Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

An integrative multidisciplinary approach to understanding cryptic divergence in Brazilian species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

An integrative multidisciplinary approach was used to delimit boundaries among cryptic species within the Anastrepha fraterculus complex in Brazil. Sexual compatibility, courtship and sexual acoustic behaviour, female morphometric variability, variation for the mitochondrial gene COI, and the presence of Wolbachia were compared among A. fraterculus populations from the Southern (Vacaria, Pelotas, Bento Gonçalves, São Joaquim) and Southeastern (Piracicaba) regions of Brazil. Our results suggest full mating compatibility among A. fraterculus populations from the Southern region and partial pre‐zygotic reproductive isolation of these populations when compared with the population from the Southeastern region. A. fraterculus populations from both regions differed in the frequency of courtship displays and aspects of the calling phase and mounting acoustic signal. Morphometric analysis showed differences between Southern region and Southeastern region samples. All populations analyzed were infected with Wolbachia. The trees generated from the COI sequencing data are broadly congruent with the behavioural and morphometric data with the exception of one Southern population. The likely mechanisms by which A. fraterculus populations might have diverged are discussed in detail based on behavioural, morphometric, molecular genetics, and biogeographical studies.     

Behavioral reproductive isolation inDrosophila silvestris,D. heteroneura,and their F1 hybrids (Diptera: Drosophilidae)

We investigated the role that courtship and aggressive interactions may have for the maintenance of reproductive isolation betweenDrosophila silvestris andD. heteroneura. We examined the behavioral bases of reproductive isolation between the parental species and we examined the courtship success of each sex of both reciprocal F₁ hybrids when paired with the parental species. We found reduced copulation success among heterotypic parental pairs compared to homotypic pairs, which was primarily due to the lack of courtship initiation betweenD. silvestris males andD. heteroneura females. When hybrid males from both reciprocal crosses were paired with parental females their copulation successes were not significantly different from that of parental males. In contrast, hybrid females from both crosses had reduced copulation success withD. silvestris males, which in turn was primarily due to a reduced success of reaching later stages of courtship. The time spent in copulation by hybrid males was intermediate between the two parental males. We studied aggression by observing the interactions of males of heterotypic pairs, both between the parental species and between the hybrids and parental males. A lack of aggressive interactions betweenD. silvestris males andD. heteroneura males in addition to the lack of courtship suggests thatD. silvestris males do not respond toD. heteroneura individuals of either sex. Hybrid males were equally successful in winning fights with bothD. silvestris andD. heteroneura males. These results indicate that the behavioral isolation betweenD. silvestris andD. heteroneura may be largely a consequence of the earliest stages of interactions. The two species may differ either in activity levels or in morphological or chemical traits that are important for species and mate recognition. The relatively high copulation and aggressive success of hybrids indicates that sexual selection against hybrids alone is unlikely to be a sufficient force to reduce gene flow and maintain species distinctions.     

A narrow hybrid zone between the grasshoppers Stenobothrus clavatus and Stenobothrus rubicundus (Orthoptera: Gomphocerinae): female preferences for courtship songs

Two grasshopper species, Stenobothrus rubicundus and Stenobothrus clavatus, were previously shown to hybridize in a narrow contact zone on Mount Tomaros in northern Greece. The species are characterized by complex and completely different courtship songs. In the present study, we investigated female preferences for the courtship songs of S. rubicundus, S. clavatus and hybrids in playback experiments. Playback of the courtship songs revealed assortative preferences in females of the parental species: they significantly more often preferred the songs of conspecific males. Hybrid females showed a lower selectivity than parental females, responding somewhat equally eager to playback of the songs of S. clavatus, S. rubicundus, and natural hybrid song, although less actively to the F₁ hybrid song. The results suggest that hybrid males may lose to males of parental species, whereas hybrid females would even have an advantage over parental females. Comparison of responses of females from allopatric populations and Mount Tomaros to different song types shows no evidence for reinforcement. Asymmetry found in female preferences may have implications for the structure of the hybrid zone. © 2013 The Linnean Society of London     

Incipient speciation in the Anastrepha fraterculus cryptic species complex: reproductive compatibility between A. sp.1 aff. fraterculus and A. sp.3 aff. fraterculus

