POSTZYGOTIC ISOLATION OVER MULTIPLE GENERATIONS OF HYBRID DESCENDENTS IN A NATURAL HYBRID ZONE: HOW WELL DO SINGLE‐GENERATION ESTIMATES REFLECT REPRODUCTIVE ISOLATION?

Understanding speciation depends on an accurate assessment of the reproductive barriers separating newly diverged populations. In several taxonomic groups, prezygotic barriers, especially preferences for conspecific mates, are thought to play the dominant role in speciation. However, the importance of postzygotic barriers (i.e., low fitness of hybrid offspring) may be widely underestimated. In this study, we examined how well the widely used proxy of postzygotic isolation (reproductive output of F₁ hybrids) reflects the long‐term fitness consequences of hybridization between two closely related species of birds. Using 40 species‐specific single nucleotide polymorphism (SNP) markers, we genotyped a mixed population of collared and pied flycatchers (Ficedula albicollis and F. hypoleuca) to identify grand‐ and great grand‐offspring from interspecific crosses to derive an accurate, multigeneration estimate of postzygotic isolation. Two independent estimates of fitness show that hybridization results in 2.4% and 2.7% of the number of descendents typical of conspecific pairing. This postzygotic isolation was considerably stronger than estimates based on F₁ hybrids. Our results demonstrate that, in nature, combined selection against hybrids and backcrossed individuals may result in almost complete postzygotic isolation between two comparatively young species. If these findings are general, postzygotic barriers separating hybridizing populations may be much stronger than previously thought.     

GENETIC ARCHITECTURE AND POSTZYGOTIC REPRODUCTIVE ISOLATION: EVOLUTION OF BATESON–DOBZHANSKY–MULLER INCOMPATIBILITIES IN A POLYGENIC MODEL

The Bateson–Dobzhansky–Muller model predicts that postzygotic isolation evolves due to the accumulation of incompatible epistatic interactions, but few studies have quantified the relationship between genetic architecture and patterns of reproductive divergence. We examined how the direction and magnitude of epistatic interactions in a polygenic trait under stabilizing selection influenced the evolution of hybrid incompatibilities. We found that populations evolving independently under stabilizing selection experienced suites of compensatory allelic changes that resulted in genetic divergence between populations despite the maintenance of a stable, high‐fitness phenotype. A small number of loci were then incompatible with multiple alleles in the genetic background of the hybrid and the identity of these incompatibility loci changed over the evolution of the populations. For F₁ hybrids, reduced fitness evolved in a window of intermediate strengths of epistatic interactions, but F₂ and backcross hybrids evolved reduced fitness across weak and moderate strengths of epistasis due to segregation variance. Strong epistatic interactions constrained the allelic divergence of parental populations and prevented the development of reproductive isolation. Because many traits with varying genetic architectures must be under stabilizing selection, our results indicate that polygenetic drift is a plausible hypothesis for the evolution of postzygotic reproductive isolation.     

Speciation genes in plants

Background

Analyses of speciation genes – genes that contribute to the cessation of gene flow between populations – can offer clues regarding the ecological settings, evolutionary forces and molecular mechanisms that drive the divergence of populations and species. This review discusses the identities and attributes of genes that contribute to reproductive isolation (RI) in plants, compares them with animal speciation genes and investigates what these genes can tell us about speciation.

Scope

Forty-one candidate speciation genes were identified in the plant literature. Of these, seven contributed to pre-pollination RI, one to post-pollination, prezygotic RI, eight to hybrid inviability, and 25 to hybrid sterility. Genes, gene families and genetic pathways that were frequently found to underlie the evolution of RI in different plant groups include the anthocyanin pathway and its regulators (pollinator isolation), S RNase-SI genes (unilateral incompatibility), disease resistance genes (hybrid necrosis), chimeric mitochondrial genes (cytoplasmic male sterility), and pentatricopeptide repeat family genes (cytoplasmic male sterility).

