Development of diagnostic microsatellite markers from whole‐genome sequences of Ammodramus sparrows for assessing admixture in a hybrid zone

Studies of hybridization and introgression and, in particular, the identification of admixed individuals in natural populations benefit from the use of diagnostic genetic markers that reliably differentiate pure species from each other and their hybrid forms. Such diagnostic markers are often infrequent in the genomes of closely related species, and genomewide data facilitate their discovery. We used whole‐genome data from Illumina HiSeqS2000 sequencing of two recently diverged (600,000 years) and hybridizing, avian, sister species, the Saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) Sparrow, to develop a suite of diagnostic markers for high‐resolution identification of pure and admixed individuals. We compared the microsatellite repeat regions identified in the genomes of the two species and selected a subset of 37 loci that differed between the species in repeat number. We screened these loci on 12 pure individuals of each species and report on the 34 that successfully amplified. From these, we developed a panel of the 12 most diagnostic loci, which we evaluated on 96 individuals, including individuals from both allopatric populations and sympatric individuals from the hybrid zone. Using simulations, we evaluated the power of the marker panel for accurate assignments of individuals to their appropriate pure species and hybrid genotypic classes (F1, F2, and backcrosses). The markers proved highly informative for species discrimination and had high accuracy for classifying admixed individuals into their genotypic classes. These markers will aid future investigations of introgressive hybridization in this system and aid conservation efforts aimed at monitoring and preserving pure species. Our approach is transferable to other study systems consisting of closely related and incipient species.     

Phylogenetic reticulation in subtribe Helianthinae

Incongruence between phylogenetic estimates based on nuclear and chloroplast DNA (cpDNA) markers was used to infer that there have been at least two instances of chloroplast transfer, presumably through wide hybridization, in subtribe Helianthinae. One instance involved Simsia dombeyana, which exhibited a cpDNA restriction site phenotype that was markedly divergent from all of the other species of the genus that were surveyed but that matched the restriction site pattern previously reported for South American species of Viguiera. In contrast, analysis of sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region showed Simsia to be entirely monophyletic and placed samples of S. dombeyana as the sister group to the relatively derived S. foetida, a result concordant with morphological information. A sample of a South American species of Viguiera was placed by ITS sequence data as the sister group to a member of V. subg. Amphilepis, which was consistent with cpDNA restriction site data. Samples of Tithonia formed a single monophyletic clade based on ITS sequence data, whereas they were split between two divergent clades based on cpDNA restriction site analysis. The results suggested that cpDNA transfer has occurred between taxa diverged to the level of morphologically distinct genera, and highlight the need for careful and complete assessment of molecular data as a source of phylogenetic information.     

DNA from a 100-year-old holotype confirms the validity of a potentially extinct hummingbird species

We used mtDNA sequence data to confirm that the controversial 100-year-old holotype of the Bogotá sunangel (Heliangelus zusii) represents a valid species. We demonstrate that H. zusii is genetically well differentiated from taxa previously hypothesized to have given rise to the specimen via hybridization. Phylogenetic analyses place H. zusii as sister to a clade of mid- to high-elevation Andean species currently placed in the genera Taphrolesbia and Aglaiocercus. Heliangelus zusii, presumed extinct, has never been observed in nature by biologists. We infer that the species occupied a restricted distribution between the upper tropical and temperate zones of the northern Andes and that it was most probably driven to extinction by deforestation that accompanied human population growth during the nineteenth and early twentieth centuries. We demonstrate the feasibility of obtaining DNA from nearly microscopic tissue samples from old hummingbird specimens and suggest that these methods could be used to resolve the taxonomy of dozens of avian taxa known only from type specimens.     

Hybrid origin of Audubon's warbler

Several animal species have recently been shown to have hybrid origins, but no avian examples have been documented with molecular evidence. We investigate whether the Audubon’s warbler (Dendroica auduboni), one of four visually distinct species in the yellow‐rumped warbler complex, has originated through hybridization between two other species in this group, the myrtle warbler (D. coronata) and black‐fronted warbler (D. nigrifrons). Analysis of nuclear amplified fragment length polymorphism (AFLP) and sequence markers shows that Audubon’s warblers are genetically intermediate and carry a mixture of alleles otherwise found only in one or the other of their putative parental species. Audubon’s warblers also carry two deeply divergent mitochondrial DNA lineages, each shared with only one putative parental form. Broad clines between Audubon’s and black‐fronted warblers in AFLP markers call into question the validity of these two forms as full species; nevertheless, our results suggest that the Audubon’s warbler probably originated through hybridization between two long‐diverged species. It is likely that more cases of avian species of hybrid origin will be revealed by surveys of variation in nuclear DNA and other traits.     

