Quantified reproductive isolation in Heliconius butterflies: Implications for introgression and hybrid speciation

Heliconius butterflies have become a model for the study of speciation with gene flow. For adaptive introgression to take place, there must be incomplete barriers to gene exchange that allow interspecific hybridization and multiple generations of backcrossing. The recent publication of estimates of individual components of reproductive isolation between several species of butterflies in the Heliconius melpomene–H. cydno clade allowed us to calculate total reproductive isolation estimates for these species. According to these estimates, the butterflies are not as promiscuous as has been implied. Differences between species are maintained by intrinsic mechanisms, while reproductive isolation of geographical races within species is mainly due to allopatry. We discuss the implications of this strong isolation for basic aspects of the hybrid speciation with introgression hypothesis.     

Ecological and evolutionary genomics in the wild tomatoes (Solanum sect. Lycopersicon)

The plant group Solanum section Lycopersicon (the clade containing the domesticated tomato and its wild relatives) is ideal for integrating genomic tools and approaches into ecological and evolutionary research. Wild species within Lycopersicon span broad morphological, physiological, life history, mating system, and biochemical variation, and are separated by substantial, but incomplete postmating reproductive barriers, making this an ideal system for genetic analyses of these traits. This ecological and evolutionary diversity is matched by many logistical advantages, including extensive historical occurrence records for all species in the group, publicly available germplasm for hundreds of known wild accessions, demonstrated experimental tractability, and extensive genetic, genomic, and functional tools and information from the tomato research community. Here I introduce the numerous advantages of this system for Ecological and Evolutionary Functional Genomics (EEFG), and outline several ecological and evolutionary phenotypes and questions that can be fruitfully tackled in this system. These include biotic and abiotic adaptation, reproductive trait evolution, and the genetic basis of speciation. With the modest enhancement of some research strengths, this system is poised to join the best of our currently available model EEFG systems.     

Patterns of reproductive isolation in Mediterranean deceptive orchids

The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.     

Evolution of reproductive isolation in plants

Reproductive isolation is essential for the process of speciation and much has been learned in recent years about the ecology and underlying genetics of reproductive barriers. But plant species are typically isolated not by a single factor, but by a large number of different pre- and postzygotic barriers, and their potentially complex interactions. This phenomenon has often been ignored to date. Recent studies of the relative importance of different isolating barriers between plant species pairs concluded that prezygotic isolation is much stronger than postzygotic isolation. But studies of the patterns of reproductive isolation in plants did not find that prezygotic isolation evolves faster than postzygotic isolation, in contrast to most animals. This may be due to the multiple premating barriers that isolate most species pairs, some of which may be controlled by few genes of major effect and evolve rapidly, whereas others have a complex genetic architecture and evolve more slowly. Intrinsic postzygotic isolation in plants is correlated with genetic divergence, but some instrinsic postzygotic barriers evolve rapidly and are polymorphic within species. Extrinsic postzygotic barriers are rarely included in estimates of different components of reproductive isolation. Much remains to be learned about ecological and molecular interactions among isolating barriers. The role of reinforcement and reproductive character displacement in the evolution of premating barriers is an open topic that deserves further study. At the molecular level, chromosomal and genic isolation factors may be associated and act in concert to mediate reproductive isolation, but their interactions are only starting to be explored.     

Mating system and population structure of Acacia aroma and A. macracantha (Fabaceae)

Acacia aroma and A. macracantha are closely related species that inhabit northern and central Argentina. The reproductive barriers between them seem to be weak. They exhibit low genetic differentiation, high levels of interspecific gene flow, and extensive areas of sympatry. Isoenzymatic approaches were used to evaluate the population structure and mating system parameters in natural Argentine populations of A. aroma and A. macracantha and to provide new tools for the analysis of relationships between these two species. All studied populations had high levels of genetic variability and no significant departures from Hardy-Weinberg expectations, but the two species did not differ from each other. Most variability occured within populations. Mating system analysis showed high levels of outcrossing, no biparental inbreeding, and a high probability that individuals within progeny arrays are full rather than half sibs. In all A. aroma and A. macracantha populations, polymorphic loci had the same allelic variants, and no geographic or genetic isolation between species was found. The results favor the hypothesis that these two entities represent a single polymorphic species rather than two distinct species.     

