Patterns of reproductive isolation in Mediterranean deceptive orchids

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

Evolution of reproductive isolation in stickleback fish

To understand how new species form and what causes their collapse, we examined how reproductive isolation evolves during the speciation process, considering species pairs with little to extensive divergence, including a recently collapsed pair. We estimated many reproductive barriers in each of five sets of stickleback fish species pairs using our own data and decades of previous work. We found that the types of barriers important early in the speciation process differ from those important late. Two premating barriers—habitat and sexual isolation—evolve early in divergence and remain two of the strongest barriers throughout speciation. Premating isolation evolves before postmating isolation, and extrinsic isolation is far stronger than intrinsic. Completing speciation, however, may require postmating intrinsic incompatibilities. Reverse speciation in one species pair was characterized by significant loss of sexual isolation. We present estimates of barrier strengths before and after collapse of a species pair; such detail regarding the loss of isolation has never before been documented. Additionally, despite significant asymmetries in individual barriers, which can limit speciation, total isolation was essentially symmetric between species. Our study provides important insight into the order of barrier evolution and the relative importance of isolating barriers during speciation and tests fundamental predictions of ecological speciation.     

Premating, not postmating, barriers drive genetic dynamics in experimental hybrid populations of the endangered Sonoran topminnow

The timing and pattern of reproductive barrier formation in allopatric populations has received much less attention than the accumulation of reproductive barriers in sympatry. The theory of allopatric speciation suggests that reproductive barriers evolve simply as by-products of overall genetic divergence. However, observations of enhanced premating barriers in allopatric populations suggest that sexual selection driven by intraspecific competition for mates may enhance species-specific signals and accelerate the speciation process. In a previous series of laboratory trials, we examined the strength of premating and postmating barriers in an allopatric species pair of the endangered Sonoran topminnow, Poeciliopsis occidentalis and P. sonoriensis. Behavioral observations provided evidence of asymmetrical assortative mating, while reduced brood sizes and male-biased F(1) sex ratios suggest postmating incompatibilities. Here we examine the combined effects of premating and postmating barriers on the genetic makeup of mixed populations, using cytonuclear genotype frequencies of first- and second-generation offspring. Observed genotype frequencies strongly reflect the directional assortative mating observed in behavioral trials, illustrating how isolating barriers that act earlier in the reproductive cycle will have a greater effect on total reproductive isolation and may be more important to speciation than subsequent postmating reproductive barriers.     

Asymmetry in reproductive isolation and its effect on directional mitochondrial introgression in the parapatric ground beetles <Emphasis Type="Italic">Carabus yamato</Emphasis> and <Emphasis Type="Italic">C. albrechti</Emphasis>

Speciation studies seek to clarify the origin of reproductive isolation, the various mechanisms working from mate recognition through postzygotic stages. Asymmetric effects of isolating barriers can result in asymmetrical gene introgression during interspecific hybridization. The flightless ground beetles Carabus yamato and C. albrechti are distributed parapatrically in Japan, showing repeated asymmetrical introgression of mitochondria from C. albrechti to C. yamato. This pattern suggests that reproductive isolation between these species is strong, but incomplete and asymmetric (i.e., weaker for the cross between a C. albrechti female and a C. yamato male). To test this hypothesis, we conducted interspecific mating experiments in the laboratory. The estimates of total reproductive isolation, which occurred mainly at the premating and postmating/prezygotic stages, were high (isolation index = 0.964 for C. yamato female × C. albrechti male and 0.886 for the reciprocal cross), supporting the hypothesis of strong, but incomplete isolation. However, the observed difference between the reciprocal crosses was not sufficiently large to conclude that it caused directional introgression of mitochondria. Instead, we found asymmetry in individual isolating barriers in the postmating/prezygotic stages that coincided with the prediction, perhaps resulting from morphological mismatch of heterospecific genitalia. Although this asymmetry was compensated for by an inverse asymmetry of isolation in the postzygotic stage, the contribution of these individual barriers to total isolation may change for our expectation when considering females mating with multiple heterospecific males.     

