Indirect Evolution of Hybrid Lethality Due to Linkage with Selected Locus in Mimulus guttatus

Most species are superbly and intricately adapted to the environments in which they live. Adaptive evolution by natural selection is the primary force shaping biological diversity. Differences between closely related species in ecologically selected characters such as habitat preference, reproductive timing, courtship behavior, or pollinator attraction may prevent interbreeding in nature, causing reproductive isolation. But does ecological adaptation cause reproductive incompatibilities such as hybrid sterility or lethality? Although several genes causing hybrid incompatibilities have been identified, there is intense debate over whether the genes that contribute to ecological adaptations also cause hybrid incompatibilities. Thirty years ago, a genetic study of local adaptation to copper mine soils in the wildflower Mimulus guttatus identified a locus that appeared to cause copper tolerance and hybrid lethality in crosses to other populations. But do copper tolerance and hybrid lethality have the same molecular genetic basis? Here we show, using high-resolution genome mapping, that copper tolerance and hybrid lethality are not caused by the same gene but are in fact separately controlled by two tightly linked loci. We further show that selection on the copper tolerance locus indirectly caused the hybrid incompatibility allele to go to high frequency in the copper mine population because of hitchhiking. Our results provide a new twist on Darwin''s original supposition that hybrid incompatibilities evolve as an incidental by-product of ordinary adaptation to the environment.     

GENETIC ARCHITECTURE AND POSTZYGOTIC REPRODUCTIVE ISOLATION: EVOLUTION OF BATESON–DOBZHANSKY–MULLER INCOMPATIBILITIES IN A POLYGENIC MODEL

The Bateson–Dobzhansky–Muller model predicts that postzygotic isolation evolves due to the accumulation of incompatible epistatic interactions, but few studies have quantified the relationship between genetic architecture and patterns of reproductive divergence. We examined how the direction and magnitude of epistatic interactions in a polygenic trait under stabilizing selection influenced the evolution of hybrid incompatibilities. We found that populations evolving independently under stabilizing selection experienced suites of compensatory allelic changes that resulted in genetic divergence between populations despite the maintenance of a stable, high‐fitness phenotype. A small number of loci were then incompatible with multiple alleles in the genetic background of the hybrid and the identity of these incompatibility loci changed over the evolution of the populations. For F₁ hybrids, reduced fitness evolved in a window of intermediate strengths of epistatic interactions, but F₂ and backcross hybrids evolved reduced fitness across weak and moderate strengths of epistasis due to segregation variance. Strong epistatic interactions constrained the allelic divergence of parental populations and prevented the development of reproductive isolation. Because many traits with varying genetic architectures must be under stabilizing selection, our results indicate that polygenetic drift is a plausible hypothesis for the evolution of postzygotic reproductive isolation.     

Investigating Incipient Speciation in Arabidopsis lyrata from Patterns of Transmission Ratio Distortion

Our understanding of the development of intrinsic reproductive isolation is still largely based on theoretical models and thorough empirical studies on a small number of species. Theory suggests that reproductive isolation develops through accumulation of epistatic genic incompatibilities, also known as Bateson–Dobzhansky–Muller (BDM) incompatibilities. We can detect these from marker transmission ratio distortion (TRD) in hybrid progenies of crosses between species or populations, where TRD is expected to result from selection against heterospecific allele combinations in hybrids. TRD may also manifest itself because of intragenomic conflicts or competition between gametes or zygotes. We studied early stage speciation in Arabidopsis lyrata by investigating patterns of TRD across the genome in F₂ progenies of three reciprocal crosses between four natural populations. We found that the degree of TRD increases with genetic distance between crossed populations, but also that reciprocal progenies may differ substantially in their degree of TRD. Chromosomes AL6 and especially AL1 appear to be involved in many single- and two-locus distortions, but the location and source of TRD vary between crosses and between reciprocal progenies. We also found that the majority of single- and two-locus TRD appears to have a gametic, as opposed to zygotic, origin. Thus, while theory on BDM incompatibilities is typically illustrated with derived nuclear alleles proving incompatible in hybrid zygotes, our results suggest a prominent role for distortions emerging before zygote formation.     

