Along the speciation continuum: Quantifying intrinsic and extrinsic isolating barriers across five million years of evolutionary divergence in California jewelflowers

Understanding the relative roles of intrinsic and extrinsic reproductive barriers, and their interplay within the geographic context of diverging taxa, remains an outstanding challenge in the study of speciation. We conducted a comparative analysis of reproductive isolation in California Jewelflowers (Streptanthus, s.l., Brassicaceae) by quantifying potential barriers to gene flow at multiple life history stages in 39 species pairs spanning five million years of evolutionary divergence. We quantified nine potential pre‐ and postzygotic barriers and explored patterns of reproductive isolation in relation to genetic distance. Intrinsic postzygotic isolation was initially weak, increased at intermediate genetic distances, and reached a threshold characterized by complete genetic incompatibility. Climatic niche differences were strong at shallow genetic distances, and species pairs with overlapping ranges showed slight but appreciable phenological isolation, highlighting the potential for ecological barriers to contribute to speciation. Geographic analyses suggest that speciation is not regionally allopatric in the California Jewelflowers, as recently diverged taxa occur in relatively close proximity and display substantial range overlap. Young pairs are characterized by incomplete intrinsic postzygotic isolation, suggesting that extrinsic barriers or fine‐scale spatial segregation are more important early in the divergence process than genetic incompatibilities.     

Multiple strong postmating and intrinsic postzygotic reproductive barriers isolate florally diverse species of Jaltomata (Solanaceae)

Divergence in phenotypic traits often contributes to premating isolation between lineages, but could also promote isolation at postmating stages. Phenotypic differences could directly result in mechanical isolation or hybrids with maladapted traits; alternatively, when alleles controlling these trait differences pleiotropically affect other components of development, differentiation could indirectly produce genetic incompatibilities in hybrids. Here, we determined the strength of nine postmating and intrinsic postzygotic reproductive barriers among 10 species of Jaltomata (Solanaceae), including species with highly divergent floral traits. To evaluate the relative importance of floral trait diversification for the strength of these postmating barriers, we assessed their relationship to floral divergence, genetic distance, geographical context, and ecological differences, using conventional tests and a new linear‐mixed modeling approach. Despite close evolutionary relationships, all species pairs showed moderate to strong isolation. Nonetheless, floral trait divergence was not a consistent predictor of the strength of isolation; instead this was best explained by genetic distance, although we found evidence for mechanical isolation in one species, and a positive relationship between floral trait divergence and fruit set isolation across species pairs. Overall, our data indicate that intrinsic postzygotic isolation is more strongly associated with genome‐wide genetic differentiation, rather than floral divergence.     

Low reproductive isolation and highly variable levels of gene flow reveal limited progress towards speciation between European river and brook lampreys

Ecologically based divergent selection is a factor that could drive reproductive isolation even in the presence of gene flow. Population pairs arrayed along a continuum of divergence provide a good opportunity to address this issue. Here, we used a combination of mating trials, experimental crosses and population genetic analyses to investigate the evolution of reproductive isolation between two closely related species of lampreys with distinct life histories. We used microsatellite markers to genotype over 1000 individuals of the migratory parasitic river lamprey (Lampetra fluviatilis) and freshwater‐resident nonparasitic brook lamprey (Lampetra planeri) distributed in 10 sympatric and parapatric population pairs in France. Mating trials, parentage analyses and artificial fertilizations demonstrated a low level of reproductive isolation between species even though size‐assortative mating may contribute to isolation. Most parapatric population pairs were strongly differentiated due to the joint effects of geographic distance and barriers to migration. In contrast, we found variable levels of gene flow between sympatric populations ranging from panmixia to moderate differentiation, which indicates a gradient of divergence with some population pairs that may correspond to alternative morphs or ecotypes of a single species and others that remain partially isolated. Ecologically based divergent selection may explain these variable levels of divergence among sympatric population pairs, but incomplete genome swamping following secondary contact could have also played a role. Overall, this study illustrates how highly differentiated phenotypes can be maintained despite high levels of gene flow that limit the progress towards speciation.     