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.     

Review. Specificity in pollination and consequences for postmating reproductive isolation in deceptive Mediterranean orchids

The type of reproductive isolation prevalent in the initial stages of species divergence can affect the nature and rate of emergence of additional reproductive barriers that subsequently strengthen isolation between species. Different groups of Mediterranean deceptive orchids are characterized by different levels of pollinator specificity. Whereas food-deceptive orchid species show weak pollinator specificity, the sexually deceptive Ophrys species display a more specialized pollination strategy. Comparative analyses reveal that orchids with high pollinator specificity mostly rely on premating reproductive barriers and have very little postmating isolation. In this group, a shift to a novel pollinator achieved by modifying the odour bouquet may represent the main isolation mechanism involved in speciation. By contrast, orchids with weak premating isolation, such as generalized food-deceptive orchids, show strong evidence for intrinsic postmating reproductive barriers, particularly for late-acting postzygotic barriers such as hybrid sterility. In such species, chromosomal differences may have played a key role in species isolation, although strong postmating-prezygotic isolation has also evolved in these orchids. Molecular analyses of hybrid zones indicate that the types and strength of reproductive barriers in deceptive orchids with contrasting premating isolation mechanisms directly affect the rate and evolutionary consequences of hybridization and the nature of species differentiation.     

Strong asymmetry in the relative strengths of prezygotic and postzygotic barriers between two damselfly sister species

One of the longest debates in biology has been over the relative importance of different isolating barriers in speciation. However, for most species, there are few data evaluating their relative contributions and we can only speculate on the general roles of pre- and postzygotic isolation. Here, we quantify the absolute and cumulative contribution of 19 potential reproductive barriers between two sympatric damselfly sister species, Ischnura elegans and I. graellsii, including both premating (habitat, temporal, sexual and mechanical isolation) and postmating barriers (prezygotic: sperm insemination success and removal rate, oviposition success, fertility, fecundity; postzygotic: hybrid viability, hybrid sterility and hybrid breakdown). In sympatry, total reproductive isolation between I. elegans females and I. graellsii males was 95.2%, owing mostly to a premating mechanical incompatibility (93.4%), whereas other barriers were of little importance. Isolation between I. graellsii females and I. elegans males was also nearly complete (95.8%), which was caused by the cumulative action of multiple prezygotic (n= 4, 75.4%) and postzygotic postmating barriers (n= 5, 7.4%). Our results suggest that premating barriers are key factors in preventing gene flow between species, and that the relative strengths of premating barriers is highly asymmetrical between the reciprocal crosses.     

Postmating barriers to hybridization between an island’s native eucalypts and an introduced congener

We evaluate postmating barriers to hybridization between an exotic eucalypt and a group of native congeners on the island of Tasmania. We aimed to better understand the basis of reproductive isolation between the species, glean insights into the evolution of isolating mechanisms, and inform genetic risk management. Compatibility between the exotic plantation species Eucalyptus nitens (pollen parent) and 18 native Tasmanian taxa was assayed using experimental crossing for 17 taxa (13,458 flowers pollinated to produce 1058 female × male cross combinations), and previous data for one species. Compatibility was assessed in terms of F₁ hybrid production, as well as F₁ hybrid survival and growth after 5 years. This data was combined with measurements of style length, and genetic distance from E. nitens to each maternal species, in order to determine the importance of a sequence of prezygotic and postzygotic barriers. We found that the early-acting barrier of style length (prezygotic) had the strongest isolating effect, while later-acting (postzygotic) barriers, affecting early-age growth and survival, contributed little to reproductive isolation. Style length alone explained 46 % of the variation in hybridization rate. Conversely, there was no significant relationship between genetic distance and prezygotic or postzygotic compatibility in these closely related species. This pattern is consistent with selection driving the rapid evolution of prezygotic barriers, while drift-like-processes lead to the more gradual evolution of intrinsic barriers. Although other premating and postmating barriers clearly contribute, our results highlight the important role of early-acting postmating barriers in preventing gene flow from exotic E. nitens plantations.     