Conclusions

The most surprising conclusion from this review is that identities of genes underlying both prezygotic and postzygotic RI are often predictable in a broad sense from the phenotype of the reproductive barrier. Regulatory changes (both cis and trans) dominate the evolution of pre-pollination RI in plants, whereas a mix of regulatory mutations and changes in protein-coding genes underlie intrinsic postzygotic barriers. Also, loss-of-function mutations and copy number variation frequently contribute to RI. Although direct evidence of positive selection on speciation genes is surprisingly scarce in plants, analyses of gene family evolution, along with theoretical considerations, imply an important role for diversifying selection and genetic conflict in the evolution of RI. Unlike in animals, however, most candidate speciation genes in plants exhibit intraspecific polymorphism, consistent with an important role for stochastic forces and/or balancing selection in development of RI in plants.Key words: Speciation, reproductive isolation, mating system isolation, pollinator isolation, ecological isolation, unilateral incompatibility, hybrid necrosis, hybrid sterility, hybrid inviability, hybrid breakdown, cytoplasmic male sterility, restoration     

Comparison of sexual compatibility in crosses between the southern and northern populations of the cabbage beetle Colaphellus bowringi

It is widely accepted that the genetic divergence and reproductive incompat- ibility between closely related species and/or populations is often viewed as an important step toward speciation. In this study, sexual compatibility in crosses between the southern XS population and the northern TA population of the polyandrous cabbage beetle Co- laphellus bowringi was investigated by testing their mating preferences, mating latency, copulation duration, and reproductive performances of post-mating. In choice mating ex- periments, the percentages ofmatings were significantly higher in intra-population crosses than in inter-population crosses. Both isolation index （/） and index of pair sexual isolation （/PSi） indicated partial mating incompatibility or assortative mating in crosses between the two different geographical populations. In single pair mating experiments, XS females in inter-population crosses mated significantly later and copulated significantly shorter than those in intra-population crosses. However, TA females in inter-population crosses mated significantly earlier and copulated longer than those in intra-population crosses, suggesting that larger XS males may enhance heterotypic mating. The lifetime fecundity was highest in XS homotypic matings, lowest in TA homotypic matings, and intermedi- ate in heterotypic rnatings between their parents. The inter-population crosses resulted in significantly lower egg hatching rate and shorter female longevity than intra-population crosses. These results demonstrated that there exist some incompatibilities in premating, postmating-prezygotic, and postzygotic stages between the southern XS population and northern TA population of the cabbage beetle Colaphellus bowringi.     

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.     

Field studies reveal strong postmating isolation between ecologically divergent butterfly populations

Gene flow between populations that are adapting to distinct environments may be restricted if hybrids inherit maladaptive, intermediate phenotypes. This phenomenon, called extrinsic postzygotic isolation (EPI), is thought to play a critical role in the early stages of speciation. However, despite its intuitive appeal, we know surprisingly little about the strength and prevalence of EPI in nature, and even less about the specific phenotypes that tend to cause problems for hybrids. In this study, we searched for EPI among allopatric populations of the butterfly Euphydryas editha that have specialized on alternative host plants. These populations recall a situation thought typical of the very early stages of speciation. They lack consistent host-associated genetic differentiation at random nuclear loci and show no signs of reproductive incompatibility in the laboratory. However, they do differ consistently in diverse host-related traits. For each of these traits, we first asked whether hybrids between populations that use different hosts (different-host hybrids) were intermediate to parental populations and to hybrids between populations that use the same host (same-host hybrids). We then conducted field experiments to estimate the effects of intermediacy on fitness in nature. Our results revealed strong EPI under field conditions. Different-host hybrids exhibited an array of intermediate traits that were significantly maladaptive, including four behaviors. Intermediate foraging height slowed the growth of larvae, while intermediate oviposition preference, oviposition site height, and clutch size severely reduced the growth and survival of the offspring of adult females. We used our empirical data to construct a fitness surface on which different-host hybrids can be seen to fall in an adaptive valley between two peaks occupied by same-host hybrids. These findings demonstrate how ecological selection against hybrids can create a strong barrier to gene flow at the early stages of adaptive divergence.     

