Intrinsic reproductive isolating mechanisms in the maintenance of a hybrid zone between ecologically divergent subspecies

Understanding factors involved in maintaining stable hybrid zones is important for predicting the ultimate fate of the interacting taxa, but the relative importance of mechanisms such as ecological selection and intrinsic reproductive isolation remains unclear. Most studies of reproductive isolation in hybrid zones have focused either on zones with strongly bimodal patterns in genotype or phenotype frequencies, with relatively strong isolation, or unimodal zones with relatively weak isolation, whereas less is known about more intermediate classes of hybrid zone. Here, we utilize a hybrid zone of this intermediate type occurring between northern and southern subspecies of Atlantic killifish, Fundulus heteroclitus, to identify isolating mechanisms playing a role in maintaining this type of zone. The two subspecies differ in environmental tolerance, and we found some evidence of microhabitat preference between subspecies within a small tidal creek at the centre of the hybrid zone. There was also an association between sex, mitochondrial genotype and habitat within this creek. Fertilization success did not differ between consubspecific and heterosubspecific crosses, but hatching success was significantly lower for crosses involving southern males and northern females, and crosses between southern females and northern males had altered developmental rates. Southern females and northern males showed patterns consistent with positive assortative mating. Together, these results indicate a role for a combination of factors including assortative mating and/or early hybrid inviability in the maintenance of this hybrid zone and suggest that hybrid zones with intermediate levels of reproductive isolation are likely to be maintained by multiple interacting isolating mechanisms.     

The role of salivary androgen-binding protein in reproductive isolation between two subspecies of house mouse: Mus musculus musculus and Mus musculus domesticus

Previous behavioural studies using inbred lines have suggested that the gene ( Abpa ) for the alpha subunit of salivary androgen-binding protein (ABP) plays a role in prezygotic isolation between house mouse Mus musculus subspecies. We tested this hypothesis in animals from wild allopatric (121 individuals from four samples) and parapatric (320 animals from 15 samples) populations sampled on the Czech–Bavarian transect across the hybrid zone between M. m. domesticus and M. m. musculus . The study did not reveal a consistent statistically significant trend of homosubspecific preferences in individual allopatric and parapatric populations. Nonetheless, the whole pattern of preference was skewed toward homosubspecific preference mostly on the M. m. musculus side of the hybrid zone. The pattern of homosubspecific preferences was stronger for the time spent sniffing than it was for the first choice of the signal (the ratio of homosubspecific vs. heterosubspecific preferences for both sexes was 6 : 2 in allopatric and 21 : 9 in parapatric populations, while the same rates were 4 : 4 and 16 : 14 for the first choice). To the extent that Y-maze tests reflect preference under wild conditions, we suggest that this slight preference may not in itself be sufficient to impede gene flow between the two subspecies and thus act as a reproductive barrier. ABP most probably participates in a complex system of subspecies-specific recognition in the hybrid zone, but the picture is far too complex at this time to allow a conclusive evaluation of the importance of this role.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 349–361.     

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.     

Mechanisms of reproductive isolation of interspecific hybridization between Trillium camschatcense and T. tschonoskii (Melanthiaceae)

Reproductive isolation plays a significant role in the prevention of gene flow between different plant species. Isolation factors can vary, acting either pre‐ or postzygotically. Trillium camschatcense and T. tschonoskii are herbaceous perennials which frequently grow together in Hokkaido, Japan. Natural hybrid formation, T. × hagae, between these species is common, and occurs asymmetrically with T. camschatcense as the maternal parent and T. tschonoskii as the paternal. Here, we examined the efficiency of each reproductive isolation factor to clarify which factor was responsible for the frequency and asymmetry of the hybridization. We found that prezygotic barriers, self fertilization and conspecific pollen precedence, are major isolation factors in both parental species, and that T. tschonoskii as a maternal parent has more effective prezygotic barriers than T. camschatcense. In addition, hybrids with T. tschonoskii as the maternal parent were not observed to reach the flowering stage. We concluded that prezygotic isolation factors in the both species act as main barriers to prevent natural hybridization, and that asymmetry of the isolating barriers between these species would promote T. camschatcense as the maternal parent of the hybrids.     

