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.     

Interpreting the estimated timing of migration events between hybridizing species

The question of whether speciation can occur in the presence of gene flow has long been a contentious one. However, measuring the amount and timing of gene flow remains challenging. The computer program IMa2 allows researchers to estimate the timing of migration events for each locus during analyses, and these estimates have been used to infer the timing of introgression and mode of speciation. We use simulated data sets to examine the degree to which gene-flow timing estimates can be used for these purposes, and what demographic conditions and data sets may be most amenable to gene-flow timing estimation. We find that the 90% highest posterior density (HPD) interval of gene-flow timing is almost always substantially wider than the actual window of gene flow, and increasing the information content of the data set in terms of number of loci, number of sequences sampled or locus length (and thus number of variable sites) has little impact on the posterior distribution over the range of values we tested. Even when simulated gene flow only occurred over the most recent 0.01% of the species' history, the HPD interval usually encompasses the inferred divergence time. Our results indicate that gene-flow timing estimates made using the method currently implemented in IMa2 cannot reliably be used to make inferences about the timing of introgression between diverged species or to distinguish between speciation with gene flow and allopatric speciation followed by one or more episodes of gene flow.     

Patterns of nucleotide variation and reproductive isolation between a Mimulus allotetraploid and its progenitor species

Here we report our characterization of a widespread, highly selfing Mimulus allotetraploid formed by interspecific hybridization between M. nasutus and M. guttatus. Nucleotide variation at two nuclear loci (mCYCA and mAP3) within and among tetraploid populations resolves two haplotype clusters for each locus: one shares near identity with sequences from M. nasutus and the other group shares substantial variation with M. guttatus. With respect to the two loci studied, each allotetraploid individual is a 'fixed heterozygote' carrying sequences from both clusters. Moreover, mCYCA variation is consistent with at least two evolutionary origins for the Mimulus allotetraploid. We show that the allotetraploid is strongly reproductively isolated from M. nasutus and M. guttatus; interploidy crosses produce almost no viable seeds. By extension, we infer strong triploid block and argue that Mimulus allotetraploid formation might proceed in one step via the union of unreduced gametes in an M. nasutus-M. guttatus F(1) hybrid. We also discuss the potential roles of mating system and flowering asynchrony in allotetraploid establishment.     

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.     

Evidence for bimodal hybrid zones between two species of char (Pisces: Salvelinus) in northwestern North America

Dolly Varden (Salvelinus malma, Pisces: Salmonidae) and bull trout (Salvelinus confluentus) have widely overlapping, but largely parapatric ranges in watersheds in northwestern North America from Washington State to northern British Columbia. Genetic analysis of natural populations using diagnostic molecular markers revealed widespread local sympatry and hybridization with hybrids comprising 0-25% of the local samples. In a detailed analysis of hybridization using four nuclear DNA markers and mitochondrial DNA within the Thutade Lake watershed, northcentral British Columbia, hybrid genotypes constituted up to 9% of the population of juvenile char. There were significant deviations from Hardy-Weinberg, gametic, and cytonuclear equilibria, and local samples showed bimodal frequency distributions of genotypes. Pure parental and inferred backcross genotypes were most common, and F1 and F(n) hybrids were comparatively rare. Interspecific hybridization was asymmetrical, with most F1 hybrids (five of six) bearing S. confluentus mtDNA. The introgression of nuclear and mitochondrial alleles was asymmetrical, with S. confluentus mtDNA and Growth Hormone 2 introgressing into S. malma significantly more than either introgression of the three other nuclear loci, or introgression of S. malma alleles into S. confluentus. Substantial prezygotic isolation between the species likely depends on the large body size difference between them in sympatry: S. malma have small bodies and a stream resident life history (12-21 cm adult fork length at maturity), while S. confluentus are larger and adfluvial, i.e., they migrate to Thutade Lake where they grow to maturity before returning to tributary streams to spawn (40-90 cm at maturity). These traits may limit interspecific pairings because of size assortative pairing and size-dependent reproductive habitat use.     