The accumulation of reproductive barriers during speciation: postmating barriers in two behaviorally isolated species of darters (Percidae: Etheostoma)

Identifying the manner in which reproductive barriers accumulate during lineage divergence is central to establishing general principles of species formation. One outstanding question is which isolating mechanisms form the first complete barrier to gene flow in a given lineage or under a particular set of conditions. To identify these initial reproductive barriers requires examining lineages in very early stages of divergence, before multiple reproductive barriers have evolved to completion. We quantified the strength of three postmating barriers in a pair of darter species and compared these estimates to each other and to the strength of behavioral isolation (BI) reported in a previous study. Results reveal no evidence of gametic incompatibility but intermediate levels of conspecific sperm precedence and hybrid inviability. As BI is nearly complete, our analysis comparing the strength of multiple reproductive barriers implicates the evolution of mate choice as central to both the origin and maintenance of these species. Further examination of ecological isolation and hybrid sterility is necessary to determine the role of these barriers in darter speciation.     

Multiple strong postmating and intrinsic postzygotic reproductive barriers isolate florally diverse species of Jaltomata (Solanaceae)

Divergence in phenotypic traits often contributes to premating isolation between lineages, but could also promote isolation at postmating stages. Phenotypic differences could directly result in mechanical isolation or hybrids with maladapted traits; alternatively, when alleles controlling these trait differences pleiotropically affect other components of development, differentiation could indirectly produce genetic incompatibilities in hybrids. Here, we determined the strength of nine postmating and intrinsic postzygotic reproductive barriers among 10 species of Jaltomata (Solanaceae), including species with highly divergent floral traits. To evaluate the relative importance of floral trait diversification for the strength of these postmating barriers, we assessed their relationship to floral divergence, genetic distance, geographical context, and ecological differences, using conventional tests and a new linear‐mixed modeling approach. Despite close evolutionary relationships, all species pairs showed moderate to strong isolation. Nonetheless, floral trait divergence was not a consistent predictor of the strength of isolation; instead this was best explained by genetic distance, although we found evidence for mechanical isolation in one species, and a positive relationship between floral trait divergence and fruit set isolation across species pairs. Overall, our data indicate that intrinsic postzygotic isolation is more strongly associated with genome‐wide genetic differentiation, rather than floral divergence.     

THE DISTRIBUTION OF POSTMATING REPRODUCTIVE ISOLATING GENES IN POPULATIONS OF THE YELLOW MONKEY FLOWER,MIMULUS GUTTATUS

Postmating reproductive isolating barriers are generally believed to arise as the chance by-product of genetic differentiation. The classical view is that these barriers normally involve differentiation at many loci, and therefore require long periods of allopatric isolation. The formal genetics of, and the distribution of genes responsible for, such barriers are known in very few cases. This paper examines the distribution of the genes responsible for two different postmating barriers in 18 populations of the yellow monkey flower, Mimulus guttatus. The genetic relatedness of the populations was measured by a morphometrical analysis. Widespread polymorphism was found for three of the four components of the two genetic systems responsible for the two barriers, with at least 13 populations possessing genes for one or both of the barriers. In one system (the C7/U8 system; Christie and Macnair, 1984), the distribution of the two component genes was correlated with the morphometrical similarity and geographical location of the populations. This pattern could be produced by a historical association or by an adaptive response. In the other system (the Cerig/C10 system; Macnair and Christie, 1983), the genes were more widely dispersed, and there was no obvious morphometrical or geographical association. Populations possessing the complementary factors causing partial reproductive isolation are not always widely separated geographically. These results indicate that the spread of postmating reproductive isolating genes through drift, selection, or hitchhiking could readily cause reproductive isolation to evolve in this species.>     

Both morph‐ and species‐dependent asymmetries affect reproductive barriers between heterostylous species

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre‐ versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre‐ and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co‐occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species‐dependent asymmetries, morph‐dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short‐styled flowers to stigmas of long‐styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long‐styled flowers to stigmas of short‐styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long‐styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph‐ and species‐dependent asymmetries shape boundaries between heterostylous species.     

