chosen accordingly.     

Evolution of a hybrid zone of two willow species (Salix L.) in the European Alps analyzed by RAD-seq and morphometrics

Natural hybridization of plants can result in many outcomes with several evolutionary consequences, such as hybrid speciation and introgression. Natural hybrid zones can arise in mountain systems as a result of fluctuating climate during the exchange of glacial and interglacial periods, where species retract and expand their territories, resulting in secondary contacts. Willows are a large genus of woody plants with an immense capability of interspecific crossing. In this study, the sympatric area of two diploid sister species, S. foetida and S. waldsteiniana in the eastern European Alps, was investigated to study the genomic structure of populations within and outside their contact zone and to analyze congruence of morphological phenotypes with genetic data. Eleven populations of the two species were sampled across the Alps and examined using phylogenetic network and population genetic structure analyses of RAD Seq data and morphometric analyses of leaves. The results showed that a homoploid hybrid zone between the two species was established within their sympatric area. Patterns of genetic admixture in homoploid hybrids indicated introgression with asymmetric backcrossing to not only one of the parental species but also one hybrid population forming a separate lineage. The lack of F1 hybrids indicated a long-term persistence of the hybrid populations. Insignificant isolation by distance suggests that gene flow can act over large geographical scales. Morphometric characteristics of hybrids supported the molecular data and clearly separated populations of the parental species, but showed intermediacy in the hybrid zone populations with a bias toward S. waldsteiniana. The homoploid hybrid zone might have been established via secondary contact hybridization, and its establishment was fostered by the low genetic divergence of parental species and a lack of strong intrinsic crossing barriers. Incomplete ecological separation and the ability of long-distance dispersal of willows could have contributed to the spatial expansion of the hybrid zone.     

Extensive hybridization and past introgression between divergent lineages in a quasi‐clonal hermaphroditic fish: Ramifications for species concepts and taxonomy

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self‐fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a ‘Central clade’ closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self‐fertilization for 1–3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.     

Comparative analysis of introgression at three marker classes: a case study in a stocked population of brown trout

Comparative analysis of protein loci, microsatellite and mtDNA markers revealed generally comparable estimates for introgression and apparent admixture rates in stocked brown trout populations at two sites in the River Doubs (Rhône dainage, Switzerland), which are 10 km apart and which belong to the same management unit. At one site, a significant deviation between mtDNA and nuclear markers could be explained by stocking of F1 hybrids originating from crosses between hatchery females and males from the local population. Substantial differences between diagnostic protein loci and protein loci having non-fixed private alleles indicated that caution must be exercised when using genetic markers not strictly diagnostic for the distinction of the populations under investigation. Congruent estimates of introgression and apparent admixture rates between diagnostic protein loci and presumed diagnostic microsatellite loci suggest that the latter can be regarded as reliable genetic markers for the estimation of introgression in Mediterranean brown trout populations stocked with trout of Atlantic origin. Significant differences in introgression and apparent admixture rates between the two sites and between age-classes of one study site were observed. Introgression is suggested to depend on environmental factors. Significantly lower introgression rates in age-class 2+ years as compared to juvenile trout might further indicate that introduced Atlantic brown trout and hybrids decrease in proportion between age-classes 1+ and 2+ years.     

An unusual barrier to gene flow: perpetually immature larvae from inter-population crosses in the flour beetle, Tribolium castaneum

We genetically characterize an unusual hybrid incompatibility phenotype manifest in F₁ offspring of crosses between two populations of Tribolium castaneum. Hybrid larvae cease development at the third larval instar, persisting as ‘perpetually immature larvae’ thereafter. Although unable to produce viable adult hybrid offspring with one another, each population produces abundant, fertile hybrids with other populations, indicating a recent origin of the incompatibility and facilitating genetic studies. We mapped the paternal component of the hybrid phenotype to a single region, which exhibits two characteristics common to hybrid incompatibility: marker transmission ratio distortion within crosses and elevated genetic divergence between populations. The incompatible variation and an elevation in between‐population genetic divergence is associated with a region containing the T. castaneum ecdysone receptor homologue, a major regulatory switch, controlling larval moults, pupation and metamorphosis. This contributes to understanding the genetics of speciation in the Coleoptera, one of the most speciose of all arthropod taxa.     

