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.     

Relative abundance and the species-specific reinforcement of male mating preference in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most studies of reinforcement have focused on the evolution of either female choice or male mating cues, following the long-held view in sexual selection theory that mating mistakes are typically more costly for females than for males. However, factors such as conspecific sperm precedence can buffer females against the cost of mating mistakes, suggesting that in some hybrid zones mating mistakes may be more costly for males than for females. Thus, the historical bias in reinforcement research may underestimate its frequency. In this study, we present evidence that reinforcement has driven the evolution of male choice in a hybrid zone between the highly promiscuous leaf beetles Chrysochus cobaltinus and C. auratus, the hybrids of which have extremely low fitness. In addition, there is evidence for male choice in these beetles and that male mating mistakes may be costly, due to reduced opportunities to mate with conspecific females. The present study combines laboratory and field methods to quantify the strength of sexual isolation, test the hypothesis of reproductive character displacement, and assess the link between relative abundance and the strength of selection against hybridization. We document that, while sexual isolation is weak, it is sufficient to produce positive assortative mating. In addition, reproductive character displacement was only detected in the relatively rare species. The strong postzygotic barriers in this system are sufficient to generate the bimodality that characterizes this hybrid zone, but the weak sexual isolation is not, calling into question whether strong prezygotic isolation is necessary for the maintenance of bimodality. Growing evidence that the cost of mating mistakes is sufficient to shape the evolution of male mate choice suggests that the reinforcement of male mate choice may prove to be a widespread occurrence.     

Linking intra‐ and interspecific assortative mating: Consequences for asymmetric sexual isolation

Assortative mating is of interest because of its role in speciation and the maintenance of species boundaries. However, we know little about how within‐species assortment is related to interspecific sexual isolation. Most previous studies of assortative mating have focused on a single trait in males and females, rather than utilizing multivariate trait information. Here, we investigate how intraspecific assortative mating relates to sexual isolation in two sympatric and congeneric damselfly species (genus Calopteryx). We connect intraspecific assortment to interspecific sexual isolation by combining field observations, mate preference experiments, and enforced copulation experiments. Using canonical correlation analysis, we demonstrate multivariate intraspecific assortment for body size and body shape. Males of the smaller species mate more frequently with heterospecific females than males of the larger species, which showed less attraction to small heterospecific females. Field experiments suggest that sexual isolation asymmetry is caused by male preferences for large heterospecific females, rather than by mechanical isolation due to interspecific size differences or female preferences for large males. Male preferences for large females and male–male competition for high quality females can therefore counteract sexual isolation. This sexual isolation asymmetry indicates that sexual selection currently opposes a species boundary.     

Tests of reproductive isolation among species in the <Emphasis Type="Italic">Fundulus notatus</Emphasis> (Cyprinodontiformes: Fundulidae) species complex

We assessed prezygotic (probability of spawning) and postzygotic (hatching success) reproductive isolation among the three ecologically and morphologically similar species in the Fundulus notatus species complex. We employed a multi-generation breeding experiment to test the hypotheses that karyotypic differences, body size differences, or geographic isolation among populations will increase pre or postzygotic reproductive barriers. Overall, prezygotic barriers were strong and postzygotic barriers weak in crosses of non-hybrid heterospecifics (F1 hybrid crosses) while prezygotic barriers were weaker and postzygotic barriers stronger in crosses involving hybrid individuals (F2 hybrid crosses and backcrosses). Prezygotic barriers among the two smaller species (Fundulus notatus and F. euryzonus) broke down rapidly; first generation hybrids spawned (F2 hybrid crosses and backcrosses) as frequently as parental forms in intraspecific crosses. There was no increase in postzygotic barriers among species with cytogenetic differences. There were increased prezygotic, but not postzygotic, barriers among geographically isolated populations of one species. While pure males and females were just as likely to spawn with hybrids, some types of hybrid females suffered from increased sterility, but not inviability, over hybrid males. Female sterility was only seen in hybrids with a Fundulus euryzonus parent, while other female hybrids produced viable eggs.     