Population structure attributable to reproductive time: isolation by time and adaptation by time

Many populations are composed of a mixture of individuals that reproduce at different times, and these times are often heritable. Under these conditions, gene flow should be limited between early and late reproducers, even within populations having a unimodal temporal distribution of reproductive activity. This temporal restriction on gene flow might be called "isolation by time" (IBT) to acknowledge its analogy with isolation by distance (IBD). IBD and IBT are not exactly equivalent, however, owing to differences between dispersal in space and dispersal in time. We review empirical studies of natural populations that provide evidence for IBT based on heritabilities of reproductive time and on molecular genetic differences associated with reproductive time. When IBT is present, variation in selection through the reproductive season may lead to adaptive temporal variation in phenotypic traits [adaptation by time (ABT)]. We introduce a novel theoretical model that shows how ABT increases as (i) selection on the trait increases; (ii) environmental influences on reproductive time decrease; (iii) the heritability of reproductive time increases; and (iv) the temporal distribution of reproductive activity becomes increasingly uniform. We then review empirical studies of natural populations that provide evidence for ABT by documenting adaptive temporal clines in phenotypic traits. The best evidence for IBT and ABT currently comes from salmonid fishes and flowering plants, but we expect that future work will show these processes are more widespread.     

Experimental evolution: Assortative mating and sexual selection,independent of local adaptation,lead to reproductive isolation in the nematode Caenorhabditis remanei

Using experimental evolution, we investigated the contributions of ecological divergence, sexual selection, and genetic drift to the evolution of reproductive isolation in Caenorhabditis remanei. The nematodes were reared on two different environments for 100 generations. They were assayed for fitness on both environments after 30, 64, and 100 generations, and hybrid fitness were analyzed after 64 and 100 generations. Mating propensity within and between populations was also analyzed. The design allowed us to determine whether local adaptation was synchronous with pre‐ and postzygotic reproductive isolation. Prezygotic isolation evolved quickly but was unconnected with adaptation to the divergent environments. Instead, prezygotic isolation was driven by mate preferences favoring individuals from the same replicate population. A bottleneck treatment, meant to enhance the opportunity for genetic drift, had no effect on prezygotic isolation. Postzygotic isolation occurred in crosses where at least one population had a large fitness advantage in its “home” environment. Taken together, our results suggest that prezygotic isolation did not depend on drift or adaptation to divergent environments, but instead resulted from differences in sexual interactions within individual replicates. Furthermore, our results suggest that postzygotic isolation can occur between populations even when only one population has greater fitness in its home environment.     

Evaluation of pre‐ and post‐zygotic mating barriers,hybrid fitness and phylogenetic relationship between Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi (Cyprinodontiformes,Teleostei)

The euryhaline fish Cyprinodon variegatus variegatus (Cvv) is capable of tolerating ambient salinities ranging from 0.3 to 167 g l^?1, but incapable of long‐term survival in freshwater (< 2 mM Na⁺). However, a population of this species, now designated as a subspecies (Cyprinodon variegatus hubbsi; Cvh), has been isolated in several freshwater (0.4–1 mM Na⁺) lakes in central Florida for the past ~150 ky. We previously demonstrated that Cvh has a significantly higher affinity for Na⁺ uptake suggesting that it has adapted to its dilute freshwater environment. We here evaluate whether Cvh should be considered a separate species by characterizing pre‐ and post‐zygotic isolation, Na⁺ transport characteristics of the two populations and their hybrids, and developing a molecular phylogeny of Cvv and Cvh populations in Florida using mtDNA sequence data. We found evidence of partial prezygotic isolation with Cvv females mating almost exclusively (89%) with con‐specific males in choice mating experiments. Partial post‐zygotic isolation was also observed with significant (59–89%) reductions in hatching success of hybrid embryos compared with con‐specific embryos. Na⁺ uptake kinetics in hybrids (both Cvv x Cvh and Cvh x Cvv) bred and raised under common garden conditions were intermediate to Cvh (high affinity) and Cvv (low affinity) indicating that observed differences are genetically based. Similar observations were made with respect to short‐term (96 h) survival of juveniles acutely transferred from 7 mM Na⁺ to a range of more dilute (0.1–2 mM Na⁺) freshwater. Finally, although phylogenetic analysis of Cvv and Cvh populations using mtDNA sequence for ND2 were unable to fully resolve a polytomy between Cvh and Cvv populations from northeastern Florida, these data do not falsify the hypothesis that Cvh is of monophyletic origin. Overall, the available data suggest that Cvh should be considered a separate species or at a minimum an evolutionarily significant unit.     