Estimation of introgression in cutthroat trout populations using microsatellites

Introgressive hybridization, mediated by anthropogenic activity, poses a threat to numerous and diverse taxa. The management of introgressed individuals or populations within species of conservation concern is currently the subject of scientific and political debate. We investigate the utility of 10 non-diagnostic microsatellite loci for investigating admixture from introduced Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss) within 25 putative Rio Grande cutthroat trout (O. c. virginalis) populations. We apply five different approaches (correspondence analysis, maximum-likelihood assignment tests, an admixture estimator based on allele frequencies, an admixture estimator based on coalescent theory and an admixture estimator implementing a Bayesian method) and use two alternative O. c. virginalis reference samples. All approaches were capable of identifying one population that consisted entirely of introduced O. c. bouvieri, and three out of five approaches enabled us to discriminate those populations with relatively high levels of non-native introgression from those populations with little or none. Actual estimates of admixture coefficients within a test population, varied, however, with the approach and reference sample used. These results have important implications for policies dividing populations into different management categories according to the estimated proportion of non-native genetic material that they contain.     

An evaluation of introgression of Atlantic coast striped bass mitochondrial DNA in a Gulf of Mexico population using formalin-preserved museum collections

Striped bass Morone saxatilis populations in drainages along the Gulf of Mexico coast (Gulf) were depleted in the 1950s and 1960s, probably because of anthropogenic influences. It is believed that only the Apalachicola-Chattahoochee-Flint (A-C-F) river system continually supported a naturally reproducing population of Gulf lineage. Striped bass juveniles of Atlantic coast (Atlantic) ancestry were introduced to restore population abundances in the A-C-F from the late 1960s to the mid 1970s and in many other Gulf rivers from the 1960s to the present. We previously identified mtDNA polymorphisms that were unique to ? 60% of striped bass from the A-C-F and which confirmed the continued successful natural reproduction of striped bass of Gulf maternal ancestry within the system. However, the genetic relatedness of the extant A-C-F population to ‘pure’ Gulf striped bass was not addressed. In this study, we determined the frequency of a diagnostic mtDNA XbaI polymorphism in samples of ‘pure’ Gulf striped bass that were collected from the A-C-F prior to the introduction of Atlantic fish, that were obtained from museum collections, and that were originally preserved in formalin. PCR primers were developed that allowed for amplification of a 191-bp mtDNA fragment that contained the diagnostic XbaI restriction site. Using RFLP and direct sequence analyses of the PCR amplicons, we found no significant differences in mtDNA XbaI genotype frequencies between the archived samples and extant A-C-F samples collected over a 15-year period. This indicates that significant maternally mediated introgression of Atlantic mtDNA genomes into the A-C-F gene pool has not occurred. Additionally, we found no evidence of the unique Gulf mtDNA genotype in striped bass from extant populations in Texas, Louisiana and the Mississippi River. These results highlight the importance of the A-C-F as a repository of striped bass to restore extirpated Gulf populations and the potential use of museum collections in retrospective population studies.     

Evolutionary dynamics of hybridization and introgression following the recent colonization of Glossy Ibis (Aves: Plegadis falcinellus) into the New World

Geographic range shifts can cause secondary contact and hybridization between closely related species, revealing mechanisms of species formation and integrity. These dynamics typically play out in restricted geographic regions, but highly vagile species may experience major distributional changes resulting in broad areas of contact. The Glossy Ibis (Plegadis falcinellus) is a dispersive waterbird of the Old World and Australia that colonized eastern North America in the early 19th century and came into contact with the native White‐faced Ibis (P. chihi). Putative hybrids between the two species have been observed across North America. To examine the population genomic consequences of this natural invasion, we sequenced 4,616 ultraconserved elements from 66 individuals sampled across the distributions of falcinellus, chihi, and the Puna Ibis (P. ridgwayi) of South America. We found genomic differentiation among the three species. Loci with high sequence divergence were often shared across all pairwise species comparisons, were associated with regions of high nucleotide diversity, and were concentrated on the Z chromosome. We detected signals of genetic admixture between chihi and falcinellus in individuals both near and far from their core area of sympatry. Genomic cline analyses revealed evidence of greater introgression into falcinellus from chihi, but we found little evidence for selection against hybrids. We also found signals of admixture between ridgwayi and South American populations of chihi. Our results indicate vagile species can experience pervasive introgression upon secondary contact, although we suggest these dynamics may be more ephemeral than the stable hybrid zones often observed in less dispersive organisms.     