No evidence of intrinsic reproductive isolation between two reciprocally non-monophyletic,ecologically differentiated mountain plants at an early stage of speciation

Adaptation to dissimilar habitats can trigger phenotypic and genetic differences between populations, which may, in the absence of gene flow, ultimately lead to ecological speciation. Reproductive isolation of diverging populations is a critical step at the onset of speciation. An excellent example for exploring the extent of reproductive isolation at early stages of speciation is provided by Heliosperma pusillum and H. veselskyi (Caryophyllaceae), two reciprocally non-monophyletic, ecologically differentiated species from the Alps. Interspecific gene flow—as revealed by recent genetic studies—is rare even between geographically close populations. Cross pollinations and fitness experiments revealed no evidence of intrinsic reproductive barriers, since fitness parameters measured under uniform conditions were not lower in inter- than in intraspecific crosses. Further, morphometric analyses of the offspring clearly showed that the differentiation of parental species is heritable. As parental phenotypes are likely adaptive, the intermediate morphology of hybrids may lead to reduced hybrid fitness in parental habitats. Altogether, H. pusillum and H. veselskyi provide an increasingly well characterised model system offering exciting insights into early stages of ecological speciation.     

Lineages of Silene nutans developed rapid,strong, asymmetric postzygotic reproductive isolation in allopatry

Reproductive isolation can rise either as a consequence of genomic divergence in allopatry or as a byproduct of divergent selection in parapatry. To determine whether reproductive isolation in gynodioecious Silene nutans results from allopatric divergence or from ecological adaptation following secondary contact, we investigated the pattern of postzygotic reproductive isolation and hybridization in natural populations using two phylogeographic lineages, western (W1) and eastern (E1). Experimental crosses between the lineages identified strong, asymmetric postzygotic isolation between the W1 and the E1 lineages, independent of geographic overlap. The proportion of ovules fertilized, seeds aborted, and seeds germinated revealed relatively little effect on the fitness of hybrids. In contrast, hybrid mortality was high and asymmetric: while half of the hybrid seedlings with western lineage mothers died, nearly all hybrid seedlings with E1 mothers died. This asymmetric mortality mirrored the proportion of chlorotic seedlings, and is congruent with cytonuclear incompatibility. We found no evidence of hybridization between the lineages in regions of co‐occurrence using nuclear and plastid markers. Together, our results are consistent with the hypothesis that strong postzygotic reproductive isolation involving cytonuclear incompatibilities arose in allopatry. We argue that the dynamics of cytonuclear gynodioecy could facilitate the evolution of reproductive isolation.     

The molecular and evolutionary basis of reproductive isolation in plants

Reproductive isolation is defined as processes that prevent individuals of different populations from mating, survival or producing fertile offspring. Reproductive isolation is critical for driving speciation and maintaining species identity, which has been a fundamental concern in evolutionary biology. In plants, reproductive isolation can be divided into prezygotic and postzygotic reproductive barriers, according to its occurrence at different developmental stages. Postzygotic reproductive isolation caused by reduced fitness in hybrids is frequently observed in plants, which hinders gene flow between divergent populations and has substantial effects on genetic differentiation and speciation, and thus is a major obstacle for utilization of heterosis in hybrid crops. During the past decade, China has made tremendous progress in molecular and evolutionary basis of prezygotic and postzygotic reproductive barriers in plants. Present understandings in reproductive isolation especially with new data in the last several years well support three evolutionary genetic models, which represent a general mechanism underlying genomic differentiation and speciation. The updated understanding will offer new approaches for the development of wide-compatibility or neutral varieties, which facilitate breeding of hybrid rice as well as other hybrid crops.     

An unusual barrier to gene flow: perpetually immature larvae from inter-population crosses in the flour beetle, Tribolium castaneum

We genetically characterize an unusual hybrid incompatibility phenotype manifest in F₁ offspring of crosses between two populations of Tribolium castaneum. Hybrid larvae cease development at the third larval instar, persisting as ‘perpetually immature larvae’ thereafter. Although unable to produce viable adult hybrid offspring with one another, each population produces abundant, fertile hybrids with other populations, indicating a recent origin of the incompatibility and facilitating genetic studies. We mapped the paternal component of the hybrid phenotype to a single region, which exhibits two characteristics common to hybrid incompatibility: marker transmission ratio distortion within crosses and elevated genetic divergence between populations. The incompatible variation and an elevation in between‐population genetic divergence is associated with a region containing the T. castaneum ecdysone receptor homologue, a major regulatory switch, controlling larval moults, pupation and metamorphosis. This contributes to understanding the genetics of speciation in the Coleoptera, one of the most speciose of all arthropod taxa.     

