Reinforced postmating reproductive isolation barriers in Neurospora,an Ascomycete microfungus

Maladaptive hybridization promotes reinforcement, selection for stringent reproductive isolation barriers during speciation. Reinforcement is suspected when barriers between sympatric populations are stronger than allopatric barriers, and particularly when stronger barriers evolve in the species and sex suffering the greatest costs of hybridization. Canonically, reinforcement involves premating barriers. Selection for postmating barriers is controversial, but theoretically possible. We examined geographical patterns in reproductive isolation barriers between Neurospora crassa and Neurospora intermedia, fungi with pheromone‐mediated mate recognition and maternal care. We find that isolation is stronger between sympatric populations than allopatric populations, and stronger barriers are associated with the species (N. crassa) and mating role (maternal) suffering the greater costs of hybridization. Notably, reinforced isolation involves a postmating barrier, abortion of fruitbodies. We hypothesize that fruitbody abortion is selectively advantageous if it increases the likelihood that maternal Neurospora individuals successfully mate conspecifically after maladaptive hybrid fertilization.     

Rapid evolution of spermathecal duct length in the Allonemobius socius complex of crickets: species, population and Wolbachia effects

The three species in the Allonemobius socius complex of crickets have recently diverged and radiated across North America. Interestingly, the only barriers to gene flow between these species in zones of secondary contact appear to be associated with fertilization traits - e.g., conspecific sperm precedence and the ability of males to induce females to lay eggs. Other traits, such as the length of female's reproductive tract, may also influence fertilization success and be associated with species boundaries. However, the underlying variation in this duct has not been assessed across populations and species. Moreover, the effects of reproductive parasites like Wolbachia on these morphological features have yet to be addressed, even though its infections are concentrated in reproductive tissues. I evaluated both the natural variation in and the effects of Wolbachia infection on spermathecal duct length among several populations of two species in the Allonemobius socius complex. My results suggest the following: (1) spermathecal duct length varies between species and is associated with species boundaries, (2) there is considerable variation among populations within species, (3) there is a Wolbachia infection-by-population interaction effect on the length of the spermathecal duct, and (4) experimental curing of Wolbachia recovers the uninfected morphology. These findings suggest the following hypotheses: (1) spermathecal duct length, like other fertilization traits in Allonemobius, is evolving rapidly and influences reproductive isolation and (2) Wolbachia-induced modifications of this duct could influence the dynamics of male-female coevolution. Further experiments are needed, however, to explicitly test these latter two hypotheses.     

Persistent postmating,prezygotic reproductive isolation between populations

Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.     

LACK OF CALLING SONG DISPLACEMENT BETWEEN TWO CLOSELY RELATED GROUND CRICKETS

Thorough examinations of purported cases of reproductive character displacement are critical for reaching an understanding of the role of reinforcement in the evolution of reproductive barriers between closely related species. In this paper, we report the results of an extensive investigation of male calling song variation in the ground crickets, Allonemobius fasciatus and A. socius. Contrary to the results of an earlier study, we uncovered little evidence of displacement of songs in areas of overlap. We discuss explanations for the lack of displacement as well as for the discrepancies between the results of the current study and those of the earlier study.     

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.     

Reproductive isolation between wild and domesticated chaya (Cnidoscolus aconitifolius) in sympatry

• Reproductive isolation is a necessary condition for plant domestication in their domestication centre where crops co‐occur with their wild progenitors. However, the identification of reproductive barriers and their relative contribution to reproductive isolation have been overlooked in plants under domestication.
• We assessed pre‐ and post‐pollination reproductive barriers and their relative contribution to reproductive isolation between wild and domesticated chaya (Cnidoscolus aconitifolius) in its domestication centre.
• We found that wild and domesticated chaya both exhibit a high degree of reproductive isolation. However, the reproductive isolation barriers exhibited some asymmetry: while pre‐pollination barriers (differential pollen production and pollinator specificity) were only detected in wild plants, post‐pollination barriers (pollen–pistil incompatibility and/or failure to set fruit) were observed in both wild and domesticated plants.
• We conclude that complete reproductive isolation has evolved in sympatry in co‐occurring domesticated and wild chaya.

