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.     

Hybrid Sterility over Tens of Meters Between Ecotypes Adapted to Serpentine and Non-Serpentine Soils

The development of hybrid sterility is an important step in the process of speciation, however the role of adaptive evolution in triggering these postzygotic barriers is poorly understood. We show that, in the California endemic plant Collinsia sparsiflora, ecotypic adaptation to two distinct soil types is associated with the expression of intrinsic F₁ hybrid sterility between ecotypes, over spatial scales of less than 1?km. First, we show that hybrids between soil-adapted ecotypes are less fertile than hybrids within soil ecotypes. Second, we show that between-ecotype postzygotic incompatibility is insensitive to soil growth environment, and can therefore operate under conditions relevant to both ecotypes in the wild. Third, we confirm there is little genetic differentiation between ecotypes using molecular markers, indicating that these postzygotic barriers are recently evolved. Finally, we explore specific soil attributes that might be the source of selective differentiation that confers hybrid sterility. Our results indicate that hybrid barriers are developing in response to strong adaptive differentiation between adjacent and very recently diverged lineages, despite likely ongoing gene exchange.     

Evolution of postzygotic reproductive isolation in galliform birds: analysis of first and second hybrid generations and backcrosses

The process of speciation is a crucial aspect of evolutionary biology. In this study, we analysed the patterns of evolution of postzygotic reproductive isolation in Galliformes using information on hybridization and genetic distance among species. Four main patterns arose: (1) hybrid inviability and sterility in F₁ hybrids increase as species diverge; (2) the presence of geographical overlap does not affect the evolution of postzygotic isolation; (3) the galliforms follow Haldane's rule; (4) hybrid inviability is higher in F₂ than in F₁ hybrids, but does not appear to be increased in the backcrosses. This study contributes to the growing evidence suggesting that the patterns of evolution of postzygotic isolation and the process of speciation are shared among avian groups (and animals in general). In particular, our results support the notion of F₂ hybrid inviability as being key for the maintenance of species genetic integrity when prezygotic isolation barriers are overcome in closely related species, in which postzygotic isolation in the F₁ hybrid might still not be fully developed. To the contrary, hybrids from backcrosses did not show serious inviability problems (at least not more than F₁ hybrids), demonstrating that they could generate gene flow among bird species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 528–542.     

Homoploid hybridization of plants in the Hengduan mountains region

The Hengduan Mountains Region (HMR) is a major global biodiversity hotspot. Complex tectonic and historical climatic conditions created opportunities for natural interspecific hybridization. Likewise, anthropogenic disturbance potentially raises the frequency of hybridization. Among species studies to date, the frequency of homoploid hybridization appears in the HMR. Of nine taxa in which natural hybridization has been detected, three groups are involved in homoploid hybrid speciation, and species pairs from the remaining six genera suggest that continuous gene flow occurs in hybrid zones. Reproductive isolation may greatly affect the dynamic and architecture of hybrid zones in the HMR. Asymmetrical hybridization and introgression can primarily be attributed to both prezygotic and postzygotic barriers. The frequent observation of such asymmetry may imply that reproductive barrier contributes to maintaining species boundaries in the alpine region. Ecological isolations with environmental disturbance may promote breeding barriers between parental species and hybrids. Hybrid zones may be an important phase for homoploid hybrid speciation. Hybrid zones potentially provided abundant genetic resources for the diversification of the HMR flora. The ecological and molecular mechanisms of control and mediation for natural hybridization will help biologists to understand the formation of biodiversity in the HMR. More researches from ecological and molecular aspects were required in future studies.     

The molecular and evolutionary basis of reproductive isolation in plants

Reproductive isolation is defined as processes that prevent individuals of different populations from mating, survival or producing fertile offspring. Reproductive isolation is critical for driving speciation and maintaining species identity, which has been a fundamental concern in evolutionary biology. In plants, reproductive isolation can be divided into prezygotic and postzygotic reproductive barriers, according to its occurrence at different developmental stages. Postzygotic reproductive isolation caused by reduced fitness in hybrids is frequently observed in plants, which hinders gene flow between divergent populations and has substantial effects on genetic differentiation and speciation, and thus is a major obstacle for utilization of heterosis in hybrid crops. During the past decade, China has made tremendous progress in molecular and evolutionary basis of prezygotic and postzygotic reproductive barriers in plants. Present understandings in reproductive isolation especially with new data in the last several years well support three evolutionary genetic models, which represent a general mechanism underlying genomic differentiation and speciation. The updated understanding will offer new approaches for the development of wide-compatibility or neutral varieties, which facilitate breeding of hybrid rice as well as other hybrid crops.     

