A captive breeding experiment reveals no evidence of reproductive isolation among lineages of a polytypic poison frog

Reproductive isolation is central to the generation of biodiversity, yet a clear understanding of the contributions of alternative reproductive barriers to this process remains elusive. Studies of young lineages that have diverged in ecologically important traits can offer insights into the chronology and relative importance of various isolating mechanisms during speciation. In poison frogs (Dendrobatidae), within‐species lineages often differ dramatically in coloration, a trait subject to natural and sexual selection. Coloration in the strawberry poison frog (Oophaga pumilio) is particularly diverse and previous work suggests the potential for reproductive isolation. We used a captive breeding experiment to assess the extent of reproductive isolation among three allopatric, genetically distinct O. pumilio lineages that differ in coloration. We compared reproduction of within‐ and between‐lineage pairs, predicting that if lineages are isolated, within‐lineage pairs would be most successful. We also examined the fertility and productivity of F1 backcrosses of admixed offspring. We found no evidence suggesting behavioural pre‐zygotic or post‐zygotic reproductive isolation, indicating that isolation would not be maintained by intrinsic mechanisms in the event of secondary contact. Future work should address costs of between‐lineage matings exerted by extrinsic natural and/or sexual selection against admixed offspring.     

Variation in reproductive isolation across a species range

Reproductive isolation is often variable within species, a phenomenon that while largely ignored by speciation studies, can be leveraged to gain insight into the potential mechanisms driving the evolution of genetic incompatibilities. We used experimental greenhouse crosses to characterize patterns of reproductive isolation among three divergent genetic lineages of Campanulastrum americanum that occur in close geographic proximity in the Appalachian Mountains. Substantial, asymmetrical reproductive isolation for survival due to cytonuclear incompatibility was found among the lineages (up to 94% reduction). Moderate reductions in pollen viability, as well as cytoplasmic male sterility, were also found between some Mountain populations. We then compared these results to previously established patterns of reproductive isolation between these Mountain lineages and a fourth, widespread Western lineage to fully characterize reproductive isolation across the complete geographic and genetic range of C. americanum. Reproductive isolation for survival and pollen viability was consistent across studies, indicating the evolution of the underlying genetic incompatibilities is primarily determined by intrinsic factors. In contrast, reproductive isolation for germination was only found when crossing Mountain populations with the Western lineage, suggesting the underlying genetic incompatibility is likely influenced by environmental or demographic differences between the two lineages. Cytoplasmic male sterility was also limited in occurrence, being restricted to a handful of Mountain populations in a narrow geographic range. These findings illustrate the complexity of speciation by demonstrating multiple, independent genetic incompatibilities that lead to a mosaic of genetic divergence and reproductive isolation across a species range.     

Habitat preference and flowering‐time variation contribute to reproductive isolation between diploid and autotetraploid Anacamptis pyramidalis

Tetraploid lineages are typically reproductively isolated from their diploid ancestors by post‐zygotic isolation via triploid sterility. Nevertheless, polyploids often also exhibit ecological divergence that could contribute to reproductive isolation from diploid ancestors. In this study, we disentangled the contribution of different forms of reproductive isolation between sympatric diploid and autotetraploid individuals of the food‐deceptive orchid Anacamptis pyramidalis by quantifying the strength of seven reproductive barriers: three prepollination, one post‐pollination prezygotic and three post‐zygotic. The overall reproductive isolation between the two cytotypes was found very high, with a preponderant contribution of two prepollination barriers, that is phenological and microhabitat differences. Although the contribution of post‐zygotic isolation (triploid sterility) is confirmed in our study, these results highlight that prepollination isolation, not necessarily involving pollinator preference, can represent a strong component of reproductive isolation between different cytotypes. Thus, in the context of polyploidy as quantum speciation, that generates reproductive isolation via triploid sterility, ecological divergence can strengthen the reproductive isolation between cytotypes, reducing the waste of gametes in low fitness interploidy crosses and thus favouring the initial establishment of the polyploid lineage. Under this light, speciation by polyploidy involves ecological processes and should not be strictly considered as a nonecological form of speciation.     