Evidence for introgression in azaleas (Rhododendron; Ericaceae): Chloroplast DNA and morphological variation in a hybrid swarm on Stone Mountain,Georgia

Hybridization and introgression have been suggested by previous workers as important factors in the evolution of the southeastern azaleas. However, because of high levels of intraspecific variation and lack of intensive sampling, documentation of this phenomenon has been difficult. We investigated a population of azaleas on Stone Mountain, Georgia, composed of Rhododendron flammeum, R. canescens, and their interspecific hybrids. After analysis of chloroplast DNA, we found evidence of extensive localized cytoplasmic introgression into R. flammeum. Many individuals in the sample population that are morphologically indistinguishable from R. flammeum possess the chloroplast genome of R. canescens. This suggests that some of the observed variation in some populations of R. flammeum may be due to past introgression from R. canescens.     

Testing an hypothesis of hybrid zone movement for toads in France

Hybrid zone movement may result in substantial unidirectional introgression of selectively neutral material from the local to the advancing species, leaving a genetic footprint. This genetic footprint is represented by a trail of asymmetric tails and displaced cline centres in the wake of the moving hybrid zone. A peak of admixture linkage disequilibrium is predicted to exist ahead of the centre of the moving hybrid zone. We test these predictions of the movement hypothesis in a hybrid zone between common (Bufo bufo) and spined toads (B. spinosus), using 31 nuclear and one mtDNA SNPs along a transect in the northwest of France. Average effective selection in Bufo hybrids is low and clines vary in shape and centre. A weak pattern of asymmetric introgression is inferred from cline discordance of seven nuclear markers. The dominant direction of gene flow is from B. spinosus to B. bufo and is in support of southward movement of the hybrid zone. Conversely, a peak of admixture linkage disequilibrium north of the hybrid zone suggests northward movement. These contrasting results can be explained by reproductive isolation of the B. spinosus and B. bufo gene pools at the southern (B. spinosus) side of the hybrid zone. The joint occurrence of asymmetric introgression and admixture linkage disequilibrium can also be explained by the combination of low dispersal and random genetic drift due to low effective population sizes.     

Genetic structure in Red Junglefowl (Gallus gallus) populations: Strong spatial patterns in the wild ancestors of domestic chickens in a core distribution range

Red Junglefowl (Gallus gallus) are among the few remaining ancestors of an extant domesticated livestock species, the domestic chicken, that still occur in the wild. Little is known about genetic diversity, population structure, and demography of wild Red Junglefowl in their natural habitats. Extinction threats from habitat loss or genetic alteration from domestic introgression exacerbate further the conservation status of this progenitor species. In a previous study, we reported extraordinary adaptive genetic variation in the MHC B‐locus in wild Red Junglefowl and no evidence of allelic introgression between wild and domestic chickens was observed. In this study, we characterized spatial genetic variation and population structure in naturally occurring populations of Red Junglefowl in their core distribution range in South Central Vietnam. A sample of 212 Red Junglefowl was obtained from geographically and ecologically diverse habitats across an area of 250 × 350 km. We used amplified fragment‐length polymorphism markers obtained from 431 loci to determine whether genetic diversity and population structure varies. We found that Red Junglefowl are widely distributed but form small and isolated populations. Strong spatial genetic patterns occur at both local and regional scales. At local scale, population stratification can be identified to approximately 5 km. At regional scale, we identified distinct populations of Red Junglefowl in the southern lowlands, northern highlands, and eastern coastal portions of the study area. Both local and long‐distance genetic patterns observed in wild Red Junglefowl may reflect the species’ ground‐dwelling and territorial characteristics, including dispersal barriers imposed by the Annamite Mountain Range. Spatially explicit analyses with neutral genetic markers can be highly informative and here elevates the conservation profile of the wild ancestors of domesticated chickens.