Transitions between self-compatibility and self-incompatibility and the evolution of reproductive isolation in the large and diverse tropical genus Dendrobium (Orchidaceae)

Background and Aims The evolution of interspecific reproductive barriers is crucial to understanding species evolution. This study examines the contribution of transitions between self-compatibility (SC) and self-incompatibility (SI) and genetic divergence in the evolution of reproductive barriers in Dendrobium, one of the largest orchid genera. Specifically, it investigates the evolution of pre- and postzygotic isolation and the effects of transitions between compatibility states on interspecific reproductive isolation within the genus.Methods The role of SC and SI changes in reproductive compatibility among species was examined using fruit set and seed viability data available in the literature from 86 species and ∼2500 hand pollinations. The evolution of SC and SI in Dendrobium species was investigated within a phylogenetic framework using internal transcribed spacer sequences available in GenBank.Key Results Based on data from crossing experiments, estimations of genetic distance and the results of a literature survey, it was found that changes in SC and SI significantly influenced the compatibility between species in interspecific crosses. The number of fruits produced was significantly higher in crosses in which self-incompatible species acted as pollen donor for self-compatible species, following the SI × SC rule. Maximum likelihood and Bayesian tests did not reject transitions from SI to SC and from SC to SI across the Dendrobium phylogeny. In addition, postzygotic isolation (embryo mortality) was found to evolve gradually with genetic divergence, in agreement with previous results observed for other plant species, including orchids.Conclusions Transitions between SC and SI and the gradual accumulation of genetic incompatibilities affecting postzygotic isolation are important mechanisms preventing gene flow among Dendrobium species, and may constitute important evolutionary processes contributing to the high levels of species diversity in this tropical orchid group.     

Experimental sympatry suggests geographic isolation as an essential reproductive barrier between two sister species of Pedicularis

Although mechanical isolation mediated by shared pollinators has been considered as a classic model of pollinator-mediated floral isolation in Pedicularis, a superdiverse genus in Hengduan Mountains, southwest China, there has been no empirical study of interspecific pollen flow between closely related species. We examined reproductive barriers at six different stages between Pedicularis cranolopha and Pedicularis tricolor, two sister species. The two sister species were geographically isolated from each other based on our field survey and the records of herbarium specimens. Translocation experiments showed that flowering phenology partly overlapped and bumblebee pollinators did not discriminate between flowers of the two species. Bumblebee interspecific moves could mediate interspecific pollination as traced using fluorescent powder, in which pollen analogs placed on one species were transferred to the stigmas of the other species in experimental plots containing both species. Heterospecific pollen tubes grew in the style as well as conspecific pollen in hand-pollination experiments. Reciprocal hybridization between the two species could produce (partially) viable seeds, suggesting weak post-pollination barriers. Our results showed that geographic isolation was an important barrier of two species, and the total reproductive isolation between two species was incomplete when without geographical isolation. The formation of Big Snow Mountains could introduce an important pre-zygotic reproductive barrier between the two sister species of Pedicularis; such geographical isolation could be responsible for allopatric speciation, giving a clue to understanding the rapid radiation on mountain areas.     

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.     

Floral odour and reproductive isolation in two species of Silene

Mechanisms preventing interspecific pollination are important in closely related plant species, in particular when post-zygotic barriers are weak or absent. We investigated the role of floral odour in reproductive isolation between the two closely related species Silene latifolia and S. dioica. First, we tested whether floral odour composition and emission differed between the species. We found significant odour differences, but contrary to expectations, both species showed a rhythmic emission of the same compounds between day and night. Second, in a field experiment, odour of the two species was made more similar by applying phenylacetaldehyde to flowers. This manipulation led to higher pollen-analogue transfer between species, revealing that floral odour differences are important for maintaining reproductive isolation. We conclude that differences in single key compounds can reduce pollen transfer across species boundaries by pollinators and demonstrate that odour differences are an important component of premating floral isolation between closely related plant species.     

Genetic variation for postzygotic reproductive isolation between Caenorhabditis briggsae and Caenorhabditis sp. 9

The process of speciation is key to the origins of biodiversity, and yet the Caenorhabditis nematode model system has contributed little to this topic. Genetic studies of speciation in the genus are now feasible, owing to crosses between the recently discovered Caenorhabditis sp. 9 and the well-known C. briggsae producing fertile F(1) hybrid females. We dissected patterns of postzygotic reproductive isolation between these species by crossing eight isogenic strains of C. briggsae reciprocally with six strains of C. sp. 9. We determined that overall patterns of reproductive isolation are robust across these genetic backgrounds. However, we also quantified significant heritable variation within each species for interspecific hybrid incompatibilities for total adult progeny, egg-to-adult viability, and the percentage of male progeny. This demonstrates that intraspecific variation for interspecific hybrid incompatibility occurs despite extensive, albeit incomplete, reproductive isolation. Therefore, this emerging general phenomenon of variable reproductive isolation is not restricted to highly interfertile, early-stage incipient species, but also applies to species in the latest stages of the speciation process. Furthermore, we confirm Haldane's rule and demonstrate strongly asymmetric parent-of-origin effects (Darwin's corollary) that consistently manifest more extremely when hermaphroditic C. briggsae serves as maternal parent. These findings highlight Caenorhabditis as an emerging system for understanding the genetics of general patterns of reproductive isolation.     