Immigrant inviability promotes reproductive isolation among host‐associated populations of the gall wasp Belonocnema treatae

Ecological speciation describes the evolutionary process whereby divergent natural selection between environments generates reproductive isolation. Studying the magnitude of sequential reproductive barriers between ecologically divergent populations improves our understanding of the way these barriers evolve and how each contributes to the speciation process. Immigrant inviability describes the lower fitness of immigrants in non‐native environments and is an important, but long underexplored, reproductive barrier. In this study, we test the role of immigrant inviability among host‐associated populations of the gall wasp Belonocnema treataeMayr (Hymenoptera: Cynipini: Cynipidae) by measuring the ability of gall wasps to initiate and complete gall formation, while avoiding host immune responses, on closely related native and non‐native live oaks, Quercus virginianaMill., Quercus fusiformisSmall, and Quercus geminataSmall (Fagaceae). In general, we found evidence for immigrant inviability when B. treatae populations colonized non‐native host species. However, patterns were variable among years, suggesting that episodic events may play an important role in connecting ecologically divergent populations.     

Chromosomal variation segregates within incipient species and correlates with reproductive isolation

Reproductive isolation is a critical step in the process of speciation. Among the most important factors driving reproductive isolation are genetic incompatibilities. Whether these incompatibilities are already present before extrinsic factors prevent gene flow between incipient species remains largely unresolved in natural systems. This question is particularly challenging because it requires that we catch speciating populations in the act before they reach the full‐fledged species status. We measured the extent of intrinsic postzygotic isolation within and between phenotypically and genetically divergent lineages of the wild yeast Saccharomyces paradoxus that have partially overlapping geographical distributions. We find that hybrid viability between lineages progressively decreases with genetic divergence. A large proportion of postzygotic inviability within lineages is associated with chromosomal rearrangements, suggesting that chromosomal differences substantially contribute to the early steps of reproductive isolation within lineages before reaching fixation. Our observations show that polymorphic intrinsic factors may segregate within incipient species before they contribute to their full reproductive isolation and highlight the role of chromosomal rearrangements in speciation. We propose different hypotheses based on adaptation, biogeographical events and life history evolution that could explain these observations.     

Divergence before the host shift? Prezygotic reproductive isolation among three varieties of a specialist fly on a single host plant

1. Although divergence via host‐plant shifting is a common theme in the speciation of some phytophagous insects, it is not clear whether host shifts are typically initiators of speciation or if they instead contribute to divergence events already in progress. While host shifts appear to be generally associated with speciation events for flies in the genus Strauzia, three sympatric varieties of the sunflower fly [Strauzia longipennis (Wiedemann)] co‐occur on the same host plant in the Midwestern United States and may have evolved reproductive barriers without a host shift. 2. The strength of two prezygotic reproductive barriers was compared among the three S. longipennis varieties: one barrier that is often associated with divergent ecological selection (allochronic isolation), and another that is more likely to be independent of ecological selection (pre‐copulatory sexual isolation). The presence and relative strength of each barrier between fly varieties were evaluated using microsatellites, no choice mating experiments, studies of allochronic isolation, and field collection data. 3. Evidence for both allochronic isolation and pre‐copulatory sexual isolation was detected between the three varieties of S. longipennis. The measure of isolation calculated for each barrier between the three varieties was lower than measures calculated between different species of Strauzia found on different hosts, suggesting that subsequent host shifts may increase the degree of reproductive isolation. For Strauzia and other specialist insects, some reproductive isolation may evolve prior to, and indeed may facilitate, host shifts.     