     

Reproductive isolation and the maintenance of species boundaries in two serpentine endemic Jewelflowers

Speciation occurs when reproductive barriers substantially reduce gene flow between lineages. Understanding how specific barriers contribute to reproductive isolation offers insight into the initial forces driving divergence and the evolutionary and ecological processes responsible for maintaining diversity. Here, we quantified multiple pre‐ and post‐pollination isolating barriers in a pair of closely related California Jewelflowers (Streptanthus, Brassicaceae) living in an area of sympatry. S. breweri and S. hesperidis are restricted to similar serpentine habitats; however, populations are spatially isolated at fine‐scales and rarely co‐occur in intermixed stands. Several intrinsic postzygotic barriers were among the strongest we quantified, yet, postzygotic barriers currently contribute little to overall reproductive isolation due to the cumulative strength of earlier‐acting extrinsic barriers, including spatial isolation, and flowering time and pollinator differences. Data from multiple years suggest that pre‐pollination barriers may have different strengths depending on annual environmental conditions. Similarly, crossing data suggest that the strength of intrinsic isolation may vary among different population pairs. Estimates of total reproductive isolation in S. breweri and S. hesperidis are robust to uncertainty and variability in individual barrier strength estimates, demonstrating how multiple barriers can act redundantly to prevent gene flow between close relatives living in sympatry.     

The effectiveness of pre‐ and post‐zygotic barriers in avoiding hybridization between two snapdragons (Antirrhinum L.: Plantaginaceae)

Reproductive barriers play an important role in the maintenance of species boundaries. However, to date, few studies have provided a detailed analysis of reproductive isolation barriers between species or examined their importance in maintaining species identity. This is the first detailed study into pre‐ and post‐zygotic reproductive isolation barriers in Antirrhinum, based on a mixed population with two species that rarely co‐occur. The study revealed that pollinator constancy and preference and poor hybrid seed viability were the most important reproductive isolating mechanisms. Reproductive isolation was practically complete by both pre‐ and post‐zygotic barriers. Average pre‐zygotic isolation was greater than post‐zygotic isolation, in accordance with the trend observed in flowering plants in which reproductive isolation is principally caused by pre‐zygotic mechanisms. However, average post‐zygotic isolation was also high, in contrast to what was expected among Antirrhinum spp. This case highlights the importance of quantifying the reproductive isolation barriers thoroughly to understand how and why species boundaries are maintained. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 159–172.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.     

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene)*

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near‐complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.     

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.     

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.     

Rapid evolution of prezygotic barriers in non‐territorial damselflies

A central question in evolutionary biology concerns the accumulation of reproductive barriers during speciation. However, separating the reproductive barriers that have led to speciation from those that have secondarily accumulated (i.e. after initial divergence) is a widely recognized problem. Ideal candidate species for overcoming this problem are young species, where time for additional barriers to accrue has been limited. In the present study, we add to previous studies investigating the strength of reproductive barriers between the parapatric damselflies Ischnura elegans and Ischnura graellsii by quantifying seven prezygotic barriers between the allopatric pairs of I. elegans and Ischnura genei, as well as I. graellsii and I. genei. Specifically, we measured four premating (temporal, sexual, mechanical _I, and mechanical _II) and three postmating (oviposition success, fecundity, and fertility) barriers using experimental approaches and, for first time, we investigated the mechanisms causing mechanical isolation, which is the strongest reproductive barrier in ischnurans. The findings of the present study support the notion that premating barriers are generally strong and contribute significantly to total reproductive isolation in young lineages (65–98%), although they never solely lead to complete isolation. Asymmetry was generally stronger in premating than in postmating barriers, and was driven mostly through asymmetry in mechanical isolation, which is caused by morphological divergence of secondary sexual appendages. We found that barriers act multiplicatively in all species combinations tested, with the exception of sexual isolation, which was not detected. Our results are consistent with a recent allopatric speciation scenario driven by differences in male anal appendages, either impeding copulation or affecting female preferences. Taken together, the results from this and previous studies in diverse odonate genera suggest that premating barriers have evolved rapidly in ischnuran damselflies and, although reproductive isolation in ischnurans is more commonly the result of several barriers acting together, morphological divergence of secondary sexual appendages appears to be a common factor facilitating premating isolation in this group. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 485–496.     