Testing for intraspecific postzygotic isolation between cryptic lineages of Pseudacris crucifer

Phenotypically cryptic lineages appear common in nature, yet little is known about the mechanisms that initiate and/or maintain barriers to gene flow, or how secondary contact between them might influence evolutionary trajectories. The consequences of such contact between diverging lineages depend on hybrid fitness, highlighting the potential for postzygotic isolating barriers to play a role in the origins of biological species. Previous research shows that two cryptic, deeply diverged intraspecific mitochondrial lineages of a North American chorus frog, the spring peeper (Pseudacris crucifer), meet in secondary contact in Southwestern Ontario, Canada. Our study quantified hatching success, tadpole survival, size at metamorphosis, and development time for experimentally generated pure lineage and hybrid tadpoles. Results suggest that lineages differ in tadpole survival and that F₁ hybrids may have equal fitness and higher than average mass at metamorphosis compared with pure parental crosses. These findings imply hybrid early life viability may not be the pivotal reproductive isolation barrier helping to maintain lineage boundaries. However, we observed instances of tadpole gigantism, failure to metamorphose, and bent tails in some tadpoles from hybrid families. We also speculate and provide some evidence that apparent advantages or similarities of hybrids compared with pure lineage tadpoles may disappear when tadpoles are raised with competitors of different genetic makeup. This pilot study implies that ecological context and consideration of extrinsic factors may be a key to revealing mechanisms causing negative hybrid fitness during early life stages, a provocative avenue for future investigations on barriers to gene flow among these intraspecific lineages.     

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.     

Alternative forms for genomic clines

Understanding factors regulating hybrid fitness and gene exchange is a major research challenge for evolutionary biology. Genomic cline analysis has been used to evaluate alternative patterns of introgression, but only two models have been used widely and the approach has generally lacked a hypothesis testing framework for distinguishing effects of selection and drift. I propose two alternative cline models, implement multivariate outlier detection to identify markers associated with hybrid fitness, and simulate hybrid zone dynamics to evaluate the signatures of different modes of selection. Analysis of simulated data shows that previous approaches are prone to false positives (multinomial regression) or relatively insensitive to outlier loci affected by selection (Barton's concordance). The new, theory‐based logit‐logistic cline model is generally best at detecting loci affecting hybrid fitness. Although some generalizations can be made about different modes of selection, there is no one‐to‐one correspondence between pattern and process. These new methods will enhance our ability to extract important information about the genetics of reproductive isolation and hybrid fitness. However, much remains to be done to relate statistical patterns to particular evolutionary processes. The methods described here are implemented in a freely available package “HIest” for the R statistical software (CRAN; http://cran.r-project.org/ ).     

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

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

Ecological divergence associated with mating system causes nearly complete reproductive isolation between sympatric Mimulus species

Speciation often involves the evolution of numerous prezygotic and postzygotic isolating barriers between divergent populations. Detailed knowledge of the strength and nature of those barriers provides insight into ecological and genetic factors that directly or indirectly influenced their origin, and may help predict whether they will be maintained in the face of sympatric hybridization and introgression. We estimated the magnitude of pre- and postzygotic barriers between naturally occurring sympatric populations of Mimulus guttatus and M. nasutus. Prezygotic barriers, including divergent flowering phenologies, differential pollen production, mating system isolation, and conspecific pollen precedence, act asymmetrically to completely prevent the formation of F(1) hybrids among seeds produced by M. guttatus (F(1)g), and reduce F(1) hybrid production among seeds produced by M. nasutus (F(1)n) to only about 1%. Postzygotic isolation is also asymmetric: in field experiments, F(1)g but not F(1)n hybrids had significantly reduced germination rates and survivorship compared to parental species. Both hybrid classes had flower, pollen, and seed production values within the range of parental values. Despite the moderate degree of F(1)g hybrid inviability, postzygotic isolation contributes very little to the total isolation between these species in the wild. We also found that F(1) hybrid flowering phenology overlapped more with M. guttatus than M. nasutus. These results, taken together, suggest greater potential for introgression from M. nasutus to M. guttatus than for the reverse direction. We also address problems with commonly used indices of isolation, discuss difficulties in calculating meaningful measures of reproductive isolation when barriers are asymmetric, and propose novel measures of prezygotic isolation that are consistent with postzygotic measures.     