Behavioural mechanisms of sexual isolation involving multiple modalities and their inheritance

Speciation research dissects the genetics and evolution of reproductive barriers between parental species. Hybrids are the “gatekeepers” of gene flow, so it is also important to understand the behavioural mechanisms and genetics of any potential isolation from their parental species. We tested the role of multiple behavioural barriers in reproductive isolation among closely related field crickets and their hybrids (Teleogryllus oceanicus and Teleogryllus commodus). These species hybridize in the laboratory, but the behaviour of hybrids is unusual and there is little evidence for gene flow in the wild. We found that heterospecific pairs exhibited reduced rates of courtship behaviour due to discrimination by both sexes, and that this behavioural isolation was symmetrical. However, hybrids were not sexually selected against and exhibited high rates of courtship behaviour even though hybrid females are sterile. Using reciprocal hybrid crosses, we characterized patterns of interspecific divergence and inheritance in key sexual traits that might underlie the mating patterns we found: calling song, courtship song and cuticular hydrocarbons (CHCs). Song traits exhibited both sex linkage and transgressive segregation, whereas CHCs exhibited only the latter. Calculations of the strength of isolation exerted by these sexual traits suggest that close‐range signals are as important as long‐distance signals in contributing to interspecific sexual isolation. The surprisingly weak mating barriers observed between hybrids and parental species highlight the need to examine reproductive isolating mechanisms and their genetic bases across different potential stages of introgressive hybridization.     

Evolutionary insights into Rhinolophus episcopus (Chiroptera,Rhinolophidae) in China: Isolation by distance,environment, or sensory system?

Evolutionary processes can be influenced by several factors, such as geographic isolation, environmental selection, and sensory variation. For most nocturnal bats, echolocation is the primary sensory system used to prey and communicate, and plays important roles in chiropteran diversification and evolution. Understanding the relative contribution of geography, the environment, and this sensory system to population genetic divergence can elucidate the processes involved in bat incipient speciation and evolution. In this study, we collected spatial and environmental information, echolocation calls, as well as the previously published genetic data (six microsatellite loci and the mitochondrial cytochrome b gene) of widely distributed Rhinolophus episcopus populations to test three hypotheses for nuclear and mitochondrial divergence (isolation by distance, isolation by environment, and isolation by sensory variation) and unveil the factors that drive intraspecific genetic differentiation. The moderate level of nuclear differentiation was correlated with geographic/spatial distance and acoustic variation, whereas the relatively high level of mitochondrial differentiation was mainly associated with acoustic divergence. No significant correlation was observed between genetic divergence and environmental variables. Among the three factors, acoustic divergence explained the highest percentage of both nuclear and mitochondrial divergence. Thus, our results indicate that sensory variation may have played important roles in driving population isolation early in bat speciation, which is consistent with the hypothesis of isolation by sensory variation. Our study emphasizes the need to consider more factors, especially sensory traits, and combine multiple statistical methods in landscape genetic studies to test their potential contributions to driving population divergence.     

Strong postmating reproductive isolation in Mimulus section Eunanus

Postmating reproductive isolation can help maintain species boundaries when premating barriers to reproduction are incomplete. The strength and identity of postmating reproductive barriers are highly variable among diverging species, leading to questions about their genetic basis and evolutionary drivers. These questions have been tackled in model systems but are less often addressed with broader phylogenetic resolution. In this study we analyse patterns of genetic divergence alongside direct measures of postmating reproductive barriers in an overlooked group of sympatric species within the model monkeyflower genus, Mimulus. Within this Mimulus brevipes species group, we find substantial divergence among species, including a cryptic genetic lineage. However, rampant gene discordance and ancient signals of introgression suggest a complex history of divergence. In addition, we find multiple strong postmating barriers, including postmating prezygotic isolation, hybrid seed inviability and hybrid male sterility. M. brevipes and M. fremontii have substantial but incomplete postmating isolation. For all other tested species pairs, we find essentially complete postmating isolation. Hybrid seed inviability appears linked to differences in seed size, providing a window into possible developmental mechanisms underlying this reproductive barrier. While geographic proximity and incomplete mating isolation may have allowed gene flow within this group in the distant past, strong postmating reproductive barriers today have likely played a key role in preventing ongoing introgression. By producing foundational information about reproductive isolation and genomic divergence in this understudied group, we add new diversity and phylogenetic resolution to our understanding of the mechanisms of plant speciation.     