Evolution of postzygotic reproductive isolation in a guild of deceptive orchids

The evolution of reproductive barriers is of central importance for speciation. Here, we investigated three components of postzygotic isolation-embryo mortality, hybrid inviability, and hybrid sterility-in a group of food-deceptive Mediterranean orchids from the genera Anacamptis, Neotinea, and Orchis. In these orchids, pollinator-mediated isolation is weak, which suggests that postpollination barriers exist. Based on crossing experiments and a literature survey, we found that embryo mortality caused complete reproductive isolation among 36.3% of the species pairs, and hybrid inviability affected 55.6% of the potentially hybridizing species pairs. Hybrid sterility was assessed experimentally for seven species pairs. A strong reduction of fertility in all investigated hybrids was found, together with clear differences between male and female components of hybrid sterility. Postzygotic isolation was found to evolve gradually with genetic divergence, and late postzygotic isolation (i.e., hybrid inviability and sterility) evolved faster than embryo mortality, which is an earlier postzygotic isolation stage. These results reveal that intrinsic postzygotic isolation strongly contributes to maintaining species boundaries among Mediterranean food-deceptive orchids while establishing a prominent role for these reproductive barriers in the early stage of species isolation.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION IN LOUISIANA IRISES: HYBRID FITNESS IN NATURE

Negative epistasis in hybrid genomes commonly results in postzygotic isolation between divergent lineages. However, some genomic regions may be selectively neutral or adaptive in hybrids and thus may potentially cross species barriers. We examined postzygotic isolation between ecologically similar species of Louisiana Iris: Iris brevicaulis and I. fulva to determine the potential for adaptive introgression in nature. Line-cross analyses allowed us a general overview of the gene action responsible for fitness-related traits. We then used a QTL mapping approach to detect genomic regions responsible for variation in these traits. Although hybrid classes suffered reduced fitness for many traits, hybrid means were equivalent to at least one of the parental species in overall estimates of maternal and paternal fitness during the two years of the field study. The genetic architecture underlying the fitness-related traits varied across field site and year of the study, thus emphasizing the importance of the environment in determining the degree of postzygotic isolation and potential for introgression across natural hybrid zones.     

Mechanisms of prezygotic reproductive isolation between two sympatric species, Gelsemium rankinii and G. sempervirens (Gelsemiaceae), in the southeastern United States

Natural hybridization plays a critical role in speciation, the maintenance of reproductive isolation, and genetic introgression. While many plant species have hybrid swarms in areas of sympatry, the lack of hybrids among closely related sympatrically distributed species suggests that strong pre- and/or postzygotic barriers exist to hybridization. Gelsemium sempervirens and G. rankinii (Gelsemiaceae) are sympatrically distributed southeastern sister taxa that have strong postzygotic barriers to hybrid formation and high levels of genetic differentiation. In this study, two sympatric populations in Lowndes County, Georgia were surveyed from 1999-2005 to assess the role of temporal and pollinator isolation as potential prezygotic barriers. The populations had mostly non-overlapping flowering periods in 2003-2005, with significant differences in time of peak flowering and length of flowering. Both species shared a similar community of flower visitors, with the apid bee Habropoda laboriosa the dominant visitor to both species. A choice experiment found that H. laboriosa visited both species but preferred G. sempervirens. The primary prezygotic barrier is temporal isolation preventing hybridization in spite of the shared pollinators. This study suggests that reliance on a shared pollinator during speciation may limit opportunity for divergent selection on flowering time.     