WIDESPREAD HOST‐DEPENDENT HYBRID UNFITNESS IN THE PEA APHID SPECIES COMPLEX

Linking adaptive divergence to hybrid unfitness is necessary to understand the ecological factors contributing to reproductive isolation and speciation. To date, this link has been demonstrated in few model systems, most of which encompass ecotypes that occupy relatively early stages in the speciation process. Here we extend these studies by assessing how host‐plant adaptation conditions hybrid fitness in the pea aphid, Acyrthosiphon pisum. We made crosses between and within five pea aphid biotypes adapted to different host plants and representing various stages of divergence within the complex. Performance of F1 hybrids and nonhybrids was assessed on a “universal” host that is favorable to all pea aphid biotypes in laboratory conditions. Although hybrids performed equally well as nonhybrids on the universal host, their performance was much lower than nonhybrids on the natural hosts of their parental populations. Hence, hybrids, rather than being intrinsically deficient, are maladapted to their parents’ hosts. Interestingly, the impact of this maladaptation was stronger in certain hybrids from crosses involving the most divergent biotype, suggesting that host‐dependent postzygotic isolation has continued to evolve late in divergence. Even though host‐independent deficiencies are not excluded, hybrid maladaptation to parental hosts supports the hypothesis of ecological speciation in this complex.     

Experimental evidence for reduced hybrid viability between dwarf and normal ecotypes of lake whitefish (Coregonus clupeaformis Mitchill)

Forces driving the evolution of reproductive isolation among natural populations, as well as the mechanisms involved to maintain it, are still poorly understood. Because sympatric fish ecotypes mainly differ in phenotypic traits associated with occupying distinct trophic niches, it is generally believed that reproductive isolation is mainly driven by ecological divergent selection, excluding genome incompatibility as a basis for postmating isolation. We did cross experiments between dwarf and normal ecotypes of lake whitefish (Coregonus clupeaformis Mitchill) originating from distinct glacial refugia to test the hypothesis that their geographical isolation during the Pleistocene may have led to sufficient genetic divergence for the development of reproductive isolation between them before their secondary contact. Similar fertilization success in pure and hybrid crosses indicated the absence of gametic incompatibility between the two ecotypes. In contrast, daily embryonic mortality rates were 2.4–4.7 times higher in reciprocal hybrid crosses compared to pure crosses, which supports our working hypothesis. These results, along with previous morphological and population genetic studies, indicate that both genetic and ecological mechanisms may jointly act to promote speciation among northern freshwater fish ecotypes.     

Patterns of Reproductive Isolation in Toads

Understanding the general features of speciation is an important goal in evolutionary biology, and despite significant progress, several unresolved questions remain. We analyzed an extensive comparative dataset consisting of more than 1900 crosses between 92 species of toads to infer patterns of reproductive isolation. This unique dataset provides an opportunity to examine the strength of reproductive isolation, the development and sex ratios of hybrid offspring, patterns of fertility and infertility, and polyploidization in hybrids all in the context of genetic divergence between parental species. We found that the strength of intrinsic postzygotic isolation increases with genetic divergence, but relatively high levels of divergence are necessary before reproductive isolation is complete in toads. Fertilization rates were not correlated to genetic divergence, but hatching success, the number of larvae produced, and the percentage of tadpoles reaching metamorphosis were all inversely related with genetic divergence. Hybrids between species with lower levels of divergence developed to metamorphosis, while hybrids with higher levels of divergence stopped developing in gastrula and larval stages. Sex ratios of hybrid offspring were biased towards males in 70% of crosses and biased towards females in 30% of crosses. Hybrid females from crosses between closely related species were completely fertile, while approximately half (53%) of hybrid males were sterile, with sterility predicted by genetic divergence. The degree of abnormal ploidy in hybrids was positively related to genetic divergence between parental species, but surprisingly, polyploidization had no effect on patterns of asymmetrical inviability. We discuss explanations for these patterns, including the role of Haldane''s rule in toads and anurans in general, and suggest mechanisms generating patterns of reproductive isolation in anurans.     