Selection on Plant Male Function Genes Identifies Candidates for Reproductive Isolation of Yellow Monkeyflowers

Understanding the genetic basis of reproductive isolation promises insight into speciation and the origins of biological diversity. While progress has been made in identifying genes underlying barriers to reproduction that function after fertilization (post-zygotic isolation), we know much less about earlier acting pre-zygotic barriers. Of particular interest are barriers involved in mating and fertilization that can evolve extremely rapidly under sexual selection, suggesting they may play a prominent role in the initial stages of reproductive isolation. A significant challenge to the field of speciation genetics is developing new approaches for identification of candidate genes underlying these barriers, particularly among non-traditional model systems. We employ powerful proteomic and genomic strategies to study the genetic basis of conspecific pollen precedence, an important component of pre-zygotic reproductive isolation among yellow monkeyflowers (Mimulus spp.) resulting from male pollen competition. We use isotopic labeling in combination with shotgun proteomics to identify more than 2,000 male function (pollen tube) proteins within maternal reproductive structures (styles) of M. guttatus flowers where pollen competition occurs. We then sequence array-captured pollen tube exomes from a large outcrossing population of M. guttatus, and identify those genes with evidence of selective sweeps or balancing selection consistent with their role in pollen competition. We also test for evidence of positive selection on these genes more broadly across yellow monkeyflowers, because a signal of adaptive divergence is a common feature of genes causing reproductive isolation. Together the molecular evolution studies identify 159 pollen tube proteins that are candidate genes for conspecific pollen precedence. Our work demonstrates how powerful proteomic and genomic tools can be readily adapted to non-traditional model systems, allowing for genome-wide screens towards the goal of identifying the molecular basis of genetically complex traits.     

Divergence in genital morphology may contribute to mechanical reproductive isolation in a millipede

Genitalia appear to evolve rapidly and divergently in taxa with internal fertilization. The current consensus is that intense directional sexual selection drives the rapid evolution of genitalia. Recent research on the millipede Antichiropus variabilis suggests that the male genitalia are currently experiencing stabilizing selection – a pattern of selection expected for lock‐and‐key structures that enforce mate recognition and reproductive isolation. Here, we investigate how divergence in genital morphology affects reproductive compatibility among isolated populations of A. variabilis. Females from a focal population were mated first to a male from their own population and, second, to a male from one of two populations with divergent genital morphology. We observed variation in mating behavior that might indicate the emergence of precopulatory reproductive barriers: males from one divergent population took significantly longer to recognize females and exhibited mechanical difficulty in genital insertion. Moreover, we observed very low paternity success for extra‐population males who were successful in copulating. Our data suggest that divergence in genital shape may be contributing to reproductive isolation, and incipient speciation among isolated populations of A. variabilis.     