Multiple sources of reproductive isolation in a bimodal butterfly hybrid zone

An important evolutionary question concerns whether one or many barriers are involved in the early stages of speciation. We examine pre‐ and post‐zygotic reproductive barriers between two species of butterflies (Heliconius erato chestertonii and H. e. venus) separated by a bimodal hybrid zone in the Cauca Valley, Colombia. We show that there is both strong pre‐ and post‐mating reproductive isolation, together leading to a 98% reduction in gene flow between the species. Pre‐mating isolation plays a primary role, contributing strongly to this isolation (87%), similar to previous examples in Heliconius. Post‐mating isolation was also strong, with absence of Haldane’s rule, but an asymmetric reduction in fertility (< 11%) in inter‐specific crosses depending on maternal genotype. In summary, this is one of the first examples of post‐zygotic reproductive isolation playing a significant role in early stages of parapatric speciation in Heliconius and demonstrates the importance of multiple barriers to gene flow in the speciation process.     

Ecological speciation in sympatric palms: 2. Pre‐ and post‐zygotic isolation

We evaluated reproductive isolation in two species of palms (Howea) that have evolved sympatrically on Lord Howe Island (LHI, Australia). We estimated the strength of some pre‐ and post‐zygotic mechanisms in maintaining current species boundaries. We found that flowering time displacement between species is consistent across in and ex situ common gardens and is thus partly genetically determined. On LHI, pre‐zygotic isolation due solely to flowering displacement was 97% for Howea belmoreana and 80% for H. forsteriana; this asymmetry results from H. forsteriana flowering earlier than H. belmoreana and being protandrous. As expected, only a few hybrids (here confirmed by genotyping) at both juvenile and adult stages could be detected in two sites on LHI, in which the two species grow intermingled (the Far Flats) or adjacently (Transit Hill). Yet, the distribution of hybrids was different between sites. At Transit Hill, we found no hybrid adult trees, but 13.5% of younger palms examined there were of late hybrid classes. In contrast, we found four hybrid adult trees, mostly of late hybrid classes, and only one juvenile F1 hybrid in the Far Flats. This pattern indicates that selection acts against hybrids between the juvenile and adult stages. An in situ reciprocal seed transplant between volcanic and calcareous soils also shows that early fitness components (up to 36 months) were affected by species and soil. These results are indicative of divergent selection in reproductive isolation, although it does not solely explain the current distribution of the two species on LHI.     

Competition‐driven build‐up of habitat isolation and selection favoring modified dispersal patterns in a young avian hybrid zone

Competition‐driven evolution of habitat isolation is an important mechanism of ecological speciation but empirical support for this process is often indirect. We examined how an on‐going displacement of pied flycatchers from their preferred breeding habitat by collared flycatchers in a young secondary contact zone is associated with (a) access to an important food resource (caterpillar larvae), (b) immigration of pied flycatchers in relation to habitat quality, and (c) the risk of hybridization in relation to habitat quality. Over the past 12 years, the estimated access to caterpillar larvae biomass in the habitat surrounding the nests of pied flycatchers has decreased by a fifth due to shifted establishment possibilities, especially for immigrants. However, breeding in the high quality habitat has become associated with such a high risk of hybridization for pied flycatchers that overall selection currently favors pied flycatchers that were forced to immigrate into the poorer habitats (despite lower access to preferred food items). Our results show that competition‐driven habitat segregation can lead to fast habitat isolation, which per se caused an opportunity for selection to act in favor of future “voluntarily” altered immigration patterns and possibly strengthened habitat isolation through reinforcement.     

Behavioural,ecological and genetic evidence confirm the occurrence of host‐associated differentiation in goldenrod gall‐midges

Host‐associated differentiation (HAD) is considered a step towards ecological speciation and an important mechanism promoting diversification in phytophagous insects. Although the number of documented cases of HAD is increasing, these still represent only a small fraction of species and feeding guilds among phytophagous insects, and most reports are based on a single type of evidence. Here we employ a comprehensive approach to present behavioural, morphological, ecological and genetic evidence for the occurrence of HAD in the gall midge Dasineura folliculi (Diptera: Cecidomyiidae) on two sympatric species of goldenrods (Solidago rugosa and S. gigantea). Controlled experiments revealed assortative mating and strong oviposition fidelity for the natal‐host species. Analysis of mitochondrial DNA showed an amount of genetic divergence between the two host‐associated populations compatible with cryptic species rather than host races. Lower levels of within‐host genetic divergence, gall development and natural‐enemy attack in the S. gigantea population suggest this is the derived host.     

