Differential barrier strength and allele frequencies in hybrid zones maintained by sex-biased hybrid incompatibilities

In animals, hybrid sterility and inviability between closely related species often affect only the heterogametic sex (XY). This widespread phenomenon, known as Haldane's rule, is an early speciation event found across broad taxa, but the role of heterogametic hybrid incompatibilities, as opposed to homogametic ones, as a barrier in a speciation process remains obscure. It has been hypothesized that heterogametic incompatibility may be a more efficient mechanism in driving speciation. The population dynamics after (rather than before) the occurrence of sex-biased incompatibilities may account for Haldane's rule. In this study, a recursion model of hybrid zones was developed to investigate the differences between heterogametic and homogametic incompatibilities. The selection strengths and selection patterns of sex chromosome-linked, two-locus Bateson-Dobzhansky-Muller (BDM) genetic incompatibilities were examined. It is noted that a sex-biased hybrid incompatibility in a hybrid zone confers asymmetric and uneven impedance to gene flow. The clines of different loci in such a hybrid zone displayed diverse differentiation in their width, steepness and asymmetry. Alleles involved in the incompatibility face much stronger resistance to cross a hybrid zone. Different sex-biased BDM incompatibilities also affect the flow of neutral alleles differently. Compared to a homogametic one, heterogametic incompatibility is a weaker but more asymmetric barrier. These unique patterns of gene flow may explain uneven divergence among different genomic regions during speciation between some closely related species.     

Differential introgression across newt hybrid zones: Evidence from replicated transects

Genomic heterogeneity of divergence between hybridizing species may reflect heterogeneity of introgression, but also processes unrelated to hybridization. Heterogeneous introgression and its repeatability can be directly tested in natural hybrid zones by examining multiple transects. Here, we studied hybrid zones between the European newts Lissotriton montandoni and two lineages of Lissotriton vulgaris, with replicate transects within each zone. Over 1,000 nuclear genes located on a linkage map and mitochondrial DNA were investigated using geographical and genomic clines. Overall, the five transects were all similar, showing hallmarks of strong reproductive isolation: bimodal distribution of genotypes in central populations and narrow allele frequency clines. However, the extent of introgression differed between the zones, possibly as a consequence of their different ages, as suggested by the analysis of heterozygosity runs in diagnostic markers. In three transects genomic signatures of small‐scale (~2 km) zone movements were detected. We found limited overlap of cline outliers between transects, and only weak evidence of stronger differentiation of introgression between zones than between transects within zones. Introgression was heterogeneous across linkage groups, with patterns of heterogeneity similar between transects and zones. Predefined candidates for increased or reduced introgression exhibited only a subtle tendency in the expected direction, suggesting that interspecific differentiation is not a reliable indicator for the strength of introgression. These hierarchically sampled hybrid zones of apparently different ages show how introgression unfolds with time and offer an excellent opportunity to dissect the dynamics of hybridization and architecture of reproductive isolation at advanced stages of speciation.     

Hybrid zones and the speciation continuum in Heliconius butterflies

Tropical butterflies in the genus Heliconius have long been models in the study of the stages of speciation. Heliconius are unpalatable to predators, and many species are notable for multiple geographic populations with striking warning colour pattern differences associated with Müllerian mimicry. A speciation continuum is evident in Heliconius hybrid zones. Examples range from hybrid zones across which (a) there is little genetic differentiation other than at mimicry loci, but where hybrids are common, (b) to ‘bimodal‘ hybrid zones with strong genetic divergence and few hybrids, (c) through to ‘good’ sympatric species, with hybridization extremely rare or absent. Now, in this issue of Molecular Ecology, Arias et al. ( 2012 ) have found an intermediate case in Colombian Heliconius cydno showing evidence for assortative mating and molecular differences, but where hybrids are abundant.     