The evolution of reproductive isolation in the Drosophila yakuba complex of species

In the Drosophila melanogaster subgroup, the yakuba species complex, D. yakuba, D. santomea and D. teissieri have identical mitochondrial genomes in spite of nuclear differentiation. The first two species can be readily hybridized in the laboratory and produce fertile females and sterile males. They also form hybrids in natural conditions. Nonetheless, the third species, D. teissieri, was thought to be unable to produce hybrids with either D. yakuba or D. santomea. This in turn posed the conundrum of why the three species shared a single mitochondrial genome. In this report, we show that D. teissieri can indeed hybridize with both D. yakuba and D. santomea. The resulting female hybrids from both crosses are fertile, whereas the hybrid males are sterile. We also characterize six isolating mechanisms that might be involved in keeping the three species apart. Our results open the possibility of studying the history of introgression in the yakuba species complex and dissecting the genetic basis of interspecific differences between these three species by genetic mapping.     

The genetics of reproductive isolation and the potential for gene exchange between Drosophila pseudoobscura and D. persimilis via backcross hybrid males

Hybrid male sterility, hybrid inviability, sexual isolation, and a hybrid male courtship dysfunction reproductively isolate Drosophila pseudoobscura and D. persimilis. Previous studies of the genetic bases of these isolating mechanisms have yielded only limited information about how much and what areas of the genome are susceptible to interspecies introgression. We have examined the genetic basis of these barriers to gene exchange in several thousand backcross hybrid male progeny of these species using 14 codominant molecular genetic markers spanning the five chromosomes of these species, focusing particularly on the autosomes. Hybrid male sterility, hybrid inviability, and the hybrid male courtship dysfunction were all associated with X-autosome interactions involving primarily the inverted regions on the left arm of the X-chromosome and the center of the second chromosome. Sexual isolation from D. pseudoobscura females was primarily associated with the left arm of the X-chromosome, although both the right arm and the center of the second chromosome also contributed to it. Sexual isolation from D. persimilis females was primarily associated with the second chromosome. The absence of isolating mechanisms being associated with many autosomal regions, including some large inverted regions that separate the strains, suggests that these phenotypes may not be caused by genes spread throughout the genome. We suggest that gene flow between these species via hybrid males may be possible at loci spread across much of the autosomes.     

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.     

Mechanical and tactile incompatibilities cause reproductive isolation between two young damselfly species

External male reproductive structures have received considerable attention as a cause of reproductive isolation (RI), because the morphology of these structures often evolves rapidly between populations. This rapid evolution presents the potential for mechanical incompatibilities with heterospecific female structures during mating and could thus prevent interbreeding between nascent species. Although such mechanical incompatibilities have received little empirical support as a common cause of RI, the potential for mismatch of reproductive structures to cause RI due to incompatible species‐specific tactile cues has not been tested. We tested the importance of mechanical and tactile incompatibilities in RI between Enallagma anna and E. carunculatum, two damselfly species that diverged within the past ～250,000 years and currently hybridize in a sympatric region. We quantified 19 prezygotic and postzygotic RI barriers using both naturally occurring and laboratory‐reared damselflies. We found incomplete mechanical isolation between the two pure species and between hybrid males and pure species females. Interestingly, in mating pairs for which mechanical isolation was incomplete, females showed greater resistance and refusal to mate with hybrid or heterospecific males compared to conspecific males. This observation suggests that tactile incompatibilities involving male reproductive structures can influence female mating decisions and form a strong barrier to gene flow in early stages of speciation.     

