Bidirectional introgressive hybridization between Lepus capensis and Lepus yarkandensis

Lepus capensis and Lepus yarkandensis are two well-established hare species with different habitat preferences and obvious morphological distinctions. L. capensis is common and widespread while L. yarkandensis is an endemic species with a restricted distribution in the Tarim Basin of western China. During field sampling, individuals with a distinct phenotype were found in the contact zone between the two species. To understand the origin of these unclassified individuals, we analyzed morphological and genetic data from 700 individuals including L. yarkandensis, L. capensis and the unclassified individuals. Morphological analyses of the unclassified individuals revealed that they lie between L. yarkandensis and L. capensis. Genetic analyses based on species-specific molecular markers (mitochondrial control region and SRY) showed that the unclassified populations have mtDNA and SRY genes from both L. capensis and L. yarkandensis, suggesting that the unclassified populations result from hybridization between the two species. Interestingly, many identical and/or very similar mtDNA haplotypes were shared between L. capensis populations and L. yarkandensis populations close to their contact zones, which further suggests the existence of extensive bidirectional mtDNA introgression. Similarly, we found evidence for SRY introgression, though it existed at a lower level compared to mtDNA introgression.     

Distortion of symmetrical introgression in a hybrid zone: evidence for locus-specific selection and uni-directional range expansion

The fate of species integrity upon natural hybridization depends on the interaction between selection and dispersal. The relative significance of these processes may be studied in the initial phase of contact before selection and gene flow reach equilibrium. Here we study a hybrid zone of two salamander species, Lyciasalamandra antalyana and Lyciasalamandra billae, at the initial phase of hybridization. We quantify the degree and mode of introgression using nuclear and mtDNA markers. The hybrid zone can be characterized as an abrupt transition zone, the central hybrid zone being only c. 400 m, but introgressed genes were traced up to 3 km. Introgression was traced in both sexes but gene flow may be slightly male-biased. Indirect evidence suggests that hybrid males are less viable than females. Introgression occurred at two levels: (1) locus-specific selection led to different allelic introgression patterns independent of species, while (2) asymmetrical species-level introgression occurred predominately from L. antalyana to L. billae due to range expansion of the former. This indicates that foreign genes can be incorporated into novel genomic environments, which in turn may contribute to the great diversity of morphological variants in Lyciasalamandra.     

Phylogeographic Patterns and Evolution of the Mitochondrial DNA Control Region in Two Neotropical Cats (Mammalia, Felidae)

The ocelot (Leopardus pardalis) and margay (L. wiedii) are sister-species of Neotropical cats which evolved from a lineage that migrated into South America during the formation of the Panamanian land bridge 3–5 million years ago. Patterns of population genetic divergence of each species were studied by phylogenetic analyses of mitochondrial DNA (mtDNA) control region sequences in individuals sampled across the distribution of these taxa. Abundant genetic diversity and remarkably concordant phylogeographic partitions for both species were observed, identifying parallel geographic regions which likely reflect historical faunal barriers. Inferred aspects of phylogeography, population genetic structure, and demographic history were used to formulate conservation recommendations for these species. In addition, observed patterns of sequence variation provided insight into the molecular evolution of the mtDNA control region in closely related felids. Received: 26 January 1998 / Accepted: 14 May 1998     

Morphological and phylogenetic evidence for hybridization and introgression in a sea star secondary contact zone

Abstract. Glacial cycles and other climatic events have been widely invoked as factors promoting divergence, secondary contact, and hybridization between populations of terrestrial organisms, but the origin and fate of secondary contact in the sea is much less well understood. We studied the distribution of morphological and genetic variation in a northwest Atlantic zone of secondary contact between congeneric sea stars of Asterias that probably separated after the Pliocene as part of the trans-Arctic interchange. These species have similar reproductive biology and can hybridize in the laboratory. However, multivariate analysis of morphological traits scored from sea stars inside and outside the zone of secondary contact clearly indicated two clusters of phenotypes that corresponded to the two taxonomic species. A quantitative analysis of this clustering pattern did not support the hypothesis of a third grouping that might correspond to intermediate hybrid phenotypes. Known F₁ hybrids from laboratory matings grouped with one of the two taxonomic species. However, a survey of mtDNA sequence variation clearly indicated that ∼13% of individuals of one species ( Asterias forbesi ) are descendants of hybridization events that resulted in introgression of haplotypes of Asterias rubens into populations of A. forbesi . We conclude that morphological phenotypes are inadequate to identify hybrids of Asterias and their descendants, and that hybridization and introgression might be common in this secondary contact zone.     