Distinctiveness in the face of gene flow: hybridization between specialist and generalist gartersnakes

Patterns of divergence and polymorphism across hybrid zones can provide important clues as to their origin and maintenance. Unimodal hybrid zones or hybrid swarms are composed predominantly of recombinant individuals whose genomes are patchworks of alleles derived from each parental lineage. In contrast, bimodal hybrid zones contain few identifiable hybrids; most individuals fall within distinct genetic clusters. Distinguishing between hybrid swarms and bimodal hybrid zones can be important for taxonomic and conservation decisions regarding the status and value of hybrid populations. In addition, the causes of bimodality are important in understanding the generation and maintenance of biological diversity. For example, are distinct clusters mostly reproductively isolated and co‐adapted gene complexes, or can distinctiveness be maintained by a few ‘genomic islands’ despite rampant gene flow across much of the genome? Here we focus on three patterns of distinctiveness in the face of gene flow between gartersnake taxa in the Great Lakes region of North America. Bimodality, the persistence of distinct clusters of genotypes, requires strong barriers to gene flow and supports recognition of distinct specialist (Thamnophis butleri) and generalist (Thamnophis radix) taxa. Concordance of DNA‐based clusters with morphometrics supports the hypothesis that trophic morphology is a key component of divergence. Finally, disparity in the level of differentiation across molecular markers (amplified fragment length polymorphisms) indicates that distinctiveness is maintained by strong selection on a few traits despite high gene flow currently or in the recent past.     

Speciation through cytonuclear incompatibility: Insights from yeast and implications for higher eukaryotes

Several features of the yeast mitochondrial genome, including high mutation rate, dynamic genomic structure, small effective population size, and dispensability for cellular viability, make it a promising candidate for generating hybrid incompatibility and driving speciation. Cytonuclear incompatibility, a specific type of Dobzhansky‐Muller genetic incompatibility caused by improper interactions between mitochondrial and nuclear genomes, has previously been observed in a variety of organisms, yet its role in speciation remains obscure. Recent studies in Saccharomyces yeast species provide a new insight, with experimental evidence that cytonuclear incompatibility and DNA sequence divergence are both causes of the reproductive isolation of different yeast species. Interestingly, these two mechanisms seem to be perfectly complementary to each other in terms of their effects and evolutionary trajectories. Direct molecular analyses of the incompatible genes in yeasts have started to shed light on the evolutionary forces driving speciation. Editor's suggested further reading in BioEssays The cytoplasmic structure hypothesis for ribosome assembly, vertical inheritance, and phylogeny Abstract Mitochondrial bioenergetics as a major motive force of speciation Abstract     

Using differential introgression in hybrid zones to identify genomic regions involved in speciation

Hybrids between species provide information about the evolutionary processes involved in divergence. In addition to creating hybrids in the laboratory, biologists can take advantage of natural hybrid zones to understand the factors that shape gene flow between divergent lineages. In the early stages of speciation, most regions of the genome continue to flow freely between populations. Alternatively, the subset of the genome that confers reproductive barriers between nascent species is expected to reject introgression. Now enabled by advances in genomics, this perspective is motivating detailed comparisons of gene flow across genomic regions in hybrid zones. Here, I review methods for measuring and interpreting introgression at multiple loci in hybrid zones, focusing on the problem of identifying loci that contribute to reproductive isolation. Emerging patterns from multi-locus studies of hybrid zones are highlighted, including remarkable variance in introgression across the genome. Although existing methods have been useful, there is scope for development of new analytical approaches that better connect differential patterns of gene flow in hybrid zones with current knowledge of speciation mechanisms. I outline future prospects for differential introgression studies on a genomic scale.     