PATTERNS OF SPECIATION IN DROSOPHILA

To investigate the time course of speciation, we gathered literature data on 119 pairs of closely related Drosophila species with known genetic distances, mating discrimination, strength of hybrid sterility and inviability, and geographic ranges. Because genetic distance is correlated with divergence time, these data provide a cross-section of taxa at different stages of speciation. Mating discrimination and the sterility or inviability of hybrids increase gradually with time. Hybrid sterility and inviability evolve at similar rates. Among allopatric species, mating discrimination and postzygotic isolation evolve at comparable rates, but among sympatric species strong mating discrimination appears well before severe sterility or inviability. This suggests that prezygotic reproductive isolation may be reinforced when allopatric taxa become sympatric. Analysis of the evolution of postzygotic isolation shows that recently diverged taxa usually produce sterile or inviable male but not female hybrids. Moreover, there is a large temporal gap between the evolution of male-limited and female hybrid sterility or inviability. This gap, which is predicted by recent theories about the genetics of speciation, explains the overwhelming preponderance of hybridizations yielding male-limited hybrid sterility or inviability (Haldane's rule).     

Natural variation for fertile triploid F1 hybrid formation in allohexaploid wheat speciation

The tempo, mode, and geography of allopolyploid speciation are influenced by natural variation in the ability of parental species to express postzygotic reproductive phenotypes that affect hybrid fertility. To shed light on the impact of such natural variations, we used allohexaploid Triticum aestivum wheats’ evolution as a model and analyzed the geographic and phylogenetic distributions of Aegilops tauschii (diploid progenitor) accessions involved in the expression of abnormality and fertility in triploid F₁ hybrids with Triticum turgidum (tetraploid progenitor). Artificial-cross experiments and chloroplast-DNA-based evolutionary analyses showed that hybrid-abnormality-causing accessions had limited geographic and phylogenetic distributions, indicative that postzygotic hybridization barriers are underdeveloped between these species. In contrast, accessions that are involved with fertile triploid F₁ hybrid formation have wide geographic and phylogenetic distributions, indicative of a deep evolutionary origin. Wide-spread hybrid-fertilizing accessions support the theory that T. aestivum speciation occurred at multiple sites within the species range of Ae. tauschii, in which existing conditions enabled natural hybridization with T. turgidum. Implications of our findings on how natural variation in the ability of Ae. tauschii to express those postzygotic reproductive phenotypes diversified and contributed to the speciation of T. aestivum are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ecology and genetics of speciation in Ficedula flycatchers

Birds have for long been popular study objects in speciation research. Being easy to observe in the field, they have traditionally been particularly important in studies of behavioural and ecological factors in speciation, whereas the genetic aspects of the process have been studied in other organisms, such as Drosophila. More recently, however, a stronger genetic focus has been placed on speciation research also in birds. Here, we review ecological, behavioural and genetic studies on speciation in the pied flycatcher (Ficedula hypoleuca) and the collared flycatcher (Ficedula albicollis). These well‐studied birds provide among the few proposed examples of the process of reinforcement of premating isolation, and the evidence for reinforcement is strong. They are further characterized by having strong intrinsic postzygotic barriers (female hybrid sterility), yet the two species appear to be very similar ecologically. This is in stark contrast to another well‐studied bird complex, Darwin’s finches, in which the species differ vastly in ecologically important traits but have no developmental problems arising from genetic incompatibilities, and where no evidence for reinforcement is found. In the flycatchers, sex chromosome linkage of genes affecting traits associated with both pre‐ and postzygotic barriers to gene exchange is likely to facilitate reinforcement. We discuss whether such sex‐linkage may be common in birds. The contrast between flycatchers and Darwin’s finches indicate that speciation in birds cannot always be understood mainly as a result of divergent natural selection (‘ecological speciation’), and generalizations from one system may lead us astray. We discuss to what extent insight from research on the flycatchers may point to fruitful avenues for future research on bird speciation and specifically call for a more systematic effort to simultaneously investigate ecology, behaviour and genetics of birds caught in the process of speciation.     