Signatures of hybridization and speciation in genomic patterns of ancestry*

Genomes sampled from hybrid zones between nascent species provide important clues into the speciation process. With advances in genome sequencing and single nucleotide polymorphism (SNP) genotyping, it is now feasible to measure variation in gene flow with high genomic resolution. This progress motivates the development of conceptual and analytical frameworks for hybrid zones that complement well‐established cline approaches. We extend the perspective that genomic distributions of ancestry are sensitive indicators of hybridization history. We use simulations to examine the behavior of the number of ancestry junctions—a simple summary of genomic patterns—in hybrid zones under increasingly realistic scenarios. Neutral simulations revealed that ancestry junction number is shaped by population structure, migration rate, and population size. Modeling multiple genetic architectures of hybrid dysfunction, with an emphasis on epistatic hybrid incompatibilities, showed that selection reduces junction number near loci that confer reproductive barriers. The magnitude of this signature was affected by the form of selection, dominance, and genomic location (autosome vs. sex chromosome) of incompatible loci. Our results suggest that researchers can identify loci involved in reproductive isolation by scanning hybrid genomes for local reductions in junction number. We outline necessary directions for future theory and method development to realize this goal.     

Postzygotic isolation drives genomic speciation between highly cryptic Hypocnemis antbirds from Amazonia

How species evolve reproductive isolation in the species-rich Amazon basin is poorly understood in vertebrates. Here, we sequenced a reference genome and used a genome-wide sample of SNPs to analyze a hybrid zone between two highly cryptic species of Hypocnemis warbling-antbirds—the Rondonia warbling-antbird (H. ochrogyna) and Spix's warbling-antbird (H. striata)—in a headwater region of southern Amazonia. We found that both species commonly hybridize, producing F₁s and a variety of backcrosses with each species but we detected only one F₂-like hybrid. Patterns of heterozygosity, hybrid index, and interchromosomal linkage disequilibrium in hybrid populations closely match expectations under strong postzygotic isolation. Hybrid zone width (15.4 km) was much narrower than expected (211 km) indicating strong selection against hybrids. A remarkably high degree of concordance in cline centers and widths across loci, and a lack of reduced interspecific F_st between populations close to versus far from the contact zone, suggest that genetic incompatibilities have rendered most of the genome immune to introgression. These results support intrinsic postzygotic isolation as a driver of speciation in a moderately young cryptic species pair from the Amazon and suggest that species richness of the Amazon may be grossly underestimated.     

Analysis of ancestry heterozygosity suggests that hybrid incompatibilities in threespine stickleback are environment dependent

Hybrid incompatibilities occur when interactions between opposite ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are “intrinsic,” meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis using sequence data are scarce. In this article, we compiled genetic data for F₂ hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (seminatural experimental ponds) or the laboratory (aquaria). Because selection against incompatibilities results in elevated ancestry heterozygosity, we tested the prediction that ancestry heterozygosity will be higher in pond-raised fish compared to those raised in aquaria. We found that ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Additional analyses support a phenotypic basis for incompatibility and suggest that environment-specific single-locus heterozygote advantage is not the cause of selection on ancestry heterozygosity. Our study provides evidence that, in stickleback, a coarse—albeit indirect—signal of environment-dependent hybrid incompatibility is reliably detectable and suggests that extrinsic incompatibilities can evolve before intrinsic incompatibilities.

This study shows that hybrid incompatibilities between two independent pairs of hybridizing stickleback populations only appear under relevant ecological circumstances, implying that incompatibilities evolve before they can be detected in laboratory studies of speciation.     

A fine-scale genetic analysis of hybrid incompatibilities in Drosophila

The sterility and inviability of species hybrids is thought to evolve by the accumulation of genes that cause generally recessive, incompatible epistatic interactions between species. Most analyses of the loci involved in such hybrid incompatibilities have suffered from low genetic resolution. Here I present a fine-resolution genetic screen that allows systematic counting, mapping, and characterizing of a large number of hybrid incompatibility loci in a model genetic system. Using small autosomal deletions from D. melanogaster and a hybrid rescue mutation from D. simulans, I measured the viability of hybrid males that are simultaneously hemizygous for a small region of the D. simulans autosomal genome and hemizygous for the D. melanogaster X chromosome. These hybrid males are exposed to the full effects of any recessive-recessive epistatic incompatibilities present in these regions. A screen of approximately 70% of the D. simulans autosomal genome reveals 20 hybrid-lethal and 20 hybrid-semilethal regions that are incompatible with the D. melanogaster X. In further crosses, I confirm the epistatic nature of hybrid lethality by showing that all of the incompatibilities are rescued when the D. melanogaster X is replaced with a D. simulans X. Combined with information from previous studies, these results show that the number of recessive incompatibilities is approximately eightfold larger than the number of dominant ones. Finally, I estimate that a total of approximately 191 hybrid-lethal incompatibilities separate D. melanogaster and D. simulans, indicating extensive functional divergence between these species' genomes.     