Isolation by distance,not incipient ecological speciation,explains genetic differentiation in an Andean songbird (Aves: Furnariidae: Cranioleuca antisiensis,Line‐cheeked Spinetail) despite near threefold body size change across an environmental gradient

During the process of ecological speciation, reproductive isolation results from divergent natural selection and leads to a positive correlation between genetic divergence and adaptive phenotypic divergence, that is, isolation by adaptation (IBA). In natural populations, phenotypic differentiation is often autocorrelated with geographic distance, making IBA difficult to distinguish from the neutral expectation of isolation by distance (IBD). We examined these two alternatives in a dramatic case of clinal phenotypic variation in an Andean songbird, the Line‐cheeked Spinetail (Cranioleuca antisiensis). At its geographic extremes, this species shows a near threefold difference in body mass (11.5 to 31.0 g) with marked plumage differences. We analysed phenotypic, environmental and genetic data (5,154 SNPs) from 172 individuals and 19 populations sampled along its linear distribution in the Andes. We found that body mass was tightly correlated with environmental temperature, consistent with local adaptation as per Bergmann's rule. Using a P_ST–F_ST analysis, we found additional support for natural selection driving body mass differentiation, but these results could also be explained by environment‐mediated phenotypic plasticity. When we assessed the relative support for patterns of IBA and IBD using variance partitioning, we found that IBD was the best explanation for genetic differentiation along the cline. Adaptive phenotypic or environmental divergence can reduce gene flow, a pattern interpreted as evidence of ecological speciation's role in diversification. Our results provide a counterexample to this interpretation. Despite conditions conducive to ecological speciation, our results suggest that dramatic size and environmental differentiation within C. antisiensis are not limiting gene flow.     

Two reproductively isolated cytotypes and a swarm of highly inbred, disconnected populations: a glimpse into Salicornia's evolutionary history and challenging taxonomy

The main factor of differentiation at six nuclear microsatellite and seven cpDNA loci in Salicornia from the Mediterranean and Atlantic coasts of France is cytotypic identity, suggesting the presence of a strong reproductive barrier among sympatric cytotypes. Within cytotypes, a substantial proportion of the differentiation between species is due to confounded phylogeographic signal. Conspecific individuals tend to be significantly more related than individuals from different species at the population scale, but mean kinship coefficients among pairs of conspecific and nonconspecific individuals from different populations are not significantly different, suggesting the absence of reproductive isolation among species of the same cytotype. The observed association between morphology and genetic variation within populations would thus result from the selfing mating system (F(is)) = 0.70) generating substantial linkage within the genome, linkage that would quickly disappear among unrelated individuals from different populations. Salicornia species thus function as a network of inbred populations, strongly challenging taxonomic concepts.     

Geographic isolation facilitates the evolution of reproductive isolation and morphological divergence

Geographic isolation is known to contribute to divergent evolution, resulting in unique phenotypes. Oftentimes morphologically distinct populations are found to be interfertile while reproductive isolation is found to exist within nominal morphological species revealing the existence of cryptic species. These disparities can be difficult to predict or explain especially when they do not reflect an inferred history of common ancestry which suggests that environmental factors affect the nature of ecological divergence. A series of laboratory experiments and observational studies were used to address what role biogeographic factors may play in the ecological divergence of Hyalella amphipods. It was found that geographic isolation plays a key role in the evolution of reproductive isolation and divergent morphology and that divergence cannot be explained by molecular genetic variation.     