Phylogenetic evidence of mitochondrial DNA introgression among pocket gophers in New Mexico (family Geomyidae)

Mitochondrial DNA (mtDNA) variation in the cytochrome b gene was determined for two divergent taxa of pocket gophers, Thomomys bottae actuosus and T. b. ruidosae . These two taxa hybridize in a narrow contact zone, but introgression of nuclear markers such as allozymes or chromosomes does not extend much beyond the hybrid zone (Patton et al. 1979). We found that despite their distinctness, the two subspecies shared very similar mtDNA haplotypes. By a comparison of phylogenetic histories derived from nuclear markers (allozymes) and from mtDNA haplotypes sampled in different populations of T. bottae from New Mexico, we show that apparent similarity is due to an introgression of T. b. ruidosae mtDNA into T. b. actuosus nuclear background. Evidence of introgression is not limited to the present-day contact zone between these two taxa, but extends at least 75 km away from it. The actuosus haplotype coexists along with the ruidosae mtDNA in the Gallinas Mts., which are inhabited by otherwise pure T. b. actuosus , while further north only typical actuosus haplotypes were detected. Of several potential mechanisms which could lead to such a geographical pattern of variation, we argue that a combination of range shifts due to climatic fluctuations, and genetic drift are most likely. Horizontal gene transfers due to hybridization are historical events which seem rather common among pocket gophers. Although they can be identified with careful phylogenetic study using independent data sets, the potential for misinterpreting a gene tree as an organismal tree is great in this and other groups of animals.     

Hybridization and reproductive isolation among syntopic populations of the topminnows Fundulus notatus and F. olivaceus

Fundulus notatus and Fundulus olivaceus are two closely related topminnow species that exhibit similar ecological niches and broad, largely overlapping, North American ranges extending throughout much of the Mississippi River drainage as well as the coastal drainages of the Gulf of Mexico. Previous studies have suggested that these two species are reproductively compatible despite cytogenetic differences and will hybridize when syntopic. We used nuclear and mtDNA loci to assess levels of hybridization and test for introgression in syntopic populations of these two species in four drainages in southern Illinois. Although hybridization was detected in all syntopic populations, an assessment of the proportion of hybrid individuals indicated a deficiency of hybrids relative to expectations under random mating. We determined that, although mtDNA introgression was prevalent and extended beyond the zones of contact, evidence of nuclear introgression was limited to the zone of sympatry.     

Introgression of Luxilus cornutus mtDNA into allopatric populations of Luxilus chrysocephalus (Teleostei: Cyprinidae) in Missouri and Arkansas

The cyprinid fishes Luxilus cornutus and Luxilus chrysocephalus hybridize extensively in a zone extending through the Great Lakes region with extensive introgression of L. cornutus mtDNA occurring in populations of L. chrysocephalus south of the present hybrid zone in Ohio. Western populations of these two species occur adjacent to one another in Missouri but hybridization has never been observed. In order to determine if hybridization has occurred historically in Missouri, allopatric populations of L. chrysocephalus were analysed for mtDNA introgression. Extensive introgression of L. ***cornutus mtDNA was observed in most populations of L. chrysocephalus in Missouri resulting in the elimination of L. chrysocephalus mtDNA in many populations. Luxilus cornutus mtDNA in L. chrysocephalus is found approximately 300 km south of extant L. cornutus populations in Missouri. Luxilus chrysocephalus mtDNA was replaced by four unique L. cornutus mtDNA haplotypes, with one particular haplotype becoming fixed in several L. chrysocephalus populations. The pattern of introgression suggests that historically L. cornutus occupied a more southern distribution in Missouri bringing it into contact with western populations of L. chrysocephalus and resulting in a hybrid zone.     