Evidence for no sexual isolation between Drosophila albomicans and D. nasuta

Sexual isolation, the reduced tendency to mate, is one of the reproductive barriers that prevent gene flow between different species. Various species‐specific signals during courtship contribute to sexual isolation between species. Drosophila albomicans and D. nasuta are closely related species of the nasuta subgroup within the Drosophila immigrans group and are distributed in allopatry. We analyzed mating behavior and courtship as well as cuticular hydrocarbon profiles within and between species. Here, we report that these two species randomly mated with each other. We did not observe any sexual isolation between species or between strains within species by multiple‐choice tests. Significant difference in the courtship index was detected between these two species, but males and females of both species showed no discrimination against heterospecific partners. Significant quantitative variations in cuticular hydrocarbons between these two species were also found, but the cuticular hydrocarbons appear to play a negligible role in both courtship and sexual isolation between these two species. In contrast to the evident postzygotic isolation, the lack of sexual isolation between these two species suggests that the evolution of premating isolation may lag behind that of the intergenomic incompatibility, which might be driven by intragenomic conflicts.     

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 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.     

Testing for host‐associated differentiation in two egg parasitoids of a forest herbivore

Several studies underline the importance of ecological barriers and differential selection in driving sympatric speciation. Host‐associated differentiation (HAD) has been proposed as one of the mechanisms leading to sympatric speciation. However, it is still unclear how common HAD is or which are the factors that could promote it. In particular, not much is known about HAD in predators and parasitoids of herbivorous insects. One of the characteristics postulated to pre‐dispose insects to HAD is parthenogenesis as it may favour adaptive responses to particular environments, amplifying selected gene complexes. In this study, we used amplified fragment length polymorphism (AFLP) markers to determine whether HAD is present in two parthenogenetic egg parasitoids attacking the same herbivore species – the pine processionary moth, Thaumetopoea pityocampa (Denis & Schiffermüller) (Lepidoptera: Notodontidae) – on two host Pinus species. A total of 100 loci for 59 individuals sampled in four populations of Baryscapus servadeii (Domenichini) (Hymenoptera: Eulophidae), a specialist parasitoid, and 106 loci for 117 individuals sampled in six populations of Ooencyrtus pityocampae Mercet (Hymenoptera: Encyrtidae), a generalist parasitoid, were analysed. Levels of genetic differentiation were also assessed with an outlier analysis, checking for alleles associated to host plants. No evidence of HAD was detected in any of the two parasitoid species. We hypothesize that both the lack of strict parthenogenetic reproduction and the ectophagous nature of the insect host could explain the absence of HAD. The genetic variation observed in the generalist parasitoid responded to a pattern of local adaptation, whereas no relationship with either host or geography was found in the specialist parasitoid.     

Patterns of reproductive isolation within and between two Lygaeus species characterized by sexual conflicts over mating

Theory suggests that, under some circumstances, sexual conflict over mating can lead to divergent sexually antagonistic coevolution among populations for traits associated with mating, and that this can promote reproductive isolation and hence speciation. However, sexual conflict over mating may also select for traits (e.g. male willingness to mate) that enhance gene flow between populations, limiting population divergence. In the present study, we compare pre‐ and post‐mating isolation within and between two species characterized by male–female conflict over mating rate. We quantify sexual isolation among five populations of the seed bug Lygaeus equestris collected from Italy and Sweden, and two replicates of a population of the sister‐species Lygaeus simulans, also collected from Italy. We find no evidence of reproductive isolation amongst populations of L. equestris, suggesting that sexual conflict over mating has not led to population divergence in relevant mating traits in L. equestris. However, there was strong asymmetric pre‐mating isolation between L. equestris and L. simulans: male L. simulans were able to mate successfully with female L. equestris, whereas male L. equestris were largely unable to mate with female L. simulans. We found little evidence for strong post‐mating isolation between the two species, however, with hybrid F₂ offspring being produced. Our results suggest that sexual conflict over mating has not led to population divergence, and indeed perhaps supports the contrary theoretical prediction that male willingness to mate may retard speciation by promoting gene flow.     

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.     