Ants exhibit asymmetric hybridization in a mosaic hybrid zone

Research on hybridization between species provides unparalleled insights into the pre‐ and postzygotic isolating mechanisms that drive speciation. In social organisms, colony‐level incompatibilities may provide additional reproductive barriers not present in solitary species, and hybrid zones offer an opportunity to identify these barriers. Here, we use genotyping‐by‐sequencing to sequence hundreds of markers in a hybrid zone between two socially polymorphic ant species, Formica selysi and Formica cinerea. We characterize the zone, determine the frequency of hybrid workers, infer whether hybrid queens or males are produced and investigate whether hybridization is influenced by colony social organization. We also compare cuticular hydrocarbon profiles and aggression levels between the two species. The hybrid zone exhibits a mosaic structure. The asymmetric distribution of hybrids skewed towards F. cinerea suggests a pattern of unidirectional nuclear gene flow from F. selysi into F. cinerea. The occurrence of backcrossed individuals indicates that hybrid queens and/or males are fertile, and the presence of the F. cinerea mitochondrial haplotype in 97% of hybrids shows that successful F1 hybrids will generally have F. cinerea mothers and F. selysi fathers. We found no evidence that social organization contributes to speciation, because hybrids occur in both single‐queen and multiple‐queen colonies. Strongly differentiated cuticular hydrocarbon profiles and heightened interspecific aggression further reveal that species recognition cues are both present and perceived. The discovery of fertile hybrids and asymmetrical gene flow is unusual in ants, and this hybrid zone will therefore provide an ideal system with which to investigate speciation in social insects.     

Tests of reproductive isolation among species in the <Emphasis Type="Italic">Fundulus notatus</Emphasis> (Cyprinodontiformes: Fundulidae) species complex

We assessed prezygotic (probability of spawning) and postzygotic (hatching success) reproductive isolation among the three ecologically and morphologically similar species in the Fundulus notatus species complex. We employed a multi-generation breeding experiment to test the hypotheses that karyotypic differences, body size differences, or geographic isolation among populations will increase pre or postzygotic reproductive barriers. Overall, prezygotic barriers were strong and postzygotic barriers weak in crosses of non-hybrid heterospecifics (F1 hybrid crosses) while prezygotic barriers were weaker and postzygotic barriers stronger in crosses involving hybrid individuals (F2 hybrid crosses and backcrosses). Prezygotic barriers among the two smaller species (Fundulus notatus and F. euryzonus) broke down rapidly; first generation hybrids spawned (F2 hybrid crosses and backcrosses) as frequently as parental forms in intraspecific crosses. There was no increase in postzygotic barriers among species with cytogenetic differences. There were increased prezygotic, but not postzygotic, barriers among geographically isolated populations of one species. While pure males and females were just as likely to spawn with hybrids, some types of hybrid females suffered from increased sterility, but not inviability, over hybrid males. Female sterility was only seen in hybrids with a Fundulus euryzonus parent, while other female hybrids produced viable eggs.     

POSTZYGOTIC ISOLATION OVER MULTIPLE GENERATIONS OF HYBRID DESCENDENTS IN A NATURAL HYBRID ZONE: HOW WELL DO SINGLE‐GENERATION ESTIMATES REFLECT REPRODUCTIVE ISOLATION?

Understanding speciation depends on an accurate assessment of the reproductive barriers separating newly diverged populations. In several taxonomic groups, prezygotic barriers, especially preferences for conspecific mates, are thought to play the dominant role in speciation. However, the importance of postzygotic barriers (i.e., low fitness of hybrid offspring) may be widely underestimated. In this study, we examined how well the widely used proxy of postzygotic isolation (reproductive output of F₁ hybrids) reflects the long‐term fitness consequences of hybridization between two closely related species of birds. Using 40 species‐specific single nucleotide polymorphism (SNP) markers, we genotyped a mixed population of collared and pied flycatchers (Ficedula albicollis and F. hypoleuca) to identify grand‐ and great grand‐offspring from interspecific crosses to derive an accurate, multigeneration estimate of postzygotic isolation. Two independent estimates of fitness show that hybridization results in 2.4% and 2.7% of the number of descendents typical of conspecific pairing. This postzygotic isolation was considerably stronger than estimates based on F₁ hybrids. Our results demonstrate that, in nature, combined selection against hybrids and backcrossed individuals may result in almost complete postzygotic isolation between two comparatively young species. If these findings are general, postzygotic barriers separating hybridizing populations may be much stronger than previously thought.     