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

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

FEMALE HETEROGAMETY AND SPECIATION: REDUCED INTROGRESSION OF THE Z CHROMOSOME BETWEEN TWO SPECIES OF NIGHTINGALES

Several lines of evidence suggest that the X chromosome plays a large role in intrinsic postzygotic isolation. The role of the Z chromosome in speciation is much less understood. To explore the role of the Z chromosome in reproductive isolation, we studied nucleotide variation in two closely related bird species, the Thrush Nightingale ( Luscinia luscinia ) and the Common Nightingale ( L. megarhynchos ). These species are isolated by incomplete prezygotic isolation and female hybrid sterility. We sequenced introns of four Z-linked and eight autosomal loci and analyzed patterns of polymorphism and divergence using a divergence-with-gene flow framework. Our results suggest that the nightingale species diverged approximately 1.8 Mya. We found strong evidence of gene flow after divergence in both directions, although more introgression occurred from L. megarhynchos into L. luscinia . Gene flow was significantly higher on the autosomes than on the Z chromosome. Our results support the idea that the Z chromosome plays an important role in intrinsic postzygotic isolation in birds, although it may also contribute to the evolution of prezygotic isolation through sexual selection. This highlights the similarities in the genetic basis of reproductive isolation between organisms with heterogametic males and organisms with heterogametic females during the early stages of speciation.     

Mechanical and tactile incompatibilities cause reproductive isolation between two young damselfly species

External male reproductive structures have received considerable attention as a cause of reproductive isolation (RI), because the morphology of these structures often evolves rapidly between populations. This rapid evolution presents the potential for mechanical incompatibilities with heterospecific female structures during mating and could thus prevent interbreeding between nascent species. Although such mechanical incompatibilities have received little empirical support as a common cause of RI, the potential for mismatch of reproductive structures to cause RI due to incompatible species‐specific tactile cues has not been tested. We tested the importance of mechanical and tactile incompatibilities in RI between Enallagma anna and E. carunculatum, two damselfly species that diverged within the past ～250,000 years and currently hybridize in a sympatric region. We quantified 19 prezygotic and postzygotic RI barriers using both naturally occurring and laboratory‐reared damselflies. We found incomplete mechanical isolation between the two pure species and between hybrid males and pure species females. Interestingly, in mating pairs for which mechanical isolation was incomplete, females showed greater resistance and refusal to mate with hybrid or heterospecific males compared to conspecific males. This observation suggests that tactile incompatibilities involving male reproductive structures can influence female mating decisions and form a strong barrier to gene flow in early stages of speciation.     

Reproductive isolation among allopatric Drosophila montana populations

An outstanding goal in speciation research is to trace the mode and tempo of the evolution of barriers to gene flow. Such research benefits from studying incipient speciation, in which speciation between populations has not yet occurred, but where multiple potential mechanisms of reproductive isolation (RI: i.e., premating, postmating‐prezygotic (PMPZ), and postzygotic barriers) may act. We used such a system to investigate these barriers among allopatric populations of Drosophila montana. In all heteropopulation crosses we found premating (sexual) isolation, which was either symmetric or asymmetric depending on the population pair compared. Postmating isolation was particularly strong in crosses involving males from one of the study populations, and while sperm were successfully transferred, stored, and motile, we experimentally demonstrated that the majority of eggs produced were unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of pre‐ and postmating barriers showed that populations differed in the mode and magnitude of RI barriers. Our results indicate that incipient RI among populations can be driven by different contributions of both premating and PMPZ barriers occurring between different population pairs and without the evolution of postzygotic barriers.     

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.     

Geographic contrasts between pre‐ and postzygotic barriers are consistent with reinforcement in Heliconius butterflies

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.     

SEXUAL SELECTION AGAINST HYBRIDS BETWEEN SYMPATRIC STICKLEBACK SPECIES: EVIDENCE FROM A FIELD EXPERIMENT

Sexual selection against viable, fertile hybrids may contribute to reproductive isolation between recently diverged species. If so, then sexual selection may be implicated in the speciation process. Laboratory measures of the mating success of hybrids may underestimate the amount of sexual selection against them if selection pressures are habitat specific. Male F₁ hybrids between sympatric benthic and limnetic sticklebacks (Gasterosteus aculeatus complex) do not suffer a mating disadvantage when tested in the laboratory. However, in the wild males choose different microhabitats and parental females tend to be found in the same habitats as conspecific males. This sets up the opportunity for sexual selection against male hybrids because they must compete with parental males for access to parental females. To test for sexual selection against adult F₁ hybrid males, we examined their mating success in enclosures in their preferred habitat (open, unvegetated substrate) where limnetic males and females also predominate. We found significantly reduced mating success in F₁ hybrid males compared with limnetic males. Thus, sexual selection, like other mechanisms of postzygotic isolation between young sister species, may be stronger in a wild setting than in the laboratory because of habitat-specific selection pressures. Our results are consistent with, but do not confirm, a role for sexual selection in stickleback speciation.     