Phenotypic divergence and reproductive isolation between sympatric forms of Japanese threespine sticklebacks

The threespine stickleback ( Gasterosteus aculeatus ) species complex is well suited for identifying the types of phenotypic divergence and isolating barriers that contribute to reproductive isolation at early stages of speciation. In the present study, we characterize the patterns of genetic and phenotypic divergence as well as the types of isolating barriers that are present between two sympatric pairs of threespine sticklebacks in Hokkaido, Japan. One sympatric pair consists of an anadromous and a resident freshwater form and shows divergence in body size between the forms, despite the lack of genetic differentiation between them. The second sympatric pair consists of two anadromous forms, which originated before the last glacial period and are currently reproductively isolated. These two anadromous forms have diverged in many morphological traits as well as in their reproductive behaviours. Both sexual isolation and hybrid male sterility contribute to reproductive isolation between the anadromous species pair. We discuss the shared and unique aspects of phenotypic divergence and reproductive isolation in the Japanese sympatric pairs compared with postglacial stickleback species pairs. Further studies of these divergent species pairs will provide a deeper understanding of the mechanisms of speciation in sticklebacks.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 671–685.     

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

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

Mechanical barriers to introgressive hybridization revealed by mitochondrial introgression patterns in Ohomopterus ground beetle assemblages

To reveal the role of diverged body size and genital morphology in reproductive isolation among closely related species, we examined patterns of, and factors limiting, introgressive hybridization between sympatric Ohomopterus ground beetles in central Japan using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences. We sampled 17 local assemblages that consisted of two to five species and estimated levels of interspecific gene flow using the genetic distance, D(A), and maximum-likelihood estimates of gene flow. Sharing of haplotypes or haplotype lineages was detected between six of seven species that occurred in the study areas, indicating mitochondrial introgression. The intensity and direction of mitochondrial gene flow were variable among species pairs. To determine the factors affecting introgression patterns, we tested the relationships between interspecific D(A) and five independent variables: difference in body size, difference in genital size, phylogenetic relatedness (nuclear gene sequence divergence), habitat difference, and species richness of the assemblage. Body and genital size differences contributed significantly to preventing gene flow. Thus, mechanical isolation mechanisms reduce the chance of introgressive hybridization between closely related species. Our results highlight the role of morphological divergence in speciation and assemblage formation processes through mechanical isolation.     

Divergence in style length and pollen size leads to a postmating‐prezygotic reproductive barrier among populations of Silene latifolia

A central tenet of speciation research is the need to identify reproductive isolating barriers. One approach to this line of research is to identify the phenotypes that lead to reproductive isolation. Several studies on flowering plants have shown that differences in style length contribute to reproductive isolation between species, leading us to consider whether style length could act as a reproductive barrier among populations of a single species. This could occur if style length varied sufficiently and pollen size covaried with style length. Populations of Silene latifolia exhibit variation in flower size, including style length, that is negatively correlated with annual precipitation. We show that this divergence in style length has a genetic basis and acts as a reproductive barrier: males from small‐flowered populations produced relatively small pollen grains that were poor at fertilizing ovules when crossed to females from large‐flowered populations, leading to a significant reduction in seed production. Manipulating the distance pollen tubes had to travel revealed that this failure was purely mechanical and not the result of other incompatibilities. These results show that style length acts as a postmating‐prezygotic reproductive barrier and indicate a potential link between ecotypic differentiation and reproductive isolation within a species.     