Impacts of climate warming on hybrid zone movement: Geographically diffuse and biologically porous “species borders”

Abstract The ecology and evolutionary biology of insect–plant associations has realized extensive attention, especially during the past 60 years. The classifications (categorical designations) of continuous variation in biodiversity, ranging from global patterns (e.g., latitudinal gradients in species richness/diversity and degree of herbivore feeding specialization) to localized insect–plant associations that span the biospectrum from polyphenisms, polymorphisms, biotypes, demes, host races, to cryptic species, remain academically contentious. Semantic and biosystematic (taxonomical) disagreements sometimes detract from more important ecological and evolutionary processes that drive diversification, the dynamics of gene flow and local extinctions. This review addresses several aspects of insect specialization, host‐associated divergence and ecological (including “hybrid”) speciation, with special reference to the climate warming impacts on species borders of hybridizing swallowtail butterflies (Papilionidae). Interspecific hybrid introgression may result in collapse of multi‐species communities or increase species numbers via homoploid hybrid speciation. We may see diverging, merging, or emerging genotypes across hybrid zones, all part of the ongoing processes of evolution. Molecular analyses of genetic mosaics and genomic dynamics with “divergence hitchhiking”, combined with ecological, ethological and physiological studies of “species porosity”, have already begun to unveil some answers for some important ecological/evolutionary questions. (i) How rapidly can host‐associated divergence lead to new species (and why doesn't it always do so, e.g., resulting in “incomplete” speciation)? (ii) How might “speciation genes” function, and how/where would we find them? (iii) Can oscillations from specialists to generalists and back to specialists help explain global diversity in herbivorous insects? (iv) How could recombinant interspecific hybridization lead to divergence and speciation? From ancient phytochemically defined angiosperm affiliations to recent and very local geographical mosaics, the Papilionidae (swallowtail butterflies) have provided a model for enhanced understanding of ecological patterns and evolutionary processes, including host‐associated genetic divergence, genomic mosaics, genetic hitchhiking and sex‐linked speciation genes. Apparent homoploid hybrid speciation in Papilio appears to have been catalyzed by climate warming‐induced interspecific introgression of some, but not all, species diagnostic traits, reflecting strong divergent selection (discordant), especially on the Z (= X) chromosome. Reproductive isolation of these novel recombinant hybrid genotypes appears to be accomplished via a delayed post‐diapause emergence or temporal isolation, and is perhaps aided by the thermal landscape. Changing thermal landscapes appear to have created (and may destroy) novel recombinant hybrid genotypes and hybrid species.     

A spatially explicit individual-based model of reinforcement in hybrid zones

Abstract An individual-based model consisting of two dioecious populations in a two-dimensional environmental grid was constructed. Each population began with, and never exceeded, 1000 individuals; extinction was allowed. Genomes consisting of 30 biallelic loci for male sexual advertisement call, female mate preference, and population origin were constructed, and lineages of each individual in the starting populations were followed for 2000 generations. Type and level of hybrid disadvantage, initial population distribution, patchiness of environmental resources, and level of mate choice were varied. Persistence of bimodal hybrid zones was nonexistent at low levels of hybrid disadvantage and universal at high levels of hybrid disadvantage, with a narrow threshold in which persistence was unpredictable. Persistence occurred at lower levels of hybrid disadvantage when populations were initially parapatric rather than sympatric, and environments were patchy rather than homogeneous. Increased divergence in mating systems occurred when hybrid disadvantage was high, hybrids were infertile, populations were initially parapatric, and increased female choice was allowed. Mating system divergence was much higher in interacting populations compared with noninteracting populations, indicating that reinforcement caused most of the observed divergence. When hybrids were infertile, reinforcement contributed to speciation, because under hybrid infertility the probability of persistence at low levels of hybrid disadvantage was positively related to mate choice. The results agree with previous one-dimensional spatial models in finding that population persistence is more likely in parapatric and patchy population distributions. In addition, the results show that hybrid infertility may facilitate the process of reinforcement and speciation.     

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.     