Behavioral Isolation Based on Visual Signals in a Sympatric Pair of Darter Species

Elaborate visual communication signals characterize many animal lineages. Often sex‐limited, these signals are generally assumed to result from sexual selection, and in many cases, their evolution is thought to play a central role in speciation. The co‐evolution of male visual signals and female preferences is hypothesized to result in behavioral isolation between divergent lineages; however, for many lineages characterized by elaborate visual signals, the importance of visual differences in behavioral isolation is not well established. Darters (fish genus Etheostoma) are particularly appropriate for examining the role of visual signals in behavioral isolation. They comprise one of the most diverse groups of North American freshwater fish, and nearly every species is characterized by unique nuptial coloration. Multiple darter species co‐exist in sympatric populations, indicating that reproductive barriers are central to maintaining these extraordinarily diverse color patterns. This study demonstrates the presence of behavioral isolation between a pair of distinctly colored sympatric darter species, Etheostoma barrenense and Etheostoma zonale, through experimental observations using an artificial stream. In addition, a series of dichotomous mate‐choice trials indicate that females prefer conspecific males over heterospecifics based on visual differences alone. We therefore provide the first evidence that visual signals are a critical trait maintaining behavioral isolation in darters, a lineage of fishes with spectacular diversification in visual communication.     

Phylogeography of the parasitic fly Batrachomyia in the Wet Tropics of north-east Australia, and susceptibility of host frog lineages in a mosaic contact zone

Parasites could differentially impact intraspecific host lineages due to genetic, phenotypic, ecological, or behavioural differences between the lineages, or the development of reproductive isolation between them. Batrachomyia (Diptera: Chloropidae) are flies that exclusively parasitize Australian frogs, and in the Wet Tropics rainforest of north‐east Australia larvae are largely restricted to the green‐eyed tree frog Litoria genimaculata (Anura: Hylidae). This frog species consists of two highly divergent genetic lineages that overlap in two nearby, but independent, contact zones. At one contact zone there has been extensive phenotypic divergence and speciation between the lineages whereas, at the other contact relatively lower levels of phenotypic divergence and reproductive isolation suggest that speciation has not occurred. In the present study we tested: (1) whether the deep phylogeographic divergence between northern and southern host populations is mirrored by congruent genetic structuring in the parasite populations and (2) whether the host lineages are differentially impacted by parasitism. We found that the two divergent frog lineages are parasitized by a single lineage of Batrachomyia, which exhibits strikingly little phylogeographic structuring. We found a significant difference in Batrachomyia prevalence between the host lineages at mixed lineage sites in both contact zones, with the magnitude and direction of this effect being consistent in both. The pattern did not differ between the two contacts even though recent phenotypic divergence and speciation has occurred between the lineages at one contact but not the other. Taken together, this suggests a fundamental difference in susceptibility between the genetically divergent host lineages. Using weight relative to body length as a measure of body condition, we found no differential impact of parasitism on the body condition of each host lineage, and no evidence that parasitism impacts the body condition of the host in general. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 593–603.     

Behavioural mechanisms of sexual isolation involving multiple modalities and their inheritance

Speciation research dissects the genetics and evolution of reproductive barriers between parental species. Hybrids are the “gatekeepers” of gene flow, so it is also important to understand the behavioural mechanisms and genetics of any potential isolation from their parental species. We tested the role of multiple behavioural barriers in reproductive isolation among closely related field crickets and their hybrids (Teleogryllus oceanicus and Teleogryllus commodus). These species hybridize in the laboratory, but the behaviour of hybrids is unusual and there is little evidence for gene flow in the wild. We found that heterospecific pairs exhibited reduced rates of courtship behaviour due to discrimination by both sexes, and that this behavioural isolation was symmetrical. However, hybrids were not sexually selected against and exhibited high rates of courtship behaviour even though hybrid females are sterile. Using reciprocal hybrid crosses, we characterized patterns of interspecific divergence and inheritance in key sexual traits that might underlie the mating patterns we found: calling song, courtship song and cuticular hydrocarbons (CHCs). Song traits exhibited both sex linkage and transgressive segregation, whereas CHCs exhibited only the latter. Calculations of the strength of isolation exerted by these sexual traits suggest that close‐range signals are as important as long‐distance signals in contributing to interspecific sexual isolation. The surprisingly weak mating barriers observed between hybrids and parental species highlight the need to examine reproductive isolating mechanisms and their genetic bases across different potential stages of introgressive hybridization.     