Strength and variability of postmating reproductive isolating barriers between four European white oak species

The identification and quantification of the relative importance of reproductive isolating barriers is of fundamental importance to understand species maintenance in the face of interspecific gene flow between hybridising species. Yet, such assessments require extensive experimental fertilisations that are particularly difficult when dealing with more than two hybridising and long-generation-time species such as oaks. Here, we quantify the relative contribution of four postmating reproductive isolating barriers consisting of two prezygotic barriers (gametic incompatibility, conspecific pollen precedence) and two postzygotic barriers (germination rate, early survival) from extensively controlled pollinations between four oak species (Quercus robur, Quercus petraea, Quercus pubescens and Quercus pyrenaica) that have been shown to frequently hybridise in natural populations. We found high variation in the strength of total reproductive isolation between species, ranging from total reproductive isolation to advantage toward hybrid formation. As previously found, Q. robur pollen was unable to fertilise Q. petraea due to a strong reproductive isolating mechanism. On the contrary, Q. pubescens pollen was more efficient at fertilising Q. petraea than conspecific pollen. Overall, prezygotic barriers contribute far more than postzygotic barriers to isolate species reproductively, suggesting a role for reinforcement in the development of prezygotic barriers. Conspecific pollen precedence reduced hybrid formation when pollen competition was allowed; however, presence of conspecific pollen did not totally prevent hybridization. Our results suggest that pollen competition depends on multiple ecological and environmental parameters, including species local abundance, and that it may be of uppermost importance to understand interspecific gene flow among natural multispecies populations.     

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.     

Strength, diversity and plasticity of postmating reproductive barriers between two hybridizing oak species (Quercus robur L. and Quercus petraea (Matt) Liebl.)

Very little is known about the nature and strength of reproductive isolation (RI) in Quercus species, despite extensive research on the estimation and evolutionary significance of hybridization rates. We characterized postmating pre- and postzygotic RI between two hybridizing oak species, Quercus robur and Quercus petraea, using a large set of controlled crosses between different genotypes. Various traits potentially associated with reproductive barriers were quantified at several life history stages, from pollen-pistil interactions to seed set and progeny fitness-related traits. Results indicate strong intrinsic postmating prezygotic barriers, with significant barriers also at the postzygotic level, but relatively weaker extrinsic barriers on early hybrid fitness measures assessed in controlled conditions. Using general linear modelling of common garden data with clonal replicates, we showed that most traits exhibited important genotypic differences, as well as different levels of sensitivity to micro-environmental heterogeneity. These new findings suggest a large potential genetic diversity and plasticity of reproductive barriers and are confronted with hybridization evidence in these oak species.     

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.     

COMPARATIVE APPROACHES TO THE EVOLUTION OF REPRODUCTIVE ISOLATION: A COMMENT ON SCOPECE ET AL. 2007

Speciation can be driven by the evolution of many forms of reproductive isolation. Comparative study is a powerful approach for elucidating the relative importance of individual isolating barriers in the speciation process. A recent contribution by Scopece and colleagues provides comparative data for two groups of deceptive pollination orchids and aims to test hypotheses about which forms of isolation are most important in the two clades. The authors compare pollinator isolation and postmating isolation between the two orchid groups, and conclude that food-deceptive orchid species have less isolation by pollinator specificity than sexually deceptive species, and that postmating isolation is more important in the food-deceptive clade. Although we find this approach to be novel and potentially powerful, these conclusions are called into question by the methods used to define and select species and quantify pollinator isolation. Definition and selection of taxa were performed in a biased manner that undermines the ability to infer general patterns of speciation. Furthermore, pollinator isolation was calculated inconsistently for the two groups under study, effectively nullifying the comparison.     