Extensive loss of Wnt genes in Tardigrada

Evolution of reproductive isolation is an important process, generating biodiversity and driving speciation. To better understand this process, it is necessary to investigate factors underlying reproductive isolation through various approaches but also in various taxa. Previous studies, mainly focusing on diploid animals, supported the prevalent view that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection by showing a positive relationship between the degree of reproductive isolation and genetic distance. Haplodiploid animals are expected to generate additional insight into speciation, but few studies investigated the prevalent view in haplodiploid animals. In this study, we investigate whether the relationship also holds in a haplodiploid spider mite, Amphitetranychus viennensis (Zacher). We sampled seven populations of the mite in the Palaearctic region, measured their genetic distance (mtDNA) and carried out cross experiments with all combinations. We analyzed how lack of fertilization rate (as measure of prezygotic isolation) as well as hybrid inviability and hybrid sterility (as measures of postzygotic isolation) varies with genetic distance. We found that the degree of reproductive isolation varies among cross combinations, and that all three measures of reproductive isolation have a positive relationship with genetic distance. Based on the mtDNA marker, lack of fertilization rate, hybrid female inviability and hybrid female sterility were estimated to be nearly complete (99.0–99.9% barrier) at genetic distances of 0.475–0.657, 0.150–0.209 and 0.145–0.210, respectively. Besides, we found asymmetries in reproductive isolation. The prevalent view on the evolution of reproductive barriers is supported in the haplodiploid spider mite we studied here. According to the estimated minimum genetic distance for total reproductive isolation in parent population crosses in this study and previous work, a genetic distance of 0.15–0.21 in mtDNA (COI) appears required for speciation in spider mites. Variations and asymmetries in the degree of reproductive isolation highlight the importance of reinforcement of prezygotic reproductive isolation through incompatibility and the importance of cytonuclear interactions for reproductive isolation in haplodiploid spider mites.     

Floral scent and species divergence in a pair of sexually deceptive orchids

Speciation is typically accompanied by the formation of isolation barriers between lineages. Commonly, reproductive barriers are separated into pre‐ and post‐zygotic mechanisms that can evolve with different speed. In this study, we measured the strength of different reproductive barriers in two closely related, sympatric orchids of the Ophrys insectifera group, namely Ophrys insectifera and Ophrys aymoninii to infer possible mechanisms of speciation. We quantified pre‐ and post‐pollination barriers through observation of pollen flow, by performing artificial inter‐ and intraspecific crosses and analyzing scent bouquets. Additionally, we investigated differences in mycorrhizal fungi as a potential extrinsic factor of post‐zygotic isolation. Our results show that floral isolation mediated by the attraction of different pollinators acts apparently as the sole reproductive barrier between the two orchid species, with later‐acting intrinsic barriers seemingly absent. Also, the two orchids share most of their fungal mycorrhizal partners in sympatry, suggesting little or no importance of mycorrhizal symbiosis in reproductive isolation. Key traits underlying floral isolation were two alkenes and wax ester, present predominantly in the floral scent of O. aymoninii. These compounds, when applied to flowers of O. insectifera, triggered attraction and a copulation attempt of the bee pollinator of O. aymoninii and thus led to the (partial) breakdown of floral isolation. Based on our results, we suggest that adaptation to different pollinators, mediated by floral scent, underlies species isolation in this plant group. Pollinator switches may be promoted by low pollination success of individuals in dense patches of plants, an assumption that we also confirmed in our study.     

Assortative mating in sympatric ascomycete fungi revealed by experimental fertilizations

Mate recognition mechanisms resulting in assortative mating constitute an effective reproductive barrier that may promote sexual isolation and speciation. While such mechanisms are widely documented for animals and plants, they remain poorly studied in fungi. We used two interfertile species of Epichloë (Clavicipitaceae, Ascomycota), E. typhina and E. clarkii, which are host-specific endophytes of two sympatrically occurring grasses. The life cycle of these obligatory outcrossing fungi entails dispersal of gametes by a fly vector among external fungal structures (stromata). To test for assortative mating, we mimicked the natural fertilization process by applying mixtures of spermatia from both species and examined their reproductive success. Our trials revealed that fertilization is non-random and preferentially takes place between conspecific mating partners, which is indicative of assortative mating. Additionally, the viability of hybrid and non-hybrid ascospore offspring was assessed. Germination rates were lower in E. clarkii than in E. typhina and were reduced in ascospore progeny from treatments with high proportions of heterospecific spermatia. The preferential mating between conspecific genotypes and reduced hybrid viability represent important reproductive barriers that have not been documented before in Epichloë. Insights from fungal systems will deepen our understanding of the evolutionary mechanisms leading to reproductive isolation and speciation.     

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.