Genetic evidence of hybridization of the world's most endangered tern,the Chinese Crested Tern Thalasseus bernsteini

We collected multi‐locus data to test whether hybridization has occurred between a critically endangered waterbird, the Chinese Crested Tern Thalasseus bernsteini, and the Greater Crested Tern Thalasseus bergii. Phylogenetic analysis shows these two tern species are sister species, having diverged < 1 million years ago. Our findings suggest a grave threat to the survival of the Chinese Crested Tern, and illustrate the need for conservation managers to monitor further hybridization of this endangered tern species.     

DNA barcoding of bark and ambrosia beetles reveals excessive NUMTs and consistent east‐west divergence across Palearctic forests

A comprehensive DNA barcoding library is very useful for rapid identification and detection of invasive pest species. We tested the performance of species identification in the economically most damaging group of wood‐boring insects – the bark and ambrosia beetles – with particular focus on broad geographical sampling across the boreal Palearctic forests. Neighbour‐joining and Bayesian analyses of cytochrome oxidase I (COI) sequences from 151 species in 40 genera revealed high congruence between morphology‐based identification and sequence clusters. Inconsistencies with morphological identifications included the discovery of a likely cryptic Nearctic species of Dryocoetes autographus, the possible hybrid origin of shared mitochondrial haplotypes in Pityophthorus micrographus and P. pityographus, and a possible paraphyletic Xyleborinus saxeseni. The first record of Orthotomicus suturalis in North America was confirmed by DNA barcoding. The mitochondrial data also revealed consistent divergence across the Palearctic or Holarctic, confirmed in part by data from the large ribosomal subunit (28S). Some populations had considerable variation in the mitochondrial barcoding marker, but were invariant in the nuclear ribosomal marker. These findings must be viewed in light of the high number of nuclear insertions of mitochondrial DNA (NUMTs) detected in eight bark beetle species, suggesting the possible presence of additional cryptic NUMTs. The occurrence of paralogous COI copies, hybridization or cryptic speciation demands a stronger focus on data quality assessment in the construction of DNA barcoding databases.     

Unexpected hybridization patterns in Near Eastern terrapins (Mauremys caspica,M. rivulata) indicate ancient gene flow across the Fertile Crescent

Recent studies indicate that hybridization in animals occurs more frequently than previously thought and that it may play an important evolutionary role. Chelonians are capable of extensive hybridization, raising the question how chelonian species evolve and maintain genetic integrity despite hybridization. Here, we use two sister species with parapatric distribution, Mauremys caspica and M. rivulata, as our model. These taxa are estimated to have diverged some 5.3–7.0 million years ago. Using rangewide sampling and 13 unlinked polymorphic microsatellite markers, five nuclear loci and one mitochondrial gene, we show that hybridization is rare along the contact zone of the two species in Turkey. However, we discovered an unexpected hybrid swarm in the southern Levant that has been hitherto identified with M. rivulata. This hybrid swarm is separated from the inland species M. caspica by a 700‐km‐wide distribution gap corresponding to the Syrian Desert. Ecological palaeomodelling suggests that during more humid climatic episodes in the Last Glacial Maximum and mid‐Holocene, the current contact zone extended into the southern Levant, facilitating the establishment of the now isolated hybrid swarm. Our results support that there is not necessarily a general hybridization pattern in a given species couple and that the extent of gene flow may differ considerably in different parts of the distribution range. Moreover, our results highlight that studies on hybridization should not focus only on extant contact and hybrid zones, but should use rangewide sampling to detect signals of ancient hybridization that might otherwise be missed.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence

The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.     

Amplification of sequence tagged sites in five avian species using heterologous oligonucleotides