Dobzhansky-Muller and Wolbachia-Induced Incompatibilities in a Diploid Genetic System

Genetic incompatibilities are supposed to play an important role in speciation. A general (theoretical) problem is to explain the persistence of genetic diversity after secondary contact. Previous theoretical work has pointed out that Dobzhansky-Muller incompatibilities (DMI) are not stable in the face of migration unless local selection acts on the alleles involved in incompatibility. With local selection, genetic variability exists up to a critical migration rate but is lost when migration exceeds this threshold value. Here, we investigate the effect of intracellular bacteria Wolbachia on the stability of hybrid zones formed after the Dobzhansky Muller model. Wolbachia are known to cause a cytoplasmic incompatibility (CI) within and between species. Incorporating intracellular bacteria Wolbachia can lead to a significant increase of critical migration rates and maintenance of divergence, primarily because Wolbachia-induced incompatibility acts to reduce frequencies of F1 hybrids. Wolbachia infect up to two-thirds of all insect species and it is therefore likely that CI co-occurs with DMI in nature. The results indicate that both isolating mechanisms strengthen each other and under some circumstances act synergistically. Thus they can drive speciation processes more forcefully than either when acting alone.     

Genetic Architecture Underlying Nascent Speciation—The Evolution of Eurasian Pigs under Domestication

Speciation is a process whereby the evolution of reproductive barriers leads to isolated species. Although many studies have addressed large-effect genetic footprints in the advanced stages of speciation, the genetics of reproductive isolation in nascent stage of speciation remains unclear. Here, we show that pig domestication offers an interesting model for studying the early stages of speciation in great details. Pig breeds have not evolved the large X-effect of hybrid incompatibility commonly observed between “good species.” Instead, deleterious epistatic interactions among multiple autosomal loci are common. These weak Dobzhansky–Muller incompatibilities confer partial hybrid inviability with sex biases in crosses between European and East Asian domestic pigs. The genomic incompatibility is enriched in pathways for angiogenesis, androgen receptor signaling and immunity, with an observation of many highly differentiated cis-regulatory variants. Our study suggests that partial hybrid inviability caused by pervasive but weak interactions among autosomal loci may be a hallmark of nascent speciation in mammals.     

Hybridization and speciation

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near‐instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky–Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock‐on effects on speciation both within and outside regions of hybridization.     

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.     

Recombinant hybrids retain heterozygosity at many loci: new insights into the genomics of reproductive isolation in Populus

The maintenance of species barriers in the face of gene flow is often thought to result from strong selection against intermediate genotypes, thereby preserving genetic differentiation. Most speciation genomic studies thus aim to identify exceptionally divergent loci between populations, but divergence will be affected by many processes other than reproductive isolation (RI) and speciation. Through genomic studies of recombinant hybrids sampled in the wild, genetic variation associated with RI can be observed in situ, because selection against incompatible genotypes will leave detectable patterns of variation in the hybrid genomes. To better understand the mechanisms directly involved in RI, we investigated three natural ‘replicate’ hybrid zones between two divergent Populus species via locus‐specific patterns of ancestry across recombinant hybrid genomes. As expected, genomic patterns in hybrids and their parental species were consistent with the presence of underdominant selection at several genomic regions. Surprisingly, many loci displayed greatly increased between‐species heterozygosity in recombinant hybrids despite striking genetic differentiation between the parental genomes, the opposite of what would be expected with selection against intermediate genotypes. Only a limited, reproducible set of genotypic combinations was present in hybrid genomes across localities. In the absence of clearly delimited ‘hybrid habitats’, our results suggest that complex epistatic interactions within genomes play an important role in advanced stages of RI between these ecologically divergent forest trees. This calls for more genomic studies that test for unusual patterns of genomic ancestry in hybridizing species.     

Gene flow across a hybrid zone maintained by a weak heterogametic incompatibility and positive selection of incompatible alleles