Geography is more important than host plant use for the population genetic structure of a generalist insect herbivore

Population divergence can occur due to mechanisms associated with geographic isolation and/or due to selection associated with different ecological niches. Much of the evidence for selection‐driven speciation has come from studies of specialist insect herbivores that use different host plant species; however, the influence of host plant use on population divergence of generalist herbivores remains poorly understood. We tested how diet breadth, host plant species and geographic distance influence population divergence of the fall webworm (Hyphantria cunea; FW). FW is a broadly distributed, extreme generalist herbivore consisting of two morphotypes that have been argued to represent two different species: black‐headed and red‐headed. We characterized the differentiation of FW populations at two geographic scales. We first analysed the influence of host plant and geographic distance on genetic divergence across a broad continental scale for both colour types. We further analysed the influence of host plant, diet breadth and geographic distance on divergence at a finer geographic scale focusing on red‐headed FW in Colorado. We found clear genetic and morphological distinction between red‐ and black‐headed FW, and Colorado FW formed a genetic cluster distinct from other locations. Although both geographic distance and host plant use were correlated with genetic distance, geographic distance accounted for up to 3× more variation in genetic distance than did host plant use. As a rare study investigating the genetic structure of a widespread generalist herbivore over a broad geographic range (up to 3,000 km), our study supports a strong role for geographic isolation in divergence in this system.     

Genetic and morphological divergence reveals local subdivision of perch (Perca fluviatilis L.)

The level of gene flow is an important factor influencing genetic differentiation between populations. Typically, geographic distance is considered to be the major factor limiting dispersal and should thus only influence the degree of genetic divergence at larger spatial scales. However, recent studies have revealed the possibility for small-scale genetic differentiation, suggesting that the spatial scale considered is pivotal for finding patterns of isolation by distance. To address this question, genetic and morphological differentiation were studied at two spatial scales (range 2–13 km and range 300 m to 2 km) in the perch ( Perca fluviatilis L.) from the east coast archipelago of Sweden, using seven microsatellite loci and geometric morphometrics. We found highly significant genetic differentiation between sampled locations at both scales. At the larger spatial scale, the distance per se was not affecting the level of divergence. At the small scale, however, we found subtle patterns of isolation by distance. In addition, we also found morphological divergence between locations, congruent with a spatial separation at a microgeographic scale, most likely due to phenotypic plasticity. The present study highlights the importance of geographical scale and indicates that there might be a disparity between the dispersal capacity of a species and the actual movement of genes. Thus, how we view the environment and possible barriers to dispersal might have great implications for our ability to fully understand the evolution of genetic differentiation, local adaptation, and, in the end, speciation.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 746–758.     

Glacial cycles drive rapid divergence of cryptic field vole species

Understanding the factors that contribute to the generation of reproductively isolated forms is a fundamental goal of evolutionary biology. Cryptic species are an especially interesting challenge to study in this context since they lack obvious morphological differentiation that provides clues to adaptive divergence that may drive reproductive isolation. Geographical isolation in refugial areas during glacial cycling is known to be important for generating genetically divergent populations, but its role in the origination of new species is still not fully understood and likely to be situation dependent. We combine analysis of 35,434 single‐nucleotide polymorphisms (SNPs) with environmental niche modeling (ENM) to investigate genomic and ecological divergence in three cryptic species formerly classified as the field vole (Microtus agrestis). The SNPs demonstrate high genomic divergence (pairwise F_ST values of 0.45–0.72) and little evidence of gene flow among the three field vole cryptic species, and we argue that genetic drift may have been a particularly important mechanism for divergence in the group. The ENM reveals three areas as potential glacial refugia for the cryptic species and differing climatic niches, although with spatial overlap between species pairs. This evidence underscores the role that glacial cycling has in promoting genetic differentiation and reproductive isolation by subdivision into disjunct distributions at glacial maxima in areas relatively close to ice sheets. Future investigation of the intrinsic barriers to gene flow between the field vole cryptic species is required to fully assess the mechanisms that contribute to reproductive isolation. In addition, the Portuguese field vole (M. rozianus) shows a high inbreeding coefficient and a restricted climatic niche, and warrants investigation into its conservation status.     