Hybridization following recent secondary contact results in asymmetric genotypic and phenotypic introgression between island species of Myzomela honeyeaters

Hybridization and introgression can have important evolutionary consequences for speciation, especially during early stages of secondary contact when reproductive barriers may be weak. Few studies, however, have quantified dynamics of hybridization and introgression in systems in which recent natural dispersal across a geographic barrier resulted in secondary contact. We investigated patterns of hybridization and introgression between two Myzomela honeyeaters (M. tristrami and M. cardinalis) that recently achieved secondary contact on Makira in the Solomon Islands. Hybridization in this system was hypothesized to be a byproduct of conspecific mate scarcity during early stages of colonization. Our research, however, provides evidence of ongoing hybridization more than a century after secondary contact. Mitochondrial sequencing revealed strongly asymmetric reproductive isolation that is most likely driven by postzygotic incompatibilities rather than prezygotic behavioral barriers. Nuclear introgression was observed from the native species (M. tristrami) to the colonizing species (M. cardinalis). Nuclear introgression in the reverse direction is almost exclusively limited to birds that are phenotypically M. tristrami but possess M. cardinalis mitochondrial haplotypes, consistent with introgression of plumage‐related alleles into the genomic background of M. cardinalis. These results provide unique insight into the dynamics and consequences of hybridization and introgression during early stages of secondary contact.     

Genetic differentiation without mimicry shift in a pair of hybridizing Heliconius species (Lepidoptera: Nymphalidae)

Butterflies in the genus Heliconius have undergone rapid adaptive radiation for warning patterns and mimicry, and are excellent models to study the mechanisms underlying diversification. In Heliconius, mimicry rings typically involve distantly related species, whereas closely related species often join different mimicry rings. Genetic and behavioural studies have n how reproductive isolation in many pairs of Heliconius taxa is largely mediated by natural and sexual selection on wing colour patterns. However, recent studies have uncovered new cases in which pairs of closely related species are near‐perfect mimics of each other. Here, we provide morphometric and genetic evidence for the coexistence of two closely related, hybridizing co‐mimetic species on the eastern slopes of the Andes, H. melpomene amaryllis and H. timareta ssp. nov. , which is described here as H. timareta thelxinoe . A joint analysis of multilocus genotyping and geometric morphometrics of wing shape shows a high level of differentiation between the two species, with only limited gene flow and mixing. Some degree of genetic mixing can be detected, but putative hybrids were rare, only one of 175 specimens being a clear hybrid. In contrast, we found phenotypic differentiation between populations of H. timareta thelxinoe , possibly indicative of strong selection for local mimicry in different communities. In this pair of species, the absence of breakdown of genetic isolation despite near‐identical wing patterns implies that factors other than wing patterns keep the two taxa apart, such as chemical or behavioural signals, or ecological adaptation along a strong altitudinal gradient. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 830–847.     

Effects of assortative mate choice on the genomic and morphological structure of a hybrid zone between two bird subspecies

Phenotypic differentiation plays an important role in the formation and maintenance of reproductive barriers. In some cases, variation in a few key aspects of phenotype can promote and maintain divergence; hence, the identification of these traits and their associations with patterns of genomic divergence is crucial for understanding the patterns and processes of population differentiation. We studied hybridization between the alba and personata subspecies of the white wagtail (Motacilla alba), and quantified divergence and introgression of multiple morphological traits and 19,437 SNP loci on a 3,000 km transect. Our goal was to identify traits that may contribute to reproductive barriers and to assess how variation in these traits corresponds to patterns of genome‐wide divergence. Variation in only one trait—head plumage patterning—was consistent with reproductive isolation. Transitions in head plumage were steep and occurred over otherwise morphologically and genetically homogeneous populations, whereas cline centres for other traits and genomic ancestry were displaced over 100 km from the head cline. Field observational data show that social pairs mated assortatively by head plumage, suggesting that these phenotypes are maintained by divergent mating preferences. In contrast, variation in all other traits and genetic markers could be explained by neutral diffusion, although weak ecological selection cannot be ruled out. Our results emphasize that assortative mating may maintain phenotypic differences independent of other processes shaping genome‐wide variation, consistent with other recent findings that raise questions about the relative importance of mate choice, ecological selection and selectively neutral processes for divergent evolution.     