Short of a complete genomic DNA sequence, sequence tagged sites (STSs) have emerged as major genomic reagents for the genetic analysis of little-studied ecologically and agriculturally important organisms. Here, we report STS developed for the turkey (Meleagris gallopavo), guinea fowl (Numidea meleagris), Japanese quail (Coturnix coturnix) and pigeon using primers specific for reference DNA sequences of two chicken (Gallus gallus) genes, aggrecan (agc1) and type X collagen (col10). Additional STSs were also developed for turkey, quail and chicken using primers specific for the human apobec-1 gene. The total length of the STSs developed was 5990, 2522, 4127, 1539 and 6600 bp for the turkey, guinea fowl, Japanese quail, pigeon and chicken, respectively. Based on splice site consensus GT and AG sequences, four of the seven agc1-based chicken STS appear to contain introns. The human gene-based STSs showed no significant sequence identity with the reference GenBank sequences. Maximum likelihood, maximum parsimony and neighbour-joining analysis of an agc1-based STS that was common to all five species showed phylogenetic relationships consistent with those previously defined using mitochondria DNA sequences and nuclear gene restriction maps. Additionally, several putative single nucleotide polymorphisms (SNPs) were detected within the STSs, including eight in the turkey, two in the quail, and two in the chicken when multiple sequences were evaluated from each species. This report describes new STSs that are resources for genetic and physical mapping and genome analysis within and among avian species. These resources should further aid in our understanding of the biology of agriculturally important but little-studied guinea fowl and turkey. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

MITOCHONDRIAL DNA HOMOGENEITY IN THE PHENOTYPICALLY DIVERSE REDPOLL FINCH COMPLEX (AVES: CARDUELINAE: CARDUELIS FLAMMEA-HORNEMANNI)

Breeding redpoll finches (Aves: Carduelinae) show extensive plumage and size variability and, in many cases, a plumage polymorphism that is not related to age or sex. This has been ascribed to extreme phenotypic variation within a single taxon or to moderate variability within distinct taxa coupled with hybridization. The predominant view favors the recognition of two largely sympatric species: Carduelis flammea, comprised of four well-marked subspecies—flammea, cabaret, islandica, and rostrata; and C. hornemanni, comprised of two subspecies—hornemanni and exilipes. We studied representative samples of these putative subspecies (except islandica) for variation in mitochondrial DNA (mtDNA). Using 20 informative restriction enzymes that recognized 124 sites (642 base pairs [bp] of sequence or ≈ 3.7% of the molecule), we identified 17 RFLP haplotypes in the 31 individuals surveyed. The haplotypes formed a simple phylogenetic network with most clones diverging by a single site difference from a common haplotype found in almost half of the individuals. Within populations and taxa, levels of mtDNA diversity were similar to those observed in other avian species. The pattern of mtDNA divergence among populations was statistically unrelated to their geographic or traditional taxonomic relationships, and the estimated distance between the two traditionally recognized species was very small relative to those typically observed among avian sister species.     

Genetic and morphological evidence of a geographically widespread hybrid zone between two crocodile species,Crocodylus acutus and Crocodylus moreletii

Hybrid zones represent natural laboratories to study gene flow, divergence and the nature of species boundaries between closely related taxa. We evaluated the level and extent of hybridization between Crocodylus moreletii and Crocodylus acutus using genetic and morphological data on 300 crocodiles from 65 localities. To our knowledge, this is the first genetic study that includes the entire historic range and sympatric zone of the two species. Contrary to expectations, Bayesian admixture proportions and maximum‐likelihood estimates of hybrid indexes revealed that most sampled crocodiles were admixed and that the hybrid zone is geographically extensive, extending well beyond their historical region of sympatry. We identified a few geographically isolated, nonadmixed populations of both parental species. Hybrids do not appear to be F₁s or recent backcrosses, but rather are more likely later‐generation hybrids, suggesting that hybridization has been going on for several to many generations and is mostly the result of natural processes. Crocodylus moreletii is not the sister species of C. acutus, suggesting that the hybrid zone formed from secondary contact rather than primary divergence. Nonadmixed individuals from the two species were distinguishable based on morphological characters, whereas hybrids had a complex mosaic of morphological characters that hinders identification in the wild. Very few nonadmixed C. acutus and C. moreletii populations exist in the wild. Consequently, the last nonadmixed C. moreletii populations have become critically endangered. Indeed, not only the parental species but also the naturally occurring hybrids should be considered for their potential conservation value.     

Identification,cloning and sequence analysis of a dwarf genome-specific RAPD marker in rubber tree [<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Muell.) Arg.]