Pre‐zygotic reproductive isolation between two synchronopatric Opuntia (Cactaceae) species in the Brazilian Chaco

• Opuntia (Cactaceae) is known for high rates of hybridization and ploidisation, resulting in the formation of new species. The occurrence of two sympatric and closely related species of Opuntia, O. elata and O. retrorsa, in Brazilian Chaco enabled us to test the hypothesis that pre‐zygotic reproductive isolation mechanisms operate in both species.
• We monitored the flowering period, as well as floral biology, and compared the morphological variation of floral structures through measurements, performed intra‐ and interspecific cross‐pollination tests, and recorded the guild of floral visitors and pollinators.
• Flowering was seasonal and highly synchronous. Floral biology exhibits similar strategies, and although floral morphology differs significantly in many of the compared structures, such morphological variation does not result in the selection of exclusive pollinators. Floral visitors and pollinators are oligolectic bees shared by both species. Opuntia elata and O. retrorsa are self‐compatible. While interspecific cross‐pollination (bidirectional) resulted in germination, the pollen tube did not penetrate the stigma.
• Opuntia elata and O. retrorsa are closely related; however, they are isolated and do not hybridise in Brazilian Chaco. We found that both have weak pre‐pollination barriers, but that they are strongly isolated by pollen–pistil incompatibility, i.e. post‐pollination barrier.

     

Assessing reproductive isolation using a contact zone between parapatric lake‐stream stickleback ecotypes

Ecological speciation occurs when populations evolve reproductive isolation as a result of divergent natural selection. This isolation can be influenced by many potential reproductive barriers, including selection against hybrids, selection against migrants and assortative mating. How and when these barriers act and interact in nature is understood for relatively few empirical systems. We used a mark–recapture experiment in a contact zone between lake and stream three‐spined sticklebacks (Gasterosteus aculeatus, Linnaeus) to evaluate the occurrence of hybrids (allowing inferences about mating isolation), the interannual survival of hybrids (allowing inferences about selection against hybrids) and the shift in lake‐like vs. stream‐like characteristics (allowing inferences about selection against migrants). Genetic and morphological data suggest the occurrence of hybrids and no selection against hybrids in general, a result contradictory to a number of other studies of sticklebacks. However, we did find selection against more lake‐like individuals, suggesting a barrier to gene flow from the lake into the stream. Combined with previous work on this system, our results suggest that multiple (most weakly and often asymmetric) barriers must be combining to yield substantial restrictions on gene flow. This work provides evidence of a reproductive barrier in lake–stream sticklebacks and highlights the value of assessing multiple reproductive barriers in natural contexts.     

Hybridization and sequential components of reproductive isolation between parapatric walnut‐infesting sister species Rhagoletis completa and Rhagoletis zoqui

Hybridization can provide a window into how populations diverge to form new species. Here, we confirm hybridization between Rhagoletis completa Cresson, 1929 and Rhagoletis zoqui Bush, 1966, two species of walnut husk‐infesting flies that geographically overlap in a narrow area of parapatry in Northeastern Mexico. Rhagoletis completa and R. zoqui are members of a species group (Rhagoletis suavis) that has been hypothesized to speciate in allopatry. Sexual selection has been argued to be a potentially important factor for generating pre‐mating isolation among walnut husk flies, because of the differences in wing morphology and body coloration, and the existence of sexual dimorphism within species. However, there was no evidence for pre‐mating isolation between R. completa and R. zoqui, based on choice and no‐choice mating experiments conducted on adults of fly populations outside the contact zone. There was also no support for reduced fertility of hybrid matings or for F₁ inviability; however, F₁ hybrids appeared to have lower fertility and F₂ offspring have reduced survivorship. Postzygotic isolation in later generation hybrids of mixed ancestry therefore appears to be the first intrinsic barrier to gene flow evolving between R. completa and R. zoqui. We discuss the implications of our results for allopatric speciation in walnut flies and the potential evolutionary fate of R. zoqui and R. completa if they were to come into broad geographic contact in the future. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