PATTERNS OF ALLOZYME RELATIONSHIPS COMPARED WITH MORPHOLOGY,HYBRIDIZATION, AND GEOLOGIC HISTORY IN ALLOPATRIC ISLAND-DWELLING MOSQUITOES

Allozyme relationships were compared with morphology, interspecific hybridization, and geologic history in 14 closely-related species in the Aedes (Stegomyia) scutellaris subgroup. The phylogeny generated by the electrophoretic data was generally in agreement with morphological classifications except that one morphologically distinct species pair (A. alcasidi and A. malayensis) showed only populational differentiation and several nearly identical morphological pairs in Polynesia (e.g., A. pseudoscutellaris and A. polynesiensis) were very distant genetically. A molecular clock, based on the ratio between genetic distance and divergence time (one unit of Nei's distance = 11 million years) inferred from a dated geologic event, was used to compare zoogeography, morphology, and biochemical relationships. Genetic distances between species from the Solomon Islands and the Vanuatu-Polynesia area correspond to an early Pliocene separation of these areas. An invasion of Micronesia occurred approximately 5 million years ago. Most remaining speciation occurred during the mid-Pliocene as islands moved into their present-day positions. The close relationship between southeast Asian and Philippine species could have resulted from the recent onset of isolation in the Pleistocene. Interspecific hybridization potential was not significantly correlated with Nei's genetic distance. Many quite distantly related species readily hybridize, while some closely related pairs show only unidirectional compatibility. Slight interspecific morphological divergence and the independence of hybridization capabilities from phylogeny are not unexpected in a group in which species are allopatric and occupy similar habitats.     

Elevational speciation in action? Restricted gene flow associated with adaptive divergence across an altitudinal gradient

Evolutionary theory predicts that divergent selection pressures across elevational gradients could cause adaptive divergence and reproductive isolation in the process of ecological speciation. Although there is substantial evidence for adaptive divergence across elevation, there is less evidence that this restricts gene flow. Previous work in the boreal chorus frog (Pseudacris maculata) has demonstrated adaptive divergence in morphological, life history and physiological traits across an elevational gradient from approximately 1500–3000 m in the Colorado Front Range, USA. We tested whether this adaptive divergence is associated with restricted gene flow across elevation – as would be expected if incipient speciation were occurring – and, if so, whether behavioural isolation contributes to reproductive isolation. Our analysis of 12 microsatellite loci in 797 frogs from 53 populations revealed restricted gene flow across elevation, even after controlling for geographic distance and topography. Calls also varied significantly across elevation in dominant frequency, pulse number and pulse duration, which was partly, but not entirely, due to variation in body size and temperature across elevation. However, call variation did not result in strong behavioural isolation: in phonotaxis experiments, low‐elevation females tended to prefer an average low‐elevation call over a high‐elevation call, and vice versa for high‐elevation females, but this trend was not statistically significant. In summary, our results show that adaptive divergence across elevation restricts gene flow in P. maculata, but the mechanisms for this potential incipient speciation remain open.     

Environmental selection is a main driver of divergence in house sparrows (Passer domesticus) in Romania and Bulgaria

Both neutral and adaptive evolutionary processes can cause population divergence, but their relative contributions remain unclear. We investigated the roles of these processes in population divergence in house sparrows (Passer domesticus) from Romania and Bulgaria, regions characterized by high landscape heterogeneity compared to Western Europe. We asked whether morphological divergence, complemented with genetic data in this human commensal species, was best explained by environmental variation, geographic distance, or landscape resistance—the effort it takes for an individual to disperse from one location to the other—caused by either natural or anthropogenic barriers. Using generalized dissimilarity modeling, a matrix regression technique that fits biotic beta diversity to both environmental predictors and geographic distance, we found that a small set of climate and vegetation variables explained up to ~30% of the observed divergence, whereas geographic and resistance distances played much lesser roles. Our results are consistent with signals of selection on morphological traits and of isolation by adaptation in genetic markers, suggesting that selection by natural environmental conditions shapes population divergence in house sparrows. Our study thus contributes to a growing body of evidence that adaptive evolution may be a major driver of diversification.     

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

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