High-yielding dwarf clones of Hevea brasiliensis are tolerant to wind damage and therefore useful for high-density planting. The identification of molecular markers for the dwarf character is very important for isolating true-to-type high-yielding dwarf hybrid lines in the early stage of plant breeding programs. We have identified a dwarf genome-specific random amplified polymorphic DNA (RAPD) marker in rubber tree. A total of 115 random oligonucleotide 10-mer primers were used to amplify genomic DNA by PCR, of which 19 primers produced clear and detectable bands. The primer OPB-12 generated a 1.4-kb DNA marker from both natural and controlled F₁ hybrid progenies (dwarf stature) derived from a cross between a dwarf parent and a normal cultivated clone as well as from the dwarf parent; it was absent in other parent (RRII 118). To validate this DNA marker, we analyzed 22 F₁ hybrids (13 with a dwarf stature and nine with a normal stature); the dwarf genome-specific 1.4-kb RAPD marker was present in all dwarf-stature hybrids and absent in all normal-stature hybrids. This DNA marker was cloned and characterized. DNA marker locus specificity was further confirmed by Southern blot hybridization. Our results indicate that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern based on the presence/absence of specific DNA markers than dye-stained gels or Southern blot analysis of RAPD blots using probes made from purified PCR products. Detection of RAPD markers in the hybrid progenies indicates that RAPD is a powerful tool for identifying inherited genome segments following different hybridization methods in perennial tree crops.     

Phylogenetic relationships in Maloideae (Rosaceae): evidence from sequences of the internal transcribed spacers of nuclear ribosomal DNA and its congruence with morphology

We used sequences from both internal transcribed spacers (ITS) and a small portion of the 5.8S gene of nuclear ribosomal DNA (nrDNA) for phylogenetic reconstruction of 19 genera of Maloideae and four potential outgroups from the Rosaceae. Parsimony analyses indicate that Maloideae are not monophyletic; Vauquelinia, which is traditionally placed in Spiraeoideae, and two genera of the Maloideae, Eriobotrya and Rhaphiolepis, form a well-supported clade that is the sister to the remainder of the subfamily. Although our ITS phylogenetic hypothesis is highly resolved, there is considerable homoplasy, and support, as indicated by bootstrap values and decay indices, is relatively weak for all groups except four: Eriobotrya-Rhaphiolepis-Vauquelinia, Crataegus-Mespilus, Amelanchier-Peraphyllum-Malacomeles, and Cydonia-Pseudocydonia. Our DNA sequence data do not support a broad interpretation of Sorbus. Intergeneric hybridization, which is prevalent in Maloideae, occurs between genera that are far removed from one another on our most-parsimonious trees. We infer an overall phylogeny from separate analyses of ITS DNA sequences and recently published morphological and wood anatomical studies of Maloideae and from analyses after pooling these data sets. The four most strongly supported clades of the ITS phylogeny appear in the phylogeny based on pooled data.     

Conservation genetics of the central newt (Notophthalmus viridescens) in Iowa: the importance of a biogeographic framework

In light of global declines in amphibian populations, genetic data have become increasingly important for understanding population structure and for revealing hidden diversity. At the species level, Notophthalmus viridescens is an IUCN species of “least concern”, but the subspecies N. v. louisianensis (central newt) is listed as “threatened” in Iowa, a state on the western periphery of the species range. Genetic data were collected from 282 N. v. louisianensis from 14 sites in Iowa. Sequences from 1,054 nucleotides of mitochondrial DNA from Iowa newts revealed unexpected diversity in the form of two major haplotype groups that are not sister clades, with southern Iowa N. v. louisianensis being more closely related to N. v. piaropicola (peninsula newt) from Florida than to consubspecifics in Iowa. Sequence differentiation indicates that the two lineages of newts present in Iowa diverged near the beginning of the Pleistocene. Northern and southern Iowa haplotypes were found together at one site, indicating an opportunity for hybridization near Remington’s biogeographic suture zone 1, a hotspot for hybridization in other species. Three microsatellite loci provided additional evidence for distinctness of northern and southern Iowa newt populations. This study highlights the relevance of historical biogeography to conservation, as management strategies for N. v. louisianensis in Iowa must reflect previously unrecognized diversity in this species. Nuclear and mitochondrial data indicate genetic isolation of nearby populations on the same drainage, and field data suggest the decline of one study population, emphasizing the need for identification and protection of newt breeding sites in Iowa.     