Hybrid songbirds employ intermediate routes in a migratory divide

Migratory divides are contact zones between populations that use different routes to navigate around unsuitable areas on seasonal migration. Hybrids in divides have been predicted to employ intermediate and potentially inferior routes. We provide the first direct test of this hypothesis, using light‐level geolocators to track birds breeding in a hybrid zone between Swainson's thrushes in western Canada. Compared to parental forms, hybrids exhibited increased variability in their migratory routes, with some using intermediate routes that crossed arid and mountainous regions, and some using the same routes as one parental group on fall migration and the other on spring migration. Hybrids also tended to use geographically intermediate wintering sites. Analysis of genetic variation across the hybrid zone suggests moderately strong selection against hybrids. These results indicate that seasonal migratory behaviour might be a source of selection against hybrids, supporting a possible role for migration in speciation.     

Genetics of Hybrid Sterility and Hybrid Breakdown in an Intersubspecific Rice (Oryza Sativa L.) Population

F(1) hybrid sterility and ``hybrid breakdown' of F(2) and later generations in rice (Oryza sativa L.) are common and genetically complicated. We used a restriction fragment length polymorphism linkage map and F(4) progeny testing to investigate hybrid sterility and hybrid breakdown in a cross between ``widely compatible' O. sativa ssp. japonica cultivar Lemont from the Southern U.S. and ssp. indica cultivar Teqing from China. Our results implicate different genetic mechanisms in hybrid sterility and hybrid breakdown, respectively. Hybrid sterility appeared to be due to recombination within a number of putative differentiated ``supergenes' in the rice genome, which may reflect cryptic structural rearrangements. The cytoplasmic genome had a large effect on fertility of both male and female gametes in the F(1) hybrids. There appeared to be a pair of complementary genes that behaved like ``wide compatibility' genes. This pair of genes and the ``gamete eliminator' (S(1)) or ``egg killer' (S-5) may influence the phenotypic effects of presumed supergenes in hybrids. Hybrid breakdown appeared to be largely due to incompatibilities between indica and japonica alleles at many unlinked epistatic loci in the genome. These proposed mechanisms may partly account for the complicated nature of postreproductive barriers in rice.     

The genetic architecture of hybrid incompatibilities and their effect on barriers to introgression in secondary contact

Genetic incompatibilities are an important component of reproductive isolation. Although theoretical studies have addressed their evolution, little is known about their maintenance when challenged by potentially high migration rates in secondary contact. Although theory predicts that recombination can erode barriers, many empirical systems have been found to retain species‐specific differences despite substantial gene flow. By simulating whole genomes in individuals of hybridizing species, we find that the genetic architecture of two contrasting models of epistatic hybrid incompatibilities and the context of hybridization can substantially affect species integrity and genomic heterogeneity. In line with theory, our results show that intergenomic incompatibilities break down rapidly by recombination, but can maintain genome‐wide differentiation under very limited conditions. By contrast, intragenomic interactions that arise from genetic pathways can maintain species‐specific differences even with high migration rates and gene flow, whereas introgression at large parts of the genome can simultaneously remain extensive, consistent with empirical observations. We discuss the importance of intragenomic interactions in speciation and consider how this form of epistatic fitness variation is implicated and supported by other theoretical and empirical studies. We further address the relevance of replicates and knowledge of context when investigating the genomics of speciation.     