Reproductive isolation among biological strains can be detected by analyzing reproductive barriers between populations. The Anastrepha fraterculus Wiedemann (Diptera: Tephritidae) cryptic species complex comprises eight morphotypes defined by morphometric characteristics. The present study analyzed the existence of pre‐ and post‐zygotic isolation among populations of A. sp.1 aff. fraterculus (A. sp.1) and A. sp.3 aff. fraterculus (A. sp.3) determined by tests of sexual compatibility, analyses of the temporal pattern of calling behavior, and the reproductive performance of the offspring of homotypic and heterotypic crosses. Pre‐zygotic tests indicated asymmetric matings with preferences for homotypic crosses. The A. sp.3 population demonstrated distinct characteristics, including low copulation percentages and a high proportion of homotypic copulations under crowded conditions. The females of the A. sp.1 population demonstrated lower copulation latency. The analyzed morphotypes demonstrated two peaks in pheromone emission activity, at the beginning and at the end of the morning and were differentiated in the abundance of males. The calling behavior of the hybrids between A. sp.1 male and A. sp.3 female crosses had an intermediate pattern compared to either parental type. Post‐zygotic compatibility tests indicated low viability of the cross between A. sp.3 males and A. sp.1 females. Pronounced asymmetries were found in the sex ratios of the offspring of heterotypic crosses, and only hybrids from A. sp.1 male vs. A. sp.3 female crosses produced descendants. The observed pre‐ and post‐zygotic incompatibilities reflect the probable occurrence of incipient speciation between A. sp.1 and A. sp.3. A more adequate taxonomic classification of this species complex that considers the distinct characteristics of each morphotype will be needed to improve environmentally wise control methods against this insect pest.     

FUNCTIONAL ASPECTS OF DROSOPHILA COURTSHIP

1. The elements that make up the courtship behaviour of males and of females are briefly described. It is pointed out that some of the terms used, such as female ‘repelling’ behaviour, are misleading as they do not reflect the known functions of the behaviours. 2. Evidence has been presented for a number of distinct pheromones with different functions during courtship. These claims are critically examined as the evidence is incomplete and at times conflicting. It seems unlikely that any pheromones other than those acting over a very short distance are involved in courtship. There is sound evidence for an aphrodisiac pheromone produced by all females which stimulates male courtship. A pheromone, which may be the same one, is produced by males less than 12 h old, which also stimulates male courtship. No function is ascribed to this pheromone. Fertilized females either produce less aphrodisiac pheromone or they may, in addition, produce one that inhibits male courtship. Mature males may also produce an inhibitory pheromone. Females produce a contact pheromone which is species-specific and involved in sexual isolation. It is not at present clear whether this is different from the aphrodisiac pheromone. 3. There is considerable variability in the importance of vision in courtship. Many species will mate satisfactorily in the dark, suggesting that visual stimuli are not critical. Most species use vision to orient towards one another and for males to track and follow females. Even in light-independent species such as D. melanogaster, specific visual signals may be used in courtship although they are not obligatory. Thus the red eye of the male is a sexual signal for females. Conversely, some light-dependent species do not appear to make use of visual signals as a major factor in courtship. Some, however, do perform behaviours that are clearly visual and which may act to emphasize markings on wings, head or body. 4. The majority of Drosophila species perform courtship songs by vibrating one or both wings. The songs produced by males sexually stimulate the females. They are species specific and there is considerable indirect and some direct evidence that the songs are involved in sexual isolation. Males of many species produce two different songs during courtship and it is probable that one is concerned mainly with sexual stimulation and the other with species recognition. Females of certain species of Drosophila and Zaprionus also sing during courtship and these songs may aid species recognition by males. In addition males and unreceptive females perform ‘aggressive’ songs. 5. Almost all studies of Drosophila courtship have been made in very confined conditions in the laboratory. Interpretation of some of the results obtained in this way may require modification in the light of ecological research and observation of courtships under more natural conditions.     

GENETICS OF SEXUAL ISOLATION AND COURTSHIP DYSFUNCTION IN MALE HYBRIDS OF DROSOPHILA PSEUDOOBSCURA AND DROSOPHILA PERSIMILIS

Despite the importance of sexual isolation to speciation, few studies have analyzed the genetic basis of interspecific mating discrimination, particularly using hybrid males. In this study, I investigated the genetic basis of sexual isolation using male hybrids of Drosophila pseudoobscura and D. persimilis. Hybrid male mating success was caused by interactions between the X-chromosome and autosomes (or Y-chromosome), and different arms of the X-chromosome contributed to mating success with females of each species. Further, although there was an X-chromosome component to mating success, its magnitude was not disproportionately large when compared with the proportion of the genome contained on this chromosome. Some hybrid males courted with an anomalously low intensity, so I simultaneously mapped the genetic basis of this “courtship dysfunction.” The courtship dysfunction was caused by an interaction between the left arm of the X-chromosome in D. persimilis with the autosomes or Y-chromosome from D. pseudoobscura. Anomalous courtship behavior in interspecific hybrids can obscure the conclusions of studies of the genetics of sexual isolation, so courtship intensity should be evaluated in all such investigations.     