Structure of the glomerular capillaries of the domestic chicken and desert quail

The glomerular capillary architecture of nephrons that include a loop of Henle (looped) and those that lack the loop (loopless) nephrons was examined qualitatively and quantitatively by electron microscopy in Gallus gallus and Callipepla gambelii. The glomerular capillaries of looped nephrons form a dichotomously branched network, while those of loopless nephrons are arranged loosely, and the majority are unbranched. There was no significant difference in the diameter of the glomerular capillaries between looped and loopless nephrons; however, in all cases the diameter of the afferent arteriole was significantly larger than that of the efferent arteriole. Based on size alone, the predicted blood flow rate in the efferent arteriole in 20% that of the afferent arteriole in G. gallus and 7% that of the afferent arteriole in C. gambelii. There was no significant difference in the volume density (Vv) of the glomerular capillaries between looped and loopless nephrons. However, the surface area density (Sv) of the glomerular capillaries in loopless nephrons of C. gambelii was significantly larger than for the looped nephrons, and for the loopless nephrons in G. gallus. This suggests that there may be a decrease in blood flow rate along the glomerular capillaries of the loopless nephrons in C. gambelii. Overall, the results indicate that the avian glomerular capillaries are less complex than those of mammals. Reasons may be that either avian blood is more viscous than that of mammals or that avian erythrocytes may be unable to fit physically through a tight intertwining network of capillaries due to the presence of a nucleus, which limits the tank-treading ability of avian erythrocytes. © 1995 Wiley-Liss, Inc.     

On the phylogenetic position of the scrub-birds (Passeriformes: Menurae: Atrichornithidae) of Australia

Evolutionary relationships of the scrub-birds Atrichornis were investigated using complete sequences of the recombination-activating gene RAG-1 and the proto-oncogene c-mos for two individuals of the noisy scrub-bird Atrichornis clamosus. Phylogenetic analysis revealed that Atrichornis was sister to the genus Menura (the lyrebirds) and that these two genera (the Menurae) were sister to the rest of the oscine passerines. A sister relationship between Atrichornis and Menura supports the traditional view, based on morphology and DNA hybridization, that these taxa are closely related. Similarly, a sister relationship with the remaining oscine passerines agrees with the morphological distinctiveness of Atrichornis and Menura, although this result contradicts conclusions based on DNA hybridization studies. Although Atrichornis is very well known morphologically, previous conclusions regarding its relationships were hampered by a lack of comparative knowledge of other passerines, making concurrence of the sequence data of particular significance.     

MITOCHONDRIAL-DNA DIVERSITY AND THE ORIGIN OF THE MENIDIA CLARKHUBBSI COMPLEX OF UNISEXUAL FISHES (ATHERINIDAE)

Restriction-endonuclease analysis of mitochondrial DNA (mtDNA) of the unisexual M. clarkhubbsi complex and close sexual relatives indicated that the unisexual complex arose through multiple, nonreciprocal hybridizations involving females of M. peninsulae. High-resolution analyses using restriction endonucleases that cleave at 4-bp sites revealed mtDNA sequence diversity that was low among unisexuals but high among individuals of M. peninsulae. The identification of M. peninsulae as a parent of the unisexuals conflicts with some details of previous allozyme comparisons. One possibility is that the unisexuals were derived from hybridization involving M. beryllina and a recently extinct form of M. peninsulae. In contrast to the unisexuals, contemporary hybrids of M. peninsulae and M. beryllina are formed by reciprocal matings. The origins of extant unisexual lineages from nonreciprocal hybridizations, together with their low mtDNA diversity relative to the maternal ancestor, implies strong constraints on origins of unisexuality via hybridization. Data on reproduction by contemporary F₁ hybrids reveal one form of genetic/developmental constraint: M. peninsulae and M. beryllina may now have diverged beyond the point where the hybrid origin of new unisexual lineages is possible.     

Hybridization at an ecotone: ecological and genetic barriers between three Iberian vipers

The formation of stable genetic boundaries between emerging species is often diagnosed by reduced hybrid fitness relative to parental taxa. This reduced fitness can arise from endogenous and/or exogenous barriers to gene flow. Although detecting exogenous barriers in nature is difficult, we can estimate the role of ecological divergence in driving species boundaries by integrating molecular and ecological niche modelling tools. Here, we focus on a three‐way secondary contact zone between three viper species (Vipera aspis, V. latastei and V. seoanei) to test for the contribution of ecological divergence to the development of reproductive barriers at several species traits (morphology, nuclear DNA and mitochondrial DNA). Both the nuclear and mitochondrial data show that all taxa are genetically distinct and that the sister species V. aspis and V. latastei hybridize frequently and backcross over several generations. We find that the three taxa have diverged ecologically and meet at a hybrid zone coincident with a steep ecotone between the Atlantic and Mediterranean biogeographical provinces. Integrating landscape and genetic approaches, we show that hybridization is spatially restricted to habitats that are suboptimal for parental taxa. Together, these results suggest that niche separation and adaptation to an ecological gradient confer an important barrier to gene flow among taxa that have not achieved complete reproductive isolation.