Hybridization between two sympatrically breeding species of fur seal at Iles Crozet revealed by genetic analysis

Introgressive hybridization occurs when closely related taxa overlap in distribution and is often associated with historically isolated populations coming into contact as a result of anthropogenic disturbance. There is evolutionary and conservation interest in detecting hybridization to determine its implications on future species composition, especially for threatened and recovering taxa such as subantarctic (Arctocephalus tropicalis) and Antarctic (A. gazella) fur seals, which were driven to the brink of extinction by human exploitation. Hybridization between these species has been reported at two locations and they breed sympatrically at a third site, Iles Crozet. While hybrid individuals have previously been identified based on phenotype, individuals can be difficult to classify based on these characteristics alone. Genotypic hybrid identification has been successful in several species, including fur seals. In this study we conducted an assignment test using microsatellite data to identify hybrids and to measure the frequency of hybridization at Iles Crozet. Samples were collected from 372 individuals and screened with 6 polymorphic microsatellite markers. MtDNA genotypes were also determined for individuals identified as hybrids or backcrosses based on microsatellite genotype. Phenotype, microsatellite and mtDNA genotype were then compared in order to identify hybrids. The results indicate that 1% of the population have hybrid genotypes and at a minimum, 2.4% of the population are backcrossed to parental species. We found that the two species are genetically distinct from one another and given the low rate of hybridization it is unlikely that they will fuse. These results suggest that there is a mechanism for species recognition that acts as a barrier to hybridization. It therefore seems unlikely that fur seals are threatened by significant introgression. Further investigation of fur seal mating systems would provide valuable insight into the mechanisms governing hybridization and species recognition in mate choice.     

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south‐western and south‐eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south‐western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south‐eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150‐km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2–27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population‐level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.     

Hybridization and Introgression among Species of Sunfish (Lepomis): Analysis by Mitochondrial DNA and Allozyme Markers

We explore the potential of mitochondrial DNA (mtDNA) analysis, alone and in conjunction with allozymes, to study low-frequency hybridization and introgression phenomena in natural populations. MtDNAs from small samples of nine species of sunfish (Lepomis, Centrarchidae) were purified and digested with each of 13 informative restriction enzymes. Digestion profiles for all species were highly distinct: estimates of overall fragment homology between pairs of species ranged from 0-36%. Allozymes encoded by nine nuclear genes also showed large freqency differences among species and together with mtDNA provided many genetic markers for hybrid identification. A genetic analysis of 277 sunfish from two locations in north Georgia revealed the following: (1) a low frequency of interspecific hybrids, all of which appeared to be F1's; (2) the involvement of five sympatric Lepomis species in the production of these hybrids; (3) no evidence for introgression between species in our study locales (although for rare hybridization, most later-generation backcrosses would not be reliably distinguished from parentals); (4) a tendency for hybridizations to take place preferentially between parental species differing greatly in abundance; (5) a tendency for the rare species in a hybrid cross to provide the female parent. Our data suggest that absence of conspecific pairing partners and mating stimuli for females of rarer species may be important factors in increasing the likelihood of interspecific hybridization. The maternal inheritance of mtDNA offers at least two novel advantages for hybridization analysis: (1) an opportunity to determine direction in hybrid crosses; and (2) due to the linkage among mtDNA markers, an increased potential to distinguish effects of introgression from symplesiomorphy or character convergence.     

Molecular patterns of introgression in a classic hybrid zone between the Australian tree frogs,Litoria ewingii and L. paraewingi: evidence of a tension zone

Hybrid zones provide a rare opportunity to explore the processes involved in reproductive isolation and speciation. The southern hybrid zone between the southeastern Australian tree frogs Litoria ewingii and L. paraewingi has been comprehensively studied over the last 40 years, primarily using reproductive compatibility experiments and male advertisement calls. We used mitochondrial DNA (mtDNA) and eight nuclear microsatellite markers to characterize this hybrid zone along a historically studied transect and to test various dispersal‐dependent and dispersal‐independent hybrid zone models. The species are genetically distinct and the level of hybridization within the contact zone is low, with the majority of admixed individuals representing later‐generation hybrids. Based on previous experimental genetic compatibility studies, we predicted that hybrids with L. paraewingi mtDNA would be more frequent than hybrids with L. ewingii mtDNA. Surprisingly, a greater proportion of the identified hybrids had L. ewingii mtDNA. Geographical cline analyses showed a sharp transition in allele frequencies across the transect, and both the mtDNA and microsatellite data showed concordant cline centres, but were best supported by a model that allowed width to vary. Overall, the L. ewingii–L. paraewingi hybrid zone is best characterized as a tension zone, due to the narrow cline width, concordant genetic clines and low levels of hybridization.     