Reproductive mode and speciation: the viviparity-driven conflict hypothesis

In birds and frogs, species pairs retain the capacity to produce viable hybrids for tens of millions of years, an order of magnitude longer than mammals. What accounts for these differences in relative rates of pre- and postzygotic isolation? We propose that reproductive mode is a critically important but previously overlooked factor in the speciation process. Viviparity creates a post-fertilization arena for genomic conflicts absent in egg-laying species. With viviparity, conflict can arise between: mothers and embryos; sibling embryos in the womb, and maternal and paternal genomes within individual embryos. Such intra- and intergenomic conflicts result in perpetual antagonistic coevolution, thereby accelerating interpopulation postzygotic isolation. In addition, by generating intrapopulation genetic incompatibility, viviparity-driven conflict favors polyandry and limits the potential for precopulatory divergence. Mammalian diversification is characterized by rapid evolution of incompatible feto-maternal interactions, asymmetrical postzygotic isolation, disproportionate effects of genomically-imprinted genes, and "F(2) hybrid enhancement. " The viviparity-driven conflict hypothesis provides a parsimonious explanation for these patterns in mammalian evolution.     

Dead-End Hybridization in Walnut Trees Revealed by Large-Scale Genomic Sequence Data

Although hybridization plays a large role in speciation, some unknown fraction of hybrid individuals never reproduces, instead remaining as genetic dead-ends. We investigated a morphologically distinct and culturally important Chinese walnut, Juglans hopeiensis, suspected to have arisen from hybridization of Persian walnut (J. regia) with Asian butternuts (J. cathayensis, J. mandshurica, and hybrids between J. cathayensis and J. mandshurica). Based on 151 whole-genome sequences of the relevant taxa, we discovered that all J. hopeiensis individuals are first-generation hybrids, with the time for the onset of gene flow estimated as 370,000 years, implying both strong postzygotic barriers and the presence of J. regia in China by that time. Six inversion regions enriched for genes associated with pollen germination and pollen tube growth may be involved in the postzygotic barriers that prevent sexual reproduction in the hybrids. Despite its long-recurrent origination and distinct traits, J. hopeiensis does not appear on the way to speciation.     

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.     

A genomic approach to identify hybrid incompatibility genes

Uncovering the genetic and molecular basis of barriers to gene flow between populations is key to understanding how new species are born. Intrinsic postzygotic reproductive barriers such as hybrid sterility and hybrid inviability are caused by deleterious genetic interactions known as hybrid incompatibilities. The difficulty in identifying these hybrid incompatibility genes remains a rate-limiting step in our understanding of the molecular basis of speciation. We recently described how whole genome sequencing can be applied to identify hybrid incompatibility genes, even from genetically terminal hybrids. Using this approach, we discovered a new hybrid incompatibility gene, gfzf, between Drosophila melanogaster and Drosophila simulans, and found that it plays an essential role in cell cycle regulation. Here, we discuss the history of the hunt for incompatibility genes between these species, discuss the molecular roles of gfzf in cell cycle regulation, and explore how intragenomic conflict drives the evolution of fundamental cellular mechanisms that lead to the developmental arrest of hybrids.     

Natural and sexual selection against hybrid flycatchers

While sexual selection is generally assumed to quickly cause or strengthen prezygotic barriers between sister species, its role in causing postzygotic isolation, through the unattractiveness of intermediate hybrids, is less often examined. Combining 24 years of pedigree data and recently developed species-specific molecular markers from collared (Ficedula albicollis) and pied (Ficedula hypoleuca) flycatchers and their hybrids, we were able to quantify all key components of fitness. To disentangle the relative role of natural and sexual selection acting on F1 hybrid flycatchers, we estimated various fitness components, which when combined represent the total lifetime reproductive success of F1 hybrids, and then compared the different fitness components of F1 hybrids to that of collared flycatchers. Female hybrid flycatchers are sterile, with natural selection being the selective force involved, but male hybrids mainly experienced a reduction in fitness through sexual selection (decreased pairing success and increased rate of being cuckolded). To disentangle the role of sexual selection against male hybrids from a possible effect of genetic incompatibility (on the rate of being cuckolded), we compared male hybrids with pure-bred males expressing intermediate plumage characters. Given that sexual selection against male hybrids is a result of their intermediate plumage, we expect these two groups of males to have a similar fitness reduction. Alternatively, hybrids have reduced fitness owing to genetic incompatibility, in which case their fitness should be lower than that of the intermediate pure-bred males. We conclude that sexual selection against male hybrids accounts for approximately 75% of the reduction in their fitness. We discuss how natural and sexual selection against hybrids may have different implications for speciation and conclude that reinforcement of reproductive barriers may be more likely when there is sexual selection against hybrids.