Meeting review: American Genetics Association Symposium on the genetics of speciation

Yeast can be engineered to carry human chromosomes; highly diverged ducks can produce viable, fertile offspring; and mitochondrial genes can move between widely divergent groups of plants. Some sunflower or oak species have porous genomes; mice, crickets, birds, and butterflies form hybrid zones; and bacterial lineages have been exchanging genes for several billion years. Even so, nature is discrete and full of species. Here, we discuss some of the ingredients that make nature discrete and can lead to clustering even in the presence of gene flow. Many of these results have been recently published, in this issue and elsewhere, and were discussed at the Genetics of Speciation Symposium held at the annual meeting of the American Genetics Association, Vancouver, Canada, in 2006.     

Cultural isolation is greater than genetic isolation across an avian hybrid zone

Elucidating the relationship between genetic and cultural evolution is important in understanding speciation, as learned premating barriers might be involved in maintaining species differences. Here, we test this relationship by examining a widely recognized premating barrier, bird song, in a hybrid zone between black‐throated green (Setophaga virens) and Townsend's warblers (S. townsendi). We use song analysis, genomic techniques and playback experiments to characterize the cultural and genetic backgrounds of individuals in this region, expecting that if song is an important reproductive barrier between these species, there should be a strong relationship between song and genotype. We show that songs in the hybrid zone correspond to the distinctly different songs found in allopatry but that song and genotype are not tightly coupled in sympatry. Allopatric individuals responded only to local songs, indicating that individuals may have learned to respond to songs they commonly hear. We observed discordance between song and genotype clines; a narrower cline suggests that cultural selection on song is stronger than natural selection on genotype. These findings indicate that song is unlikely to play a role in reproductive isolation between these species, and we suggest that spatial variation in song may nonetheless be maintained by frequency‐dependent cultural selection. This decoupling of genes and culture may contribute to hybridization in this region.     

A powerful regression-based method for admixture mapping of isolation across the genome of hybrids

We propose a novel method that uses natural admixture between divergent lineages (hybridization) to investigate the genetic architecture of reproductive isolation and adaptive introgression. Our method employs multinomial regression to estimate genomic clines and to quantify introgression for individual loci relative to the genomic background (clines in genotype frequency along a genomic admixture gradient). Loci with patterns of introgression that deviate significantly from null expectations based on the remainder of the genome are potentially subject to selection and thus of interest to understanding adaptation and the evolution of reproductive isolation. Using simulations, we show that different forms of selection modify these genomic clines in predictable ways and that our method has good power to detect moderate to strong selection for multiple forms of selection. Using individual-based simulations, we demonstrate that our method generally has a low false positive rate, except when genetic drift is particularly pronounced (e.g. low population size, low migration rates from parental populations, and substantial time since initial admixture). Additional individual-based simulations reveal that moderate selection against heterozygotes can be detected as much as 50 c m away from the focal locus directly experiencing selection, but is not detected at unlinked loci. Finally, we apply our analytical method to previously published data sets from a mouse ( Mus musculus and M. domesticus ) and two sunflower ( Helianthus petiolaris and H. annuus ) hybrid zones. This method should be applicable to numerous species that are currently the focus of research in evolution and ecology and should help bring about new insights regarding the processes underlying the origin and maintenance of biological diversity.