Hybridization and reproductive isolation between diploid Erythronium mesochoreum and its tetraploid congener E. albidum (Liliaceae)

Polyploidy has played an important role in angiosperm diversification, but how polyploidy contributes to reproductive isolation remains poorly understood. Most work has focused on postzygotic reproductive barriers, and the influence of ploidy differences on prezygotic barriers is understudied. To address these gaps, we quantified hybrid occurrence, interspecific self‐compatibility differences, and the contributions of multiple pre‐ and postzygotic barriers to reproductive isolation between diploid Erythronium mesochoreum (Liliaceae) and its tetraploid congener Erythronium albidum. Reproductive isolation between the study species was nearly complete, and naturally occurring hybrids were infrequent and largely sterile. Although postzygotic barriers effected substantial reproductive isolation when considered in isolation, the study species’ spatial distributions and pollinator assemblages overlapped little, such that interspecific pollen transfer is likely uncommon. We did not find evidence that E. albidum and E. mesochoreum differed in mating systems, indicating that self‐incompatibility release may not have fostered speciation in this system. Ultimately, we demonstrate that E. albidum and E. mesochoreum are reproductively isolated by multiple, hierarchically‐operating barriers, and we add to the currently limited number of studies demonstrating that early acting barriers such as pollinator‐mediated isolation can be important for effecting and sustaining reproductive isolation in diploid‐polyploid systems.     

Genetic differentiation, behavioural reproductive isolation and mixis cues in three sibling species of monogonont rotifers

Many aquatic species usually considered to be 'cosmopolitan' have been identified as cryptic species complexes, based on deep genetic differentiation. However, reproductive isolation among sibling cryptic species has rarely been studied, and interspecific hybridization is common in some taxa.We investigated isolation mechanisms and possible introgression among three cyclical parthenogenetic rotifer species in the Epiphanes senta complex that are found in very different freshwater habitats: temperate floodplains, subtropical desert rock pools and a tropical alpine lake. Whereas Epiphanes ukera is reproductively isolated from E. chihuahuaensis and E. hawaiiensis, the latter hybridize under laboratory conditions.While reproductive isolation is incomplete, RAPD profiles indicated unique genetic signatures and showed no evidence for introgression, indicating that these three species are diverging and have independent evolutionary trajectories.Testing cues for sexual reproduction in these cyclic parthenogens demonstrated that mixis in E. chihuahuaensis and E. ukera is influenced by population density, whereas E. hawaiiensis females rarely produce mictic offspring regardless of density. Different mixis cues are likely to separate sexual periods and effectively cause reproductive isolation between the species. Epiphanes ukera and E. chihuahuaensis males display mate guarding behaviour, and E. ukera males distinguish between conspecific and heterospecific females in mate choice experiments. Geographic isolation, along with different cues for mixis induction and mate recognition, act as reproductive barriers among these sibling species.     

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.     

Initial stages of reproductive isolation in two species of the endangered Sonoran topminnow

Long-term geographic isolation can result in reproductive incompatibilities due to forces such as mutation, genetic drift, and differential selection. In the Sonoran topminnow, molecular genetic studies of mtDNA, microsatellites, and MHC genes have shown that the endangered Gila and Yaqui topminnows are substantially different, suggesting that divergence took place approximately two million years ago. Here we examined hybrid crosses and backcrosses between these two allopatric taxa to evaluate the accumulation of postmating barriers to reproduction. These results are then compared with results from a previous study where male topminnows were shown to mate assortatively with conspecific females. Despite their preference for conspecific mates, both types of interspecific crosses successfully produced offspring. There was evidence of reduced hybrid fitness, including smaller mean brood size and male-biased sex ratio, for some classes of backcrosses. Brood sizes and interbrood intervals varied significantly when hybrids were subdivided into different cross categories. Our results illustrate the importance of distinctly defining hybrid classes in studies of reproductive isolation. To our knowledge, this is the first such detailed evolutionary analysis in endangered fish taxa.     