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.     

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.     

Histories of host shifts and cospeciation among free‐living parasitoids of Rhagoletis flies

Host shifts by specialist insects can lead to reproductive isolation between insect populations that use different hosts, promoting diversification. When both a phytophagous insect and its ancestrally associated parasitoid shift to the same novel host plant, they may cospeciate. However, because adult parasitoids are free living, they can also colonize novel host insects and diversify independent of their ancestral host insect. Although shifts of parasitoids to new insect hosts have been documented in ecological time, the long‐term importance of such shifts to parasitoid diversity has not been evaluated. We used a genus of flies with a history of speciation via host shifting (Rhagoletis [Diptera: Tephritidae]) and three associated hymenopteran parasitoid genera (Diachasma, Coptera and Utetes) to examine cophylogenetic relationships between parasitoids and their host insects. We inferred phylogenies of Rhagoletis, Diachasma, Coptera and Utetes and used distance‐based cophylogenetic methods (ParaFit and PACo) to assess congruence between fly and parasitoid trees. We used an event‐based method with a free‐living parasitoid cost model to reconstruct cophylogenetic histories of each parasitoid genus and Rhagoletis. We found that the current species diversity and host–parasitoid associations between the Rhagoletis flies and parasitoids are the primary result of ancient cospeciation events. Parasitoid shifts to ancestrally unrelated hosts primarily occur near the branch tips, suggesting that host shifts contribute to recent parasitoid species diversity but that these lineages may not persist over longer time periods. Our analyses also stress the importance of biologically informed cost models when investigating the coevolutionary histories of hosts and free‐living parasitoids.     

Quantifying Reproductive Barriers in a Sympatric Pair of Darter Species

Quantifying and comparing the strengths of different reproductive barriers between diverging lineages is especially useful for determining the evolutionary mechanisms driving speciation. Etheostoma barrenense and Etheostoma zonale are closely related sympatric species of darters that are sexually dimorphic and exhibit clear differences in male nuptial coloration. Prior studies demonstrated that these species exhibit complete behavioral isolation, and that both intraspecific and interspecific variation in male coloration play a role in female choice, all consistent with speciation by sexual selection on male nuptial color. Remaining unclear, however, is whether behavioral isolation is the strongest reproductive barrier between these species or, alternatively, whether additional reproductive barriers are equally strong, which could implicate mechanisms other than sexual selection in speciation. Here, we compare the relative strengths of multiple reproductive barriers between the two focal species, measuring: (1) ecological isolation, (2) gametic incompatibility, (3) hybrid inviability, (4) conspecific sperm precedence, and comparing these measures to a previously estimated strength of behavioral isolation. We find that behavioral isolation is the strongest reproductive barrier measured to date and suggest it may be the only barrier that has evolved to completion. This result provides additional empirical evidence for speciation driven by sexual selection and provides insight into the maintenance of sympatric species in nature.     

Floral isolation is the major reproductive barrier between a pair of rewarding orchid sister species

The crucial role of reproductive isolation in speciation has long been recognized; however, a limited number of studies quantify different isolation barriers and embed reproductive isolation in a phylogenetic context. In this study, we investigate reproductive isolation between the often sympatrically occurring orchid species, Gymnadenia conopsea and G. odoratissima. We examine the phylogenetic relationship between the two species and analyse floral isolation, fruit set and seed viability from interspecies crosses, as well as the ploidy level. Additionally, we quantify interspecies differences in floral signals and morphology. The results suggest that the two species have a sister–species relationship. In terms of reproductive isolation, we found complete floral isolation between the two species, but little to no post‐pollination isolation; the species also mostly had the same ploidy level in the studied populations. We also show clear distinctions in floral signals, as well as in floral size and spur length. We propose that respective adaptation to short‐ vs. long‐tongued pollinators was the driver of speciation in the here studied Gymnadenia species. Our study supports the key role of floral isolation in orchid speciation and shows that floral isolation is not restricted to highly specialized pollination systems, but can also occur between species with less specialized pollination.