The Genomic Impacts of Drift and Selection for Hybrid Performance in Maize

Although maize is naturally an outcrossing organism, modern breeding utilizes highly inbred lines in controlled crosses to produce hybrids. The U.S. Department of Agriculture’s reciprocal recurrent selection experiment between the Iowa Stiff Stalk Synthetic (BSSS) and the Iowa Corn Borer Synthetic No. 1 (BSCB1) populations represents one of the longest running experiments to understand the response to selection for hybrid performance. To investigate the genomic impact of this selection program, we genotyped the progenitor lines and >600 individuals across multiple cycles of selection using a genome-wide panel of ∼40,000 SNPs. We confirmed previous results showing a steady temporal decrease in genetic diversity within populations and a corresponding increase in differentiation between populations. Thanks to detailed historical information on experimental design, we were able to perform extensive simulations using founder haplotypes to replicate the experiment in the absence of selection. These simulations demonstrate that while most of the observed reduction in genetic diversity can be attributed to genetic drift, heterozygosity in each population has fallen more than expected. We then took advantage of our high-density genotype data to identify extensive regions of haplotype fixation and trace haplotype ancestry to single founder inbred lines. The vast majority of regions showing such evidence of selection differ between the two populations, providing evidence for the dominance model of heterosis. We discuss how this pattern is likely to occur during selection for hybrid performance and how it poses challenges for dissecting the impacts of modern breeding and selection on the maize genome.     

Variable patterns of introgression in two sculpin hybrid zones suggest that genomic isolation differs among populations

Theory predicts that reproductive isolation may be due to intrinsic genetic incompatibilities or extrinsic ecological factors. Therefore, an understanding of the genetic basis of isolation may require analyses of evolutionary processes in situ to include environmental factors. Here we study genetic isolation between populations of sculpins ( Cottus ) at 168 microsatellites. Genomic clines were fit using 480 individuals sampled across independent natural hybrid zones that have formed between one invading species and two separate populations of a resident species. Our analysis tests for deviations from neutral patterns of introgression at individual loci based on expectations given genome-wide admixture. Roughly 51% of the loci analysed displayed significant deviations. An overall deficit of interspecific heterozygotes in 26% and 21% of the loci suggests that widespread underdominance drives genomic isolation. At the same time, selection promotes introgression of almost 30% of the markers, which implies that hybridization may increase the fitness of admixed individuals. Cases of overdominance or epistatic interactions were relatively rare. Despite the similarity of the two hybrid zones in their overall genomic composition, patterns observed at individual loci show little correlation between zones and many fit different genotypic models of fitness. At this point, it remains difficult to determine whether these results are due to differences in external selection pressures or cryptic genetic differentiation of distinct parental populations. In the future, data from mapped genetic markers and on variation of ecological factors will provide additional insights into the contribution of these factors to variation in the evolutionary consequences of hybridization.     

Patterns of male sterility in a grasshopper hybrid zone imply accumulation of hybrid incompatibilities without selection

It is now widely accepted that post-zygotic reproductive isolation is the result of negative epistatic interactions between derived alleles fixed independently at different loci in diverging populations (the Dobzhansky-Muller model). What is less clear is the nature of the loci involved and whether the derived alleles increase in frequency through genetic drift, or as a result of natural or sexual selection. If incompatible alleles are fixed by selection, transient polymorphisms will be rare and clines for these alleles will be steep where divergent populations meet. If they evolve by drift, populations are expected to harbour substantial genetic variation in compatibility and alleles will introgress across hybrid zones once they recombine onto a genetic background with which they are compatible. Here we show that variation in male sterility in a naturally occurring Chorthippus parallelus grasshopper hybrid zone conforms to the neutral expectations. Asymmetrical clines for male sterility have long tails of introgression and populations distant from the zone centre show significant genetic variation for compatibility. Our data contrast with recent observations on 'speciation genes' that have diverged as a result of strong natural selection.     

Genomic analysis of a migratory divide reveals candidate genes for migration and implicates selective sweeps in generating islands of differentiation

Differential gene flow, reductions in diversity following linked selection and/or features of the genome can structure patterns of genomic differentiation during the process of speciation. Possible sources of reproductive isolation are well studied between coastal and inland subspecies groups of Swainson's thrushes, with differences in seasonal migratory behaviour likely playing a key role in reducing hybrid fitness. We assembled and annotated a draft reference genome for this species and generated whole‐genome shotgun sequence data for populations adjacent to the hybrid zone between these groups. We documented substantial genomewide heterogeneity in relative estimates of genetic differentiation between the groups. Within population diversity was lower in areas of high relative differentiation, supporting a role for selective sweeps in generating this pattern. Absolute genetic differentiation was reduced in these areas, further suggesting that recurrent selective sweeps in the ancestral population and/or between divergent populations following secondary contact likely occurred. Relative genetic differentiation was also higher near centromeres and on the Z chromosome, suggesting that features of the genome also contribute to genomewide heterogeneity. Genes linked to migratory traits were concentrated in islands of differentiation, supporting previous suggestions that seasonal migration is under divergent selection between Swainson's thrushes. Differences in migratory behaviour likely play a central role in the speciation of many taxa; we developed the infrastructure here to permit future investigations into the role several candidate genes play in reducing gene flow between not only Swainson's thrushes but other species as well.     