The modality of nine Triturus newt hybrid zones assessed with nuclear,mitochondrial and morphological data

The shape of hybrid zones provides insight into genetic isolation between the evolutionary lineages involved: the greater the limitation to gene flow, the farther along the unimodal to bimodal continuum. We study hybrid zone modality in a group of closely related species at a variety of levels of genetic divergence. We explore the degree and the geography of hybridization in 12 transects of Triturus newts, for nine of the ten secondary contact zones in Europe, using nuclear and mitochondrial encoded genetic data and morphological characteristics. Species status is verified by examining the correlation between the three marker systems in a geographical context. At 40 allozyme loci, two transects classify as unimodal, three as bimodal and two as intermediate, while the signal for hybridization is weak or absent in five transects. One zone studied in duplicate was classified as intermediate in one region and as bimodal in another region. mtDNA introgression is frequent and extends beyond nuclear introgression in two transects. Morphology provides additional evidence for hybridity, including one transect for which the signal of nuclear gene flow is weak. Compared to simulations allowing panmixia, the observed allozyme transitions at contact zones show a deficit of backcrossing to various degrees. Over all transects, there is a weak negative relationship between the level of hybridization and allozyme genetic distance for species pairs, consistent with Bateson–Dobzhansky–Muller effects. This observation, based upon highly comparable data for a single genus, supports conclusions derived from analyses over a wide variety of other taxa. © 2014 The Authors. Biological Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 604–622.     

Bayesian analysis of hybridization and introgression between the endangered european mink (Mustela lutreola) and the polecat (Mustela putorius)

Human-mediated global change will probably increase the rates of natural hybridization and genetic introgression between closely related species, and this will have major implications for conservation of the taxa involved. In this study, we analyse both mitochondrial and nuclear data to characterize ongoing hybridization and genetic introgression between two sympatric sister species of mustelids, the endangered European mink (Mustela lutreola) and the more abundant polecat (M. putorius). A total of 317 European mink, 114 polecats and 15 putative hybrid individuals were collected from different localities in Europe and genotyped with 13 microsatellite nuclear markers. Recently developed Bayesian methods for assigning individuals to populations and identifying admixture proportions were applied to the genetic data. To identify the direction of hybridization, we additionally sequenced mtDNA and Y chromosomes from 78 individuals and 29 males respectively. We found that both hybridization and genetic introgression occurred at low levels (3% and 0.9% respectively) and indicated that hybridization is asymmetric, as only pure polecat males mate with pure European mink females. Furthermore, backcrossing and genetic introgression was detected only from female first-generation (F1) hybrids of European mink to polecats. This latter result implies that Haldane's rule may apply. Our results suggest that hybridization and genetic introgression between the two species should be considered a rather uncommon event. However, the current low densities of European mink might be changing this trend.     

Assessment of gene flow across a hybrid zone in red-tailed chipmunks (Tamias ruficaudus)

The role of hybridization in animal speciation is controversial and recent research has challenged the long-standing criterion of complete reproductive isolation to attain species status. The speciation-with-gene-flow model posits that the genome is semi-permeable and hybridization may be a phase in the process of divergence. Here, we apply these concepts to a previously identified zone of mtDNA introgression between the two strongly morphologically differentiated subspecies of red-tailed chipmunk ( Tamias ruficaudus ) in the US Inland Northwest. Using multilocus genotype data from the southern, older contact zone, we demonstrate that neutral gene flow is unusually low between the subspecies across the Lochsa River. This is geographically congruent with the discontinuity in bacular morphology, indicating that the cline of mitochondrial DNA (mtDNA) haplotypes is displaced. Furthermore, we elucidate the evolutionary forces responsible by testing hypotheses of lineage sorting and hybridization. We determined that introgressive hybridization is the cause of mtDNA/morphology incongruence because there are non-zero levels of migration and gene flow. Although our estimate of the age of the hybrid zone has wide credibility intervals, the hybridization events occurred in the Late Pleistocene and the divergence occurred in the Middle Pleistocene. Finally, we assessed substructure within and adjacent to the hybrid zone and found that the hybrid zone constitutes a set of populations that are genetically differentiated from parental sets of populations; therefore, hybridization in this system is not likely an evolutionary sink, but has generated novel combinations of genotypes.     