Hybridization between incipient species is more likely to produce sterile or inviable F₁ offspring in the heterogametic (XY or ZW) sex than in the homogametic (XX or ZZ) sex, a phenomenon known as Haldane's rule. Population dynamics associated with Haldane's rule may play an important role in early speciation of sexually reproducing organisms. The dynamics of the hybrid zone maintained by incomplete hybrid inferiority (sterility/inviability) in the heterogametic sex (a ‘weak’ Haldane's rule) caused by a Bateson–Dobzhansky–Muller incompatibility was modelled. The influences and interplays of the strengths of incompatibility, dispersal, density‐dependent regulation (DDR) and local adaptation of incompatible alleles in a scenario of short‐range dispersal (the stepping‐stone model) were examined. It was found that a partial heterogametic hybrid incompatibility could efficiently impede gene flow and maintain characteristic clinal noncoincidence and discordance of alleles. Density‐dependent regulation appears to be an important factor affecting hybrid zone dynamics: it can effectively skew the effects of the partial incompatibility and dispersal as measured by effective dispersal, clinal structures and density depression. Unexpectedly, local adaptation of incompatible alleles in the parental populations, which would be critical for the establishment of the incompatibility, exerts little effect on hybrid zone dynamics. These results strongly support the plausibility of the adaptive origin of hybrid incompatibility and ecological speciation: an adaptive mutation, if it confers a marginal fitness advantage in the local population and happens to cause epistatic inferiority in hybrids, could efficiently drive further genetic divergence that may result in the gene becoming an evolutionary hotspot.     

Conspecific Sperm Precedence Is a Reproductive Barrier between Free-Spawning Marine Mussels in the Northwest Atlantic Mytilus Hybrid Zone

Reproductive isolation at the gamete stage has become a focus of speciation research because of its potential to evolve rapidly between closely related species. Conspecific sperm precedence (CSP), a type of gametic isolation, has been demonstrated in a number of taxa, both marine and terrestrial, with the potential to play an important role in speciation. Free-spawning marine invertebrates are ideal subjects for the study of CSP because of a likely central role for gametic barriers in reproductive isolation. The western Atlantic Mytilus blue mussel hybrid zone, ranging from the Atlantic Canada to eastern Maine, exhibits characteristics conducive to the study of CSP. Previous studies have shown that gametic incompatibility is incomplete, variable in strength and the genotype distribution is bimodal—dominated by the parental species, with a low frequency of hybrids. We conducted gamete crossing experiments using M. trossulus and M. edulis individuals collected from natural populations during the spring spawning season in order to detect the presence or absence of CSP within this hybrid zone. We detected CSP, defined here as a reduction in heterospecific offspring from competitive fertilizations in vitro compared to that seen in non-competitive fertilizations, in five of the twelve crosses in which conspecific crosses were detectable. This is the first finding of CSP in a naturally hybridizing population of a free-spawning marine invertebrate. Our findings support earlier predictions that CSP can promote assortative fertilization in bimodal hybrid zones, further advancing their hypothesized progression towards full speciation. Despite strong CSP numerous heterospecific fertilizations remain, reinforcing the hypothesis that compatible females are a source of hybrid offspring in mixed natural spawns.     

Long- term effects of previous experience determine nutrient discrimination abilities in birds

Background

The evolution of reproductive traits, such as hybrid incompatibility (postzygotic isolation) and species recognition (prezygotic isolation), have shown their key role in speciation. Theoretical modeling has recently predicted that close linkage between genes controlling pre- and postzygotic reproductive isolation could accelerate the conditions for speciation. Postzygotic isolation could develop during the sympatric speciation process contributing to the divergence of populations. Using hybrid fitness as a measure of postzygotic reproductive isolation, we empirically studied population divergence in perch (Perca fluviatilis L.) from two genetically divergent populations within a lake.

Results

During spawning time of perch we artificially created parental offspring and F₁ hybrids of the two populations and studied fertilization rate and hatching success under laboratory conditions. The combined fitness measure (product of fertilization rate and hatching success) of F₁ hybrids was significantly reduced compared to offspring from within population crosses.

Conclusion

Our results suggest intrinsic genetic incompatibility between the two populations and indicate that population divergence between two populations of perch inhabiting the same lake may indeed be promoted by postzygotic isolation.     