The Effects of Assortative Mating and Migration on Cytonuclear Associations in Hybrid Zones

We examine the influence of nonrandom mating and immigration on the evolutionary dynamics of cytonuclear associations in hybrid zones. Recursion equations for allelic and genotypic cytonuclear disequilibria were generated under models of (1) migration alone, assuming hybrid zone matings are random with respect to cytonuclear genotype; and (2) migration in conjunction with refined epistatic mating, in which females of the pure parental species preferentially mate with conspecific males. Major results are as follows: (a) even the slightest migration removes the dependency of the final outcome on initial conditions, producing a unique equilibrium in which both pure parental genotypes are maintained in the hybrid zone; (b) in contrast to nuclear genes, the dynamics of cytoplasmic allele frequencies appear robust to changes in the assumed mating system, yet are particularly sensitive to gene flow; (c) continued immigration can generate permanent cytonuclear disequilibria, whether mating is random or assortative; and (d) the order of population censusing (before versus after reproduction by immigrants) can have a dramatic effect on the magnitude but not the pattern of cytonuclear disequilibria. Using the maximum likelihood method, the parameter space of migration rates and assortative mating rates was examined for best fit to observed cytonuclear disequilibria data in a hybrid population of Hyla tree frogs. An epistatic mating model with a total immigration rate of about 32% per generation produces equilibrium gene frequencies and cytonuclear disequilibria consistent with the empirical observations.     

The effect of Wolbachia versus genetic incompatibilities on reinforcement and speciation

Wolbachia is a widespread group of intracellular bacteria commonly found in arthropods. In many insect species, Wolbachia induce a cytoplasmic mating incompatibility (CI). If different Wolbachia infections occur in the same host species, bidirectional CI is often induced. Bidirectional CI acts as a postzygotic isolation mechanism if parapatric host populations are infected with different Wolbachia strains. Therefore, it has been suggested that Wolbachia could promote speciation in their hosts. In this article we investigate theoretically whether Wolbachia-induced bidirectional CI selects for premating isolation and therefore reinforces genetic divergence between parapatric host populations. To achieve this we combined models for Wolbachia dynamics with a well-studied reinforcement model. This new model allows us to compare the effect of bidirectional CI on the evolution of female mating preferences with a situation in which postzygotic isolation is caused by nuclear genetic incompatibilities (NI). We distinguish between nuclear incompatibilities caused by two loci with epistatic interactions, and a single locus with incompatibility among heterozygotes in the diploid phase. Our main findings are: (1) bidirectional CI and single locus NI select for premating isolation with a higher speed and for a wider parameter range than epistatic NI; (2) under certain parameter values, runaway sexual selection leads to the increase of an introduced female preference allele and fixation of its preferred male trait allele in both populations, whereas under others it leads to divergence in the two populations in preference and trait alleles; and (3) bidirectional CI and single locus NI can stably persist up to migration rates that are two times higher than seen for epistatic NI. The latter finding is important because the speed with which mutants at the preference locus spread increases exponentially with the migration rate. In summary, our results show that bidirectional CI selects for rapid premating isolation and so generally support the view that Wolbachia can promote speciation in their hosts.     

Reproductive Isolation of Hybrid Populations Driven by Genetic Incompatibilities

Despite its role in homogenizing populations, hybridization has also been proposed as a means to generate new species. The conceptual basis for this idea is that hybridization can result in novel phenotypes through recombination between the parental genomes, allowing a hybrid population to occupy ecological niches unavailable to parental species. Here we present an alternative model of the evolution of reproductive isolation in hybrid populations that occurs as a simple consequence of selection against genetic incompatibilities. Unlike previous models of hybrid speciation, our model does not incorporate inbreeding, or assume that hybrids have an ecological or reproductive fitness advantage relative to parental populations. We show that reproductive isolation between hybrids and parental species can evolve frequently and rapidly under this model, even in the presence of substantial ongoing immigration from parental species and strong selection against hybrids. An interesting prediction of our model is that replicate hybrid populations formed from the same pair of parental species can evolve reproductive isolation from each other. This non-adaptive process can therefore generate patterns of species diversity and relatedness that resemble an adaptive radiation. Intriguingly, several known hybrid species exhibit patterns of reproductive isolation consistent with the predictions of our model.     