Current knowledge of the species complex Anastrepha fraterculus (Diptera,Tephritidae) in Brazil

The study of the species complex Anastrepha fraterculus (Af complex) in Brazil is especially important in a taxonomical, evolutionary and pest management context, because there are evidences that some of them may occur in sympatry. In this review, we analyzed the main results supporting evidences that three cryptic species occur in Brazil. The taxonomical and phylogenetic relationships based on eggshell morphology, adult morphometrics, as well as cytotaxonomy and genetic differentiations are discussed. We also review available information on sexual behavior including acoustic communication of males during courtship and sexual incompatibility; and chemical signals involved in the communication between sexes, with a special focus on sex pheromones. We examined the role of long- and short-range pheromones (male-produced volatiles and cuticular hydrocarbons, respectively), their implications in sexual isolation, and their possible use for chemotaxonomic differentiation of the putative species of the Af complex.     

Divergent host plant adaptation drives the evolution of sexual isolation in the grasshopper Hesperotettix viridis (Orthoptera: Acrididae) in the absence of reinforcement

Early stages of lineage divergence in insect herbivores are often related to shifts in host plant use and divergence in mating capabilities, which may lead to sexual isolation of populations of herbivorous insects. We examined host preferences, degree of differentiation in mate choice, and divergence in cuticular morphology using near‐infrared spectroscopy in the grasshopper Hesperotettix viridis aiming to understand lineage divergence. In Kansas (USA), H. viridis is an oligophagous species feeding on Gutierrezia and Solidago host species. To identify incipient mechanisms of lineage divergence and isolation, we compared host choice, mate choice, and phenotypic divergence among natural grasshopper populations in zones of contact with populations encountering only one of the host species. A significant host‐based preference from the two host groups was detected in host‐paired feeding preference studies. No‐choice mate selection experiments revealed a preference for individuals collected from the same host species independent of geographic location, and little mating was observed between individuals collected from different host species. Female mate choice tests between males from the two host species resulted in 100% fidelity with respect to host use. Significant differentiation in colour and cuticular composition of individuals from different host plants was observed, which correlated positively with host choice and mate choice. No evidence for reinforcement in the zone of contact was detected, suggesting that divergent selection for host plant use promotes sexual isolation in this species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 866–878.     

Courtship behaviour of the African Maize Stem Borer: Busseola fusca (Fuller) (Lepidoptera: Noctuidae) under laboratory conditions

Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is the most important African stem borer damaging maize and sorghum. Chemical mediators play an essential role in all life cycle of this moth, especially for mating recognition and host plant choice. The female sex pheromone, courtship and mating behaviours act on the reproductive isolation within insect populations. B. fusca courtship behaviour was studied to decipher each step that could account as a process for reproductive isolation. B. fusca males and females presented a very simple and fast courtship behaviour, without any particular events or male pheromone emission.     

Patterns of inheritance of mating signals in interspecific hybrids between sailfin and shortfin mollies (Poeciliidae: Poecilia: Mollienesia)

Differences in male morphology and mating behaviors are thought to confer species sexual isolation between sailfin and shortfin species of mollies. This study used interspecific crosses between the sailfin molly, P. latipinna, and the shortfin molly, P. mexicana, to investigate patterns of inheritance of morphological traits and behavioral rates of three mating behaviors in F1 hybrid males. The two parental species showed clear species differences with respect to the length of the dorsal fin and dorsal fin ray number. First generation hybrid males were intermediate between the two parental species for dorsal fin length and fin ray number, suggesting autosomal control of this trait with little effect of dominance by genes from either parental species. Parental species showed clear species differences in their rates of courtship displays. Unlike the pattern for dorsal fin morphology, F1 hybrid males showed a clear distinction in display rates with respect to the direction of the interspecific cross. Male hybrids whose sires were P. latipinna had courtship display rates that were up to three times higher than the rates of displays performed by hybrid males whose fathers were P. mexicana. The distribution of phenotypes between the parental species and that of hybrid males sired by that parental species was nearly identical. Such a pattern suggests the influence of Y-linked genes on the inheritance of courtship display rates in mollies.     