Postmating reproductive barriers contribute to the incipient sexual isolation of the United States and Caribbean Drosophila melanogaster

The nascent stages of speciation start with the emergence of sexual isolation. Understanding the influence of reproductive barriers in this evolutionary process is an ongoing effort. We present a study of Drosophila melanogaster admixed populations from the southeast United States and the Caribbean islands known to be a secondary contact zone of European‐ and African‐derived populations undergoing incipient sexual isolation. The existence of premating reproductive barriers has been previously established, but these types of barriers are not the only source shaping sexual isolation. To assess the influence of postmating barriers, we investigated putative postmating barriers of female remating and egg‐laying behavior, as well as hatchability of eggs laid and female longevity after mating. In the central region of our putative hybrid zone of American and Caribbean populations, we observed lower hatchability of eggs laid accompanied by increased resistance to harm after mating to less‐related males. These results illustrate that postmating reproductive barriers act alongside premating barriers and genetic admixture such as hybrid incompatibilities and influence early phases of sexual isolation.     

Review. The strength and genetic basis of reproductive isolating barriers in flowering plants

Speciation is characterized by the evolution of reproductive isolation between two groups of organisms. Understanding the process of speciation requires the quantification of barriers to reproductive isolation, dissection of the genetic mechanisms that contribute to those barriers and determination of the forces driving the evolution of those barriers. Through a comprehensive analysis involving 19 pairs of plant taxa, we assessed the strength and patterns of asymmetry of multiple prezygotic and postzygotic reproductive isolating barriers. We then reviewed contemporary knowledge of the genetic architecture of reproductive isolation and the relative role of chromosomal and genic factors in intrinsic postzygotic isolation. On average, we found that prezygotic isolation is approximately twice as strong as postzygotic isolation, and that postmating barriers are approximately three times more asymmetrical in their action than premating barriers. Barriers involve a variable number of loci, and chromosomal rearrangements may have a limited direct role in reproductive isolation in plants. Future research should aim to understand the relationship between particular genetic loci and the magnitude of their effect on reproductive isolation in nature, the geographical scale at which plant speciation occurs, and the role of different evolutionary forces in the speciation process.     

Sympatric reinforcement of reproductive barriers between <Emphasis Type="Italic">Neotinea tridentata</Emphasis> and <Emphasis Type="Italic">N. ustulata</Emphasis> (Orchidaceae)

Reinforcement is the process by which selection favors traits that decrease mating between two incipient species in response to costly mating or the production of maladapted hybrids, causing the evolution of greater reproductive isolation between emerging species. I have studied a pair of orchids, Neotinea tridentata and N. ustulata, to examine the level of postmating pre- and post-zygotic isolating mechanisms that maintain these species, and the degree to which the boundary may still be permeable to gene flow. In this study, I performed pollen tube growth rate experiments and I investigated pre- and post-zygotic barriers by performing hand pollination experiments in order to evaluate fruit set, embryonate seed set and seed germination rates by intra- and interspecific crosses. Fruit set, the percentage of embryonate seeds and germinability of interspecific crosses were reduced compared to intraspecific pollinations, showing significant differences between sympatric and allopatric populations. While in allopatric populations the post-pollination isolation index ranged between 0.40 and 0.11, in sympatric populations orchid pairs showed total isolation due to post-pollination prezygotic barriers, guaranteed at the level of pollen–stigma interactions. Indeed, in sympatric populations, pollen tubes reached the ovary after 24 h in only 8 out of 45 plants; in the remaining cases, the pollen tubes did not enter the ovary, and thus no fruit set occurred. This pair of orchids is characterized by postmating pre-zygotic reproductive isolation in sympatric populations that prevents the formation of hybrids. This mechanism of speciation, starting in allopatry and triggering the reinforcement mechanisms of reproductive isolation in secondary sympatry, is the most likely explanation for the pattern of evolutionary transitions found in this pair of orchids.