Genomic scans reveal multiple mito‐nuclear incompatibilities in population crosses of the copepod Tigriopus californicus

The evolution of intrinsic postzygotic isolation can be explained by the accumulation of Dobzhansky‐Muller incompatibilities (DMI). Asymmetries in the levels of hybrid inviability and hybrid sterility are commonly observed between reciprocal crosses, a pattern that can result from the involvement of uniparentally inherited factors. The mitochondrial genome is one such factor that appears to participate in DMI in some crosses but the frequency of its involvement versus biparentally inherited factors is unclear. Here we assess the relative importance of incompatibilities between nuclear factors (nuclear‐nuclear) versus those between mitochondrial and nuclear factors (mito‐nuclear) in a species that lacks sex chromosomes. We used a Pool‐seq approach to survey three crosses among genetically divergent populations of the copepod, Tigriopus californicus, for regions of the genome that are affected by hybrid inviability. Results from reciprocal crosses suggest that mito‐nuclear incompatibilities are more common than nuclear‐nuclear incompatibilities overall. These results suggest that in the presence of very high levels of nucleotide divergence between mtDNA haplotypes, mito‐nuclear incompatibilities can be important for the evolution of intrinsic postzygotic isolation. This is particularly interesting considering this species lacks sex chromosomes, which have been shown to harbor a particularly high number of nuclear‐nuclear DMI in several other species.     

Hybrid fitness in a locally adapted parasite

The parasite (Red Queen) hypothesis for the maintenance of sexual reproduction and genetic diversity assumes that host-parasite interactions result from tight genetic specificity. Hence, hybridization between divergent parasite populations would be expected to disrupt adaptive gene combinations, leading to reduced infectivity on exposure to parental sympatric hosts, as long as gene effects are nonadditive. In contrast, hybridization would not cause reduced infectivity on allopatric hosts unless the divergent parasite populations possess alleles that are intrinsically incompatible when they are combined. In three different experiments, we compared the infectivity of locally adapted parasite (trematode) populations with that of F(1) hybrid parasites when exposed to host (snail) populations that were sympatric to one of the two parasite populations. We tested for intrinsic genetic incompatibilities in two experiments by including one host population that was allopatric to both parasite populations. As predicted, when the target host populations were sympatric to the parasite populations, the hybrids were significantly less infective than the parental average, while hybrid parasites on allopatric hosts were not, thereby ruling out intrinsic genetic incompatibilities. The results are consistent with nonadditive gene effects and tightly specific host-driven selection underlying parasite divergence, as envisioned by coevolutionary theory and the Red Queen hypothesis.     

HYBRID DYSFUNCTION IN FIRE-BELLIED TOADS (BOMBINA)

Reproductive isolation between two taxa may be due to endogenous selection, which is generated by incompatibilities between the respective genomes, to exogenous selection, which is generated by differential adaptations to alternative environments, or to both. The continuing debate over the relative importance of either mode of selection has highlighted the need for unambiguous data on the fitness of hybrid genotypes. The hybrid zone between the fire-bellied toad (Bombina bombina) and the yellow-bellied toad (B. variegata) in central Europe involves adaptation to different environments, but evidence of hybrid dysfunction is equivocal. In this study, we followed the development under laboratory conditions of naturally laid eggs collected from a transect across the Bombina hybrid zone in Croatia. Fitness was significantly reduced in hybrid populations: Egg batches from the center of the hybrid zone showed significantly higher embryonic and larval mortality and higher frequencies of morphological abnormalities relative to either parental type. Overall mortality from day of egg collection to three weeks after hatching reached 20% in central hybrid populations, compared to 2% in pure populations. There was no significant difference in fitness between two parental types. Within hybrid populations, there was considerable variation in fitness, with some genotypes showing no evidence of reduced viability. We discuss the implications of these findings for our understanding of barriers to gene flow between species.