Sympatric bromeliad species (Pitcairnia spp.) facilitate tests of mechanisms involved in species cohesion and reproductive isolation in Neotropical inselbergs

The roles of intra- and interspecific gene flow in speciation and species evolution are topics of great current interest in molecular ecology and evolutionary biology. Recent modelling studies call for new empirical data to test hypotheses arising from the recent shift from a 'whole-genome reproductive isolation' view to a 'genic' view of species and speciation. Particularly scarce (and thus of particular interest) are molecular genetic data on recently radiated, naturally hybridizing species in strongly structured and species-rich environments. Here, we studied four sympatric plant species (Pitcairnia spp.; Bromeliaceae) adapted to Neotropical inselbergs (isolated outcrops resembling habitat 'islands' in tropical rainforests) using nuclear and plastid DNA. Patterns of plastid DNA haplotype sharing and nuclear genomic admixture suggest the presence of both, incomplete lineage sorting and interspecific gene flow over extended periods of time. Integrity and cohesion of inselberg species of Pitcairnia are maintained despite introgression and in the face of extremely low within-species migration rates (N(e)m < 1 migrant per generation). Cross-evaluation of our genetic data against published pollination experiments indicate that species integrity is maintained by the simultaneous action of multiple prezygotic barriers, including flowering phenology, pollinator isolation and divergent mating systems. Postzygotic Bateson-Dobzhansky-Muller incompatibilities appear to contribute to isolation, as suggested by asymmetric introgression rates of single loci. Our results suggest that incomplete lineage sorting, hybridization and introgression form integral aspects of adaptive radiation in Neotropical inselberg 'archipelagos'. Inselbergs with multiple closely related co-occurring species should be of special interest to students of speciation in mountain systems, and to ongoing conservation programmes in the Atlantic Rainforest biodiversity hotspot.     

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.     

Floral odour chemistry defines species boundaries and underpins strong reproductive isolation in sexually deceptive orchids

Background and Aims

The events leading to speciation are best investigated in systems where speciation is ongoing or incomplete, such as incipient species. By examining reproductive barriers among incipient sister taxa and their congeners we can gain valuable insights into the relative timing and importance of the various barriers involved in the speciation process. The aim of this study was to identify the reproductive barriers among sexually deceptive orchid taxa in the genus Chiloglottis.

Methods

The study targeted four closely related taxa with varying degrees of geographic overlap. Chemical, morphological and genetic evidence was combined to explore the basis of reproductive isolation. Of primary interest was the degree of genetic differentiation among taxa at both nuclear and chloroplast DNA markers. To objectively test whether or not species boundaries are defined by the chemistry that controls pollinator specificity, genetic analysis was restricted to samples of known odour chemistry.

Key Results

Floral odour chemical analysis was performed for 600+ flowers. The three sympatric taxa were defined by their specific chiloglottones, the semiochemicals responsible for pollinator attraction, and were found to be fully cross-compatible. Multivariate morphometric analysis could not reliably distinguish among the four taxa. Although varying from very low to moderate, significant levels of genetic differentiation were detected among all pairwise combinations of taxa at both nuclear and chloroplast loci. However, the levels of genetic differentiation were lower than expected for mature species. Critically, a lack of chloroplast DNA haplotype sharing among the morphologically indistinguishable and most closely related taxon pair confirmed that chemistry alone can define taxon boundaries.

Conclusions

The results confirmed that pollinator isolation, mediated by specific pollinator attraction, underpins strong reproductive isolation in these taxa. A combination of large effective population sizes, initial neutral mutations in the genes controlling floral scent, and a pool of available pollinators likely drives diversity in this system.     

Comparative genetics of hybrid incompatibility: sterility in two Solanum species crosses

The genetic basis of hybrid sterility can provide insight into the genetic and evolutionary origins of species barriers. We examine the genetics of hybrid incompatibility between two diploid plant species in the plant clade Solanum sect. Lycopersicon. Using a set of near-isogenic lines (NILs) representing the wild species Solanum pennellii (formerly Lycopersicon pennellii) in the genetic background of the cultivated tomato S. lycopersicum (formerly L. esculentum), we found that hybrid pollen and seed infertility are each based on a modest number of loci, male (pollen) and other (seed) incompatibility factors are roughly comparable in number, and seed-infertility QTL act additively or recessively. These findings are remarkably consistent with our previous analysis in a different species pair, S. lycopersicum x S. habrochaites. Data from both studies contrast strongly with data from Drosophila. Finally, QTL for pollen and seed sterility from the two Solanum studies were chromosomally colocalized, indicating a shared evolutionary history for these QTL, a nonrandom genomic distribution of loci causing sterility, and/or a proclivity of certain genes to be involved in hybrid sterility. We show that comparative mapping data can delimit the probable timing of evolution of detected QTL and discern which sterility loci likely evolved earliest among species.