Massive introgression of major histocompatibility complex (MHC) genes in newt hybrid zones

Variation in the vertebrate major histocompatibility complex (MHC) genes is crucial for fighting pathogen assault. Because new alleles confer a selective advantage, MHC should readily introgress between species, even under limited hybridization. Using replicated transects through two hybrid zones between strongly reproductively isolated European newts, Lissotriton montandoni and L. vulgaris, we demonstrated recent and ongoing MHC class I and II introgression in the Carpathian region. The extent of introgression correlated with the age of contact. In the older zone, MHC similarity between species within transects exceeded similarity between transects within species, implying pervasive introgression ‐ a massive exchange of MHC genes, not limited to specific variants. In simulations, the observed pattern emerged under the combined action of balancing selection and hybridization, but not when these processes acted separately. Thus, massive introgression at advanced stages of divergence can introduce novel and restore previously lost MHC variation, boosting the adaptive potential of hybridizing taxa. In consequence, MHC genes may be the last to stop introgressing between incipient species.     

The genomic legacy from the extinct Lepus timidus to the three hare species of Iberia: contrast between mtDNA, sex chromosomes and autosomes

Extensive interspecific genetic introgression is often reported, and appraising its genomic impact can serve to determine whether it results from selection on specific loci or from demographic processes affecting the whole genome. The three species of hares present in the Iberian Peninsula harbour high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species now extinct in the region. This could result from the invasive replacement of L. timidus by the temperate species during deglaciation but should then have left traces in the nuclear genome. We typed single nucleotide polymorphisms (SNPs) discovered by sequencing 10 autosomal loci, two X-linked and one Y-linked in species-wide samples of the four taxa. Based on lineage-diagnostic SNPs, we detected no trace of L. timidus sex chromosomes in Iberia. From the frequencies of inferred haplotypes, autosomal introgression into L. granatensis appeared mostly sporadic but always widespread instead of restricted to the north as mtDNA. Autosomal introgression into Iberian L. europaeus , inhabiting the Pyrenean foothills, was hardly detectable, despite quasi-fixation of L. timidus mtDNA. L. castroviejoi , endemic to the Cantabrian Mountains and fixed for L. timidus mtDNA, showed little traces of autosomal introgression. The absence of sex-chromosome introgression presumably resulted from X-linked hybrid male unfitness. The contrasting patterns between the autosomes and mtDNA could reflect general gender asymmetric processes such as frequency-dependent female assortative mating, lower mtDNA migration and higher male dispersal, but adaptive mtDNA introgression cannot be dismissed. Additionally, we document reciprocal introgression between L. europaeus and both L. granatensis in Iberia and L. timidus outside Iberia.     

Integrating phylogeography and paleoecology to investigate the origin and dynamics of hybrid zones: insights from two widespread North American firs

Secondary contact between closely related taxa routinely occurs during postglacial migrations. After initial contact, the location of hybrid zones may shift geographically or remain spatially stable over time in response to various selective pressures or neutral processes. Studying the extent and direction of introgression using markers having contrasted levels of gene flow can help unravel the historical dynamics of hybrid zones. Thanks to their contrasted maternal and paternal inheritance, resulting in different levels of gene flow for mitochondrial and chloroplast DNA (mtDNA and cpDNA), the Pinaceae stand out as a relevant biological model for this purpose. The objective of the study was to assess whether the hybrid zone between Abies balsamea and Abies lasiocarpa (two largely distributed Pinaceae) has moved or remained stable over time by analysing the distribution of cytoplasmic DNA variation as well as published palaeobotanical data. Interspecific gene flow was higher for cpDNA than mtDNA markers; hence, the geographic distribution of mitotypes was more congruent with species distributions than chlorotypes. This genetic signature was contrary to expectations under a moving hybrid zone scenario, as well as empirical observations in other conifers. Genetic evidence for this rare instance of stable hybrid zone was corroborated by the colonization chronology derived from published fossil data, indicating that the two fir species initially came into contact in the area corresponding to the current sympatric zone 11 kyr ago. While an explanatory analysis suggested the putative influence of various environmental factors on the relative abundance of cytoplasmic genome combinations, further research appears necessary to assess the role of both demographic history and selective factors in driving the dynamics of hybrid zones.     