Adaptive divergence and the evolution of reproductive isolation in the wild: an empirical demonstration using introduced sockeye salmon

Populations exposed to different ecological environments should diverge for phenotypic traits that influence survival and reproduction. This adaptive divergence should reduce gene flow between populations because immigrants become less fit than residents and because hybrids perform poorly in either environment (i.e., ecologically-dependent reproductive isolation). Here I demonstrate adaptive divergence and the evolution of reproductive isolation in populations of sockeye salmon (Oncorhynchus nerka) introduced from a common ancestral source into a new lake system (Lake Washington, Washington). The introduced fish founded several new populations, two of which experience very different environments during breeding and early development (Cedar River v.s. Pleasure Point beach). Over 13 generations, the two populations diverged for adult traits (female body size, male body depth; measured in the wild) and embryo traits (survival to hatching, development rate, size at emergence; measured in a common environment). The rates of divergence for these characters were similar to those observed in other examples of rapid evolution, and can best be attributed to natural selection. Partial reproductive isolation has evolved in concert with adaptive divergence: the rate of exchange of adults between the populations (determined using natural tags) is higher than the rate of gene flow (determined using DNA microsatellites). The demonstration that adaptive divergence can initiate reproductive isolation in less than 13 generations suggests that the first signs of ecological speciation may appear soon after new environments are first colonized.     

SPECIATION BY REINFORCEMENT OF PREMATING ISOLATION

The generation of premating isolation given partial or complete postzygotic isolation between populations is termed reinforcement or, in the case of complete isolation, reproductive character displacement. In this study we use computer simulations and a multilocus genetic model to reevaluate the theory of reinforcement. We consider the evolution of female preferences for a male secondary sexual trait. If the populations differ in mean female preference, there is direct selection on the preference for further divergence, which may be augmented by a correlated response to sexual selection on males. Two factors prevent divergence. First, if postzygotic isolation is not complete, gene flow can prevent divergence and lead to a hybrid swarm. This is the usual outcome whenever the average number of breeding adult offspring produced by a hybrid mating is sufficient to replace the parents. Second, one or the other population may become extinct because of the large number of hybrid matings it is involved in. The likelihood of extinction is lowered if population growth rates are high, if hybrids are inviable rather than infertile, or under some conditions when allopatric populations provide immigrants into the contact zone. Provided hybrid fitness is sufficiently low, there is a wide range of genetic and ecological conditions under which reinforcement rather easily occurs, and also a range under which it may occur because of stochastic effects on both the inheritance parameters and the population sizes.     

Along the speciation continuum: Quantifying intrinsic and extrinsic isolating barriers across five million years of evolutionary divergence in California jewelflowers

Understanding the relative roles of intrinsic and extrinsic reproductive barriers, and their interplay within the geographic context of diverging taxa, remains an outstanding challenge in the study of speciation. We conducted a comparative analysis of reproductive isolation in California Jewelflowers (Streptanthus, s.l., Brassicaceae) by quantifying potential barriers to gene flow at multiple life history stages in 39 species pairs spanning five million years of evolutionary divergence. We quantified nine potential pre‐ and postzygotic barriers and explored patterns of reproductive isolation in relation to genetic distance. Intrinsic postzygotic isolation was initially weak, increased at intermediate genetic distances, and reached a threshold characterized by complete genetic incompatibility. Climatic niche differences were strong at shallow genetic distances, and species pairs with overlapping ranges showed slight but appreciable phenological isolation, highlighting the potential for ecological barriers to contribute to speciation. Geographic analyses suggest that speciation is not regionally allopatric in the California Jewelflowers, as recently diverged taxa occur in relatively close proximity and display substantial range overlap. Young pairs are characterized by incomplete intrinsic postzygotic isolation, suggesting that extrinsic barriers or fine‐scale spatial segregation are more important early in the divergence process than genetic incompatibilities.     

Intraspecific variation in reproductive barriers between two closely related Arabidopsis sister species

Reproductive isolation (RI) is a critical component of speciation and varies strongly in timing and strength among different sister taxa, depending on, for example the geography of speciation and divergence time. However, these factors may also produce variation in timing and strength among populations within species. Here we tested for variation in the expression of RI among replicate population pairs between the sister taxa Arabidopsis lyrata subsp. lyrata and A. arenicola. While the former is predominantly outcrossing, the latter is predominantly selfing. We focused on intrinsic prezygotic and postzygotic RI as both species occur largely in allopatry. We assessed RI by performing within-population crosses and interspecific between-population crosses, and by raising offspring. RI was generally high between all interspecific population pairs, but it varied in timing and strength depending on population history. Prezygotic isolation was strongest between the closest-related population pair, while early postzygotic isolation was high for all other population pairs. Furthermore, the timing and strength of RI depended strongly on cross direction. Our study provides empirical support that reproductive barriers between species are highly variable among population pairs and asymmetric within population pairs, and this variation seems to follow patterns typically described across species pairs.     