The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures – apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.     

THE ROLES OF LIFE‐HISTORY SELECTION AND SEXUAL SELECTION IN THE ADAPTIVE EVOLUTION OF MATING BEHAVIOR IN A BEETLE

Although there is continuing debate about whether sexual selection promotes or impedes adaptation to novel environments, the role of mating behavior in such adaptation remains largely unexplored. We investigated the evolution of mating behavior (latency to mating, mating probability and duration) in replicate populations of seed beetles Callosobruchus maculatus subjected to selection on life‐history (“Young” vs. “Old” reproduction) under contrasting regimes of sexual selection (“Monogamy” vs. “Polygamy”). Life‐history selection is predicted to favor delayed mating in “Old” females, but sexual conflict under polygamy can potentially retard adaptive life‐history evolution. We found that life‐history selection yielded the predicted changes in mating behavior, but sexual selection regime had no net effect. In within‐line crosses, populations selected for late reproduction showed equally reduced early‐life mating probability regardless of mating system. In between‐line crosses, however, the effect of life‐history selection on early‐life mating probability was stronger in polygamous lines than in monogamous ones. Thus, although mating system influenced male–female coevolution, removal of sexual selection did not affect the adaptive evolution of mating behavior. Importantly, our study shows that the interaction between sexual selection and life‐history selection can result in either increased or decreased reproductive divergence depending on the ecological context.     

Postmating–prezygotic isolation between two allopatric populations of Drosophila montana: fertilisation success differs under sperm competition

Postmating but prezygotic (PMPZ) interactions are increasingly recognized as a potentially important early‐stage barrier in the evolution of reproductive isolation. A recent study described a potential example between populations of the same species: single matings between Drosophila montana populations resulted in differential fertilisation success because of the inability of sperm from one population (Vancouver) to penetrate the eggs of the other population (Colorado). As the natural mating system of D. montana is polyandrous (females remate rapidly), we set up double matings of all possible crosses between the same populations to test whether competitive effects between ejaculates influence this PMPZ isolation. We measured premating isolation in no‐choice tests, female fecundity, fertility and egg‐to‐adult viability after single and double matings as well as second‐male paternity success (P₂). Surprisingly, we found no PMPZ reproductive isolation between the two populations under a competitive setting, indicating no difficulty of sperm from Vancouver males to fertilize Colorado eggs after double matings. While there were subtle differences in how P₂ changed over time, suggesting that Vancouver males’ sperm are somewhat less competitive in a first‐male role within Colorado females, these effects did not translate into differences in overall P₂. Fertilisation success can thus differ dramatically between competitive and noncompetitive conditions, perhaps because the males that mate second produce higher quality ejaculates in response to sperm competition. We suggest that unlike in more divergent species comparisons, where sperm competition typically increases reproductive isolation, ejaculate tailoring can reduce the potential for PMPZ isolation when recently diverged populations interbreed.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. VIII. Mating success mediated by epicuticular hydrocarbons within and between isolated populations

We tested the hypothesis that intrademic sexual selection has caused sexual isolation between populations of geographically isolated populations of cactophilic Drosophila mojavensis, and was mediated by epicuticular hydrocarbons (EHCs), contact pheromones in this system. Sexual selection and sexual isolation were estimated using a Baja California and mainland population by comparing the number of mated and unmated males and females in each of four pairwise population mating trials. EHC profiles were significantly different in mated and unmated males in the interdemic (Bajafemale symbol x Mainlandmale symbol and Mainlandfemale symbol x Bajamale symbol), but not the intrademic mating trials. A small number of EHCs was identified that best discriminated among mated and unmated males, mostly alkadienes with 34 and 37 carbons. Females showed population-specific preferences for male EHC profiles. However, EHC profiles between mated and unmated males in the intrademic mating trials were not significantly different, consistent with undetectable sexual selection estimated directly from numbers of copulating pairs vs. unmated adults. Thus, sexual isolation among populations was much stronger than sexual selection within these populations of D. mojavensis.     