Structure and dynamics of hybrid zones at different stages of speciation in the common vole (Microtus arvalis)

The genetic structure and dynamics of hybrid zones provide crucial information for understanding the processes and mechanisms of evolutionary divergence and speciation. In general, higher levels of evolutionary divergence between taxa are more likely to be associated with reproductive isolation and may result in suppressed or strongly restricted hybridization. In this study, we examined two secondary contact zones between three deep evolutionary lineages in the common vole (Microtus arvalis). Differences in divergence times between the lineages can shed light on different stages of reproductive isolation and thus provide information on the ongoing speciation process in M. arvalis. We examined more than 800 individuals for mitochondrial (mtDNA), Y‐chromosome and autosomal markers and used assignment and cline analysis methods to characterize the extent and direction of gene flow in the contact zones. Introgression of both autosomal and mtDNA markers in a relatively broad area of admixture indicates selectively neutral hybridization between the least‐divergent lineages (Central and Eastern) without evidence for partial reproductive isolation. In contrast, a very narrow area of hybridization, shifts in marker clines and the quasi‐absence of Y‐chromosome introgression support a moving hybrid zone and unidirectional selection against male hybrids between the lineages with older divergence (Central and Western). Data from a replicate transect further support non‐neutral processes in this hybrid zone and also suggest a role for landscape history in the movement and shaping of geneflow profiles.     

Molecular evidence for hybridization and introgression in the neotropical coastal desert-endemic Palaua (Malveae, Malvaceae)

Recent years have seen an increasing awareness of the importance of hybridization and introgression in plant evolution. Both processes were also invoked to have had a strong impact in the evolution of Palaua, based on morphological intermediacy. Here, we used nuclear ITS and cellulose synthase DNA sequences to assess previous phylogenetic hypotheses and to uncover previously undescribed reticulate evolution in Palaua. Both nuclear regions recovered largely the same reticulate pattern, showing that interspecific crosses occurred between distantly related species of the Dissecta and Integrifolia clade. Hybridization between species occurred only in a narrow contact zone. Moreover, many sequences of Palaua dissecta were placed in the strongly divergent Integrifolia clade, a pattern that is best explained by introgression. These results support our view that the strong interannual fluctuations of humidity and habitat expansion provide opportunities for the isolation of taxa, establishment of hybrid species, and for introgression between incompletely reproductively isolated taxa following recurrent contact.     

Structure of a mosaic hybrid zone between the field crickets Gryllus firmus and G. pennsylvanicus

Hybrid zones provide insight into the nature of species boundaries and the evolution of barriers to gene exchange. Characterizing multiple regions within hybrid zones is essential for understanding both their history and current dynamics. Here, we describe a previously uncharacterized region of a well‐studied hybrid zone between two species of field crickets, Gryllus pennsylvanicus and G. firmus. We use a combination of mitochondrial DNA sequencing, morphological data, and modeling of environmental variables to identify the ecological factors structuring the hybrid zone and define patterns of hybridization and introgression. We find an association between species distribution and natural habitat; Gryllus pennsylvanicus occupies natural habitat along forest edges and natural clearings, whereas G. firmus occupies more disturbed areas in agricultural and suburban environments. Hybridization and introgression occur across patch boundaries; there is evidence of substantial admixture both in morphological characters and mtDNA, over a broad geographic area. Nonetheless, the distribution of morphological types is bimodal. Given that F₁ hybrids are viable and fertile in the lab, this suggests that strong pre‐zygotic barriers are operating in this portion of the hybrid zone.