The genetic isolation between some populations of Proasellus coxalis

We tested the reroductive compatibility existing between four geographically different populations of Proaselis coxalis (Crustacea Isopoda) collected in the river Sarno (near Naples) and on three Sicilian sites: San Domenico; Villa Grazia di Carini and Enna. By means of laboratory hybridization experiments (“no-choice” method) we investigated and characterized the reproductive isolation established between these populations under allopatric conditions. The populations San Domenico and Enna are reproductively isolated from Villa Grazia di Carini and Sarno, so that they may be considered as belonging to different species. The mechanism of reproductive isolation are of postzygotic nature only: embronic death, hbrid inviability and sterility of the surviving hybrids. These results agree with tie theory of aioatric speciation, according to which in the first phase of speciation only post-zygotic mechanisms occur as an accidental product of genetic divergence.     

Evolution of postzygotic reproductive isolation in galliform birds: analysis of first and second hybrid generations and backcrosses

The process of speciation is a crucial aspect of evolutionary biology. In this study, we analysed the patterns of evolution of postzygotic reproductive isolation in Galliformes using information on hybridization and genetic distance among species. Four main patterns arose: (1) hybrid inviability and sterility in F₁ hybrids increase as species diverge; (2) the presence of geographical overlap does not affect the evolution of postzygotic isolation; (3) the galliforms follow Haldane's rule; (4) hybrid inviability is higher in F₂ than in F₁ hybrids, but does not appear to be increased in the backcrosses. This study contributes to the growing evidence suggesting that the patterns of evolution of postzygotic isolation and the process of speciation are shared among avian groups (and animals in general). In particular, our results support the notion of F₂ hybrid inviability as being key for the maintenance of species genetic integrity when prezygotic isolation barriers are overcome in closely related species, in which postzygotic isolation in the F₁ hybrid might still not be fully developed. To the contrary, hybrids from backcrosses did not show serious inviability problems (at least not more than F₁ hybrids), demonstrating that they could generate gene flow among bird species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 528–542.     

WHEN MORPHOLOGY MISLEADS: INTERPOPULATION UNIFORMITY IN SEXUAL SELECTION MASKS GENETIC DIVERGENCE IN HARLEQUIN BEETLE-RIDING PSEUDOSCORPION POPULATIONS

Differences in secondary sexual characteristics of males often provide the most conspicuous means of distinguishing between closely related species. Does this therefore imply that the absence of differentiation in exaggerated male traits between allopatric populations provides evidence of a single, genetically cohesive species? We addressed this question with a comprehensive investigation of two populations (French Guiana and Panama) of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides. This highly sexually dimorphic pseudoscorpion is currently described as a single species, ranging throughout the Neotropics. Our morphometric analyses detected minimal differentiation between the two populations in all nine external morphological characters measured, including sexually dimorphic traits in males. Only in traits of the spermatophore was there any appreciable level of differentiation. Behavior differentiation and prezygotic reproductive isolation were also limited: 78.3% of males successfully transferred sperm to “foreign” females, and in 63.9% of these cases, females' eggs were successfully fertilized. By contrast, extensive divergence existed in two of nine electrophoretic loci, including an essentially fixed-allele difference at the Ldh locus. Most significantly, postzygotic reproductive isolation was complete, with heteropopulation zygotes invariably aborting early in development. These results strongly suggest that the two populations are, in fact, sibling species, a conclusion supported by our recently published findings on their marked divergence in minisatellite DNA. How can such interpopulation homogeneity in male sexually dimorphic traits exist in the face of strong genetic divergence? We propose that sexual selection, oscillating between favoring small and then large males, maintains such high levels of male variability within each population that it has obscured a speciation event in which genetic divergence and postzygotic incompatibility have clearly outpaced the evolution of prezygotic reproductive isolation.