Quantitative trait loci for sexual isolation between Drosophila simulans and D. mauritiana

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While recent progress has been made toward understanding the genetic basis of the postzygotic isolating mechanisms of hybrid sterility and inviability, little is known about the genetic basis of prezygotic sexual isolation. Here, we map quantitative trait loci (QTL) contributing to prezygotic reproductive isolation between the sibling species Drosophila simulans and D. mauritiana. We mapped at least seven QTL affecting discrimination of D. mauritiana females against D. simulans males, three QTL affecting D. simulans male traits against which D. mauritiana females discriminate, and six QTL affecting D. mauritiana male traits against which D. simulans females discriminate. QTL affecting sexual isolation act additively, are largely different in males and females, and are not disproportionately concentrated on the X chromosome: The QTL of greatest effect are located on chromosome 3. Unlike the genetic components of postzygotic isolation, the loci for prezygotic isolation do not interact epistatically. The observation of a few QTL with moderate to large effects will facilitate positional cloning of genes underlying sexual isolation.     

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura

Reinforcement occurs when hybridization between closely related lineages produces low‐fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male–female interactions than interactions with heterospecifics.     

Mechanisms of incomplete prezygotic reproductive isolation in an intertidal snail: testing behavioural models in wild populations

Two morphs (ecotypes) of the marine snail Littorina saxatilis coexist along Galician exposed rocky shores. They hybridize, but gene flow is impeded by a partial prezygotic reproductive barrier, and we have earlier suggested that this is a case of incipient sympatric speciation. To assess the mechanisms of prezygotic reproductive isolation, we estimated deviations from random mating (sexual selection and sexual isolation) of sympatric snails in 13 localities on the shore, and performed mate choice experiments in the laboratory. We also investigated the microdistribution of both morphs over patches of barnacles and blue mussels in the hybridization zone. We used computer simulations to separate the mechanisms contributing to reproductive isolation. On the shores sampled, male–female pairs were strongly assortative both with respect to morphs (mean Yule's V = 0.77) and size (mean Pearson's r = 0.47). In the laboratory, males of both morphs mounted other snails and mated other males and juveniles at random. However, mature females of equal sizes mated assortatively with respect to morph. The two morphs were nonrandomly distributed over barnacle and mussel patches in the hybridization zone. Monte Carlo simulations showed that this microdistribution could explain about half the morph and size relationships in male–female pairs, while a simple rejection mechanism, rejecting the first 1–3 mates if they were of contrasting morphs, accounted for the remaining part of the reproductive isolation, and for parts of the size relationships found between mates. A size discriminant mate choice mechanism may also, to a lesser extent, contribute to the sexual isolation. Sexual selection was observed for female size (larger ones being favoured) and among certain morphs, but distinct biological mechanisms may cause these processes.     

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.     

Reproductive character displacement of female mate preferences for male cuticular hydrocarbons in Drosophila subquinaria

Several lines of evidence implicate sexual isolation in both initiating and completing the speciation process. Although its existence is straightforward to demonstrate, understanding the evolution of sexual isolation requires identifying the underlying phenotypes responsible so that we can determine how these have diverged. Here, we study geographic variation in female mate preferences for male sexual displays in the fly Drosophila subquinaria. Female D. subquinaria that are sympatric with its sister species D. recens discriminate strongly against both D. recens and allopatric conspecific males, whereas females from allopatric populations do not. Furthermore, female mate preferences target at least in part a suite of cuticular hydrocarbons (CHCs) in males and geographic variation in CHCs mirrors the pattern of mate discrimination. In this study, we quantify female mate preferences for male CHCs from populations that span the geographic range of D. subquinaria. We find that the direction of linear sexual selection varies significantly between populations that are sympatric versus allopatric with D. recens in a pattern of reproductive character displacement. Differences in preference partially align with existing differences in CHCs and patterns of sexual isolation, although discrepancies remain that suggest the involvement of additional traits and/or more complex, nonlinear preference functions.