Island floras as model systems for studies of plant speciation: Prospects and challenges

Oceanic islands have long been called natural laboratories for studying evolution because they are geologically young, isolated, dynamic areas with diverse habitats over small spatial scales. Volcanic substrates of different ages permit the study of different stages of divergence and speciation within plant lineages. In addition to divergence, the dynamic island setting is conducive to hybridization. Discussion will focus on the potential of systematic/ecological studies, in combination with genomic data from high throughput sequencing and an ever‐increasing array of analytical techniques, for studying evolution in island plants. These studies may include: generation of highly resolved phylogenies to clarify the biogeography of speciation and whether divergence has occurred with or without gene flow; identification of the barriers to gene flow (extrinsic vs. intrinsic) of importance during divergence; documentation of historical and current hybridization events within island lineages; and elucidation of the genomic composition and ecology of hybrid populations in order to infer the evolutionary consequences of hybridization, such as the origin of stabilized homoploid hybrid species.     

Interspecific crossing and genetic mapping reveal intrinsic genomic incompatibility between two Senecio species that form a hybrid zone on Mount Etna,Sicily

Studies of hybridizing species can reveal much about the genetic basis and maintenance of species divergence in the face of gene flow. Here we report a genetic segregation and linkage analysis conducted on F₂ progeny of a reciprocal cross between Senecio aethnensis and S. chrysanthemifolius that form a hybrid zone on Mount Etna, Sicily, aimed at determining the genetic basis of intrinsic hybrid barriers between them. Significant transmission ratio distortion (TRD) was detected at 34 (∼27%) of 127 marker loci located in nine distinct clusters across seven of the ten linkage groups detected, indicating genomic incompatibility between the species. TRD at these loci could not be attributed entirely to post-zygotic selective loss of F₂ individuals that failed to germinate or flower (16.7%). At four loci tests indicated that pre-zygotic events, such as meiotic drive in F₁ parents or gametophytic selection, contributed to TRD. Additional tests revealed that cytonuclear incompatibility contributed to TRD at five loci, Bateson–Dobzhansky–Muller (BDM) incompatibilities involving epistatic interactions between loci contributed to TRD at four loci, and underdominance (heterozygote disadvantage) was a possible cause of TRD at one locus. Major chromosomal rearrangements were probably not a cause of interspecific incompatibility at the scale that could be examined with current map marker density. Intrinsic genomic incompatibility between S. aethnensis and S. chrysanthemifolius revealed by TRD across multiple genomic regions in early-generation hybrids is likely to impact the genetic structure of the natural hybrid zone on Mount Etna by limiting introgression and promoting divergence across the genome.     

Strong reproductive isolation and narrow genomic tracts of differentiation among three woodpecker species in secondary contact

Hybrid zones allow the measurement of gene flow across the genome, producing insight into the genomic architecture of speciation. Such analysis is particularly powerful when applied to multiple pairs of hybridizing species, as patterns of genomic differentiation can then be related to age of the hybridizing species, providing a view into the build‐up of differentiation over time. We examined 33 809 single nucleotide polymorphisms (SNPs) in three hybridizing woodpecker species: Red‐breasted, Red‐naped and Yellow‐bellied sapsuckers (Sphyrapicus ruber, Sphyrapicus nuchalis and Sphyrapicus varius), two of which (ruber and nuchalis) are much more closely related than each is to the third (varius). To identify positions of SNPs on chromosomes, we developed a localization method based on comparative genomics. We found narrow clines, bimodal distributions of hybrid indices and genomic regions with decreased rates of introgression. These results suggest moderately strong reproductive isolation among species and selection against specific hybrid genotypes. We found 19 small regions of strong differentiation between species, partly shared among species pairs, but no large regions of differentiation. An association analysis revealed a single strong‐effect candidate locus associated with plumage, possibly explaining mismatch among the three species in genomic relatedness and plumage similarity. Our comparative analysis of species pairs of different age and their hybrid zones showed that moderately strong reproductive isolation can occur with little genomic differentiation, but that reproductive isolation is incomplete even with much greater genomic differentiation, implying there are long periods of time when hybridization is possible if diverging populations are in geographic contact.