Divergent strategies in pre‐ and postzygotic reproductive isolation between two closely related Dianthus species

Quantifying the relative contribution of multiple isolation barriers to gene flow between recently diverged species is essential for understanding speciation processes. In parapatric populations, local adaptation is thought to be a major contributor to the evolution of reproductive isolation. However, extrinsic postzygotic barriers assessed in reciprocal transplant experiments are often neglected in empirical assessments of multiple isolation barriers. We analyzed multiple isolation barriers between two closely related species of the plant genus Dianthus, a genus characterized by the most rapid species diversification in plants reported so far. Although D. carthusianorum L. and D. sylvestris Wulf. can easily be hybridized in crossing experiments, natural hybrids are rare. We found that in parapatry, pollinator‐mediated prezygotic reproductive isolation barriers are important for both D. carthusianorum (0.761) and D. sylvestris (0.468). In contrast to D. carthusianorum, high hybrid viability in D. sylvestris (–0.491) was counteracted by strong extrinsic postzygotic isolation (0.900). Our study highlights the importance of including reciprocal transplant experiments for documenting extrinsic postzygotic isolation and demonstrates clearly divergent strategies and hence asymmetric pre‐ and postzygotic reproductive isolation between closely related species. It also suggests that pollinator‐mediated and ecological isolation could have interacted in synergistic ways, further stimulating rapid speciation in Dianthus.     

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

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

The colour of paternity: extra‐pair paternity in the wild Gouldian finch does not appear to be driven by genetic incompatibility between morphs

In socially monogamous species, individuals can use extra‐pair paternity and offspring sex allocation as adaptive strategies to ameliorate costs of genetic incompatibility with their partner. Previous studies on domesticated Gouldian finches (Erythrura gouldiae) demonstrated a genetic incompatibility between head colour morphs, the effects of which are more severe in female offspring. Domesticated females use differential sex allocation, and extra‐pair paternity with males of compatible head colour, to reduce fitness costs associated with incompatibility in mixed‐morph pairings. However, laboratory studies are an oversimplification of the complex ecological factors experienced in the wild and may only reflect the biology of a domesticated species. This study aimed to examine the patterns of parentage and sex ratio bias with respect to colour pairing combinations in a wild population of the Gouldian finch. We utilized a novel PCR assay that allowed us to genotype the morph of offspring before the morph phenotype develops and to explore bias in morph paternity and selection at the nest. Contrary to previous findings in the laboratory, we found no effect of pairing combinations on patterns of extra‐pair paternity, offspring sex ratio or selection on morphs in nestlings. In the wild, the effect of morph incompatibility is likely much smaller, or absent, than was observed in the domesticated birds. Furthermore, the previously studied domesticated population is genetically differentiated from the wild population, consistent with the effects of domestication. It is possible that the domestication process fostered the emergence (or enhancement) of incompatibility between colour morphs previously demonstrated in the laboratory.     

The genetic basis of intrinsic and extrinsic post-zygotic reproductive isolation jointly promoting speciation in the lake whitefish species complex (Coregonus clupeaformis)

Understanding the genetic architecture of reproductive barriers and the evolutionary forces that drove their divergence represents a considerable challenge towards understanding speciation. The objective of this study was to determine the genetic basis of intrinsic and extrinsic post-zygotic isolation in diverging populations of dwarf and normal lake whitefish with allopatric glacial origins. We found that the rate of embryonic mortality was 5.3-6.5 times higher in dwarf-normal hybrid backcrosses during development than in F1 dwarf and normal crosses. When comparing embryos that died during development against larvae that successfully hatched, patterns of Mendelian segregation at 101 loci whose linkage is known identified 13 loci distributed over seven linkage groups that exhibited significant shifts in segregation ratios leading to significant segregation distortion at these loci in the surviving progeny. Controlled crosses and quantitative trait loci analysis revealed a significant genetic basis for developmental time until emergence, a trait critical to fish larval survival in nature. Hatching backcross progeny exhibited asynchronous emergence and transgressive segregation, suggesting that extrinsic post-zygotic isolation may select against hybridization in specific environmental contexts. Evidence of a genetic basis for increased embryonic mortality followed by asynchronous emergence indicated that intrinsic and extrinsic mechanisms are not mutually exclusive in the formation and maintenance of reproductive isolation, but may be jointly promoting population divergence and ultimately speciation.