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post‐mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybrid speciation to occur. Here, we investigate the potential of behavioural mate choice to generate assortative mating among hybrids and parental species. We made three‐first‐generation hybrid crosses between different species of African cichlid fish. In three‐way mate‐choice experiments, we allowed hybrid and nonhybrid females to mate with either hybrid or nonhybrid males. We found that hybrids generally mated nonrandomly and that hybridization can lead to the expression of new combinations of traits and preferences that behaviourally isolate hybrids from both parental species. Specifically, we find that the phenotypic distinctiveness of hybrids predicts the symmetry and extent of their reproductive isolation. Our data suggest that behavioural mate choice among hybrids may facilitate the establishment of isolated hybrid populations, even in proximity to one or both parental species.     

A narrow hybrid zone between the grasshoppers Stenobothrus clavatus and Stenobothrus rubicundus: courtship song analysis

The closely related grasshopper species Stenobothrus rubicundus and Stenobothrus clavatus are known to hybridize in a very narrow contact zone on Mt. Tomaros in northern Greece. These species produce very different and complex courtship songs accompanied with visual display. We analyzed the courtship songs and underlying stridulatory movements of the hind legs in natural hybrids from Mount Tomaros. The two species were also hybridized in the laboratory and their songs were compared with the songs of the natural hybrids. Some hybrid songs were shown to have intermediate features between parental songs, whereas other hybrid songs comprised completely new elements. The clavatus‐like song elements were found to dominate in hybrid songs. These song features may influence the mating success of hybrid males in the contact zone. A comparison of hybrid songs with the song pattern of the north European S. rubicundus populations allowed us to suggest a scenario of S. rubicundus and S. clavatus origin. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

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

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

Revealing the mechanisms of sexual isolation in a case of sympatric and parallel ecological divergence

Two ecotypes of a marine intertidal snail (Littorina saxatilis), living at different microhabitats and shore levels, have evolved in sympatry and in parallel across the Galician rocky shore. These ecotypes differ in many traits (including size) due to differential adaptation. They meet, mate assortatively, and partially hybridize at the mid shore where the two microhabitats overlap. The partial sexual isolation observed is claimed to be a side‐effect of the size differences between ecotypes combined with a size assortative mating found in most populations of this species. We investigated this hypothesis using three complementary experimental approaches. First, we investigated which of the different shell variables contributed most to the variation in individual sexual isolation in the field by using two new statistics developed for that purpose: (1) pair sexual isolation and (2) r_i, which is based on the Pearson correlation coefficient. We found that size is the most important trait explaining the sexual isolation and, in particular, the males appear to be the key sex contributing to sexual isolation. Second, we compared the size assortative mating between regions: exposed rocky shore populations from north‐westwern Spain (showing incomplete reproductive isolation due to size assortative mating) and protected Spanish and Swedish populations (showing size assortative mating but not reproductive isolation between ecomorphs). Most of the variation in size assortative mating between localities was significantly explained by the within‐population level of variation on size. Third, we performed a laboratory male choice experiment, which further suggested that the choice is made predominantly on the basis of size. These results confirm the mechanism proposed to explain the sexual isolation in the Galician hybrid zone and thus support this case as a putative example of parallel incipient speciation. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 513–526.     

Beyond magic traits: Multimodal mating cues in Heliconius butterflies

Species coexistence involves the evolution of reproductive barriers opposing gene flow. Heliconius butterflies display colorful patterns affecting mate choice and survival through warning signaling and mimicry. These patterns are called “magic traits” for speciation because divergent natural selection may promote mimicry shifts in pattern whose role as mating cue facilitates reproductive isolation. By contrast, between comimetic species, natural selection promotes pattern convergence. We addressed whether visual convergence interferes with reproductive isolation by testing for sexual isolation between two closely related species with similar patterns, H. timareta thelxinoe and H. melpomene amaryllis. Experiments with models confirmed visual attraction based on wing phenotype, leading to indiscriminate approach. Nevertheless, mate choice experiments showed assortative mating. Monitoring male behavior toward live females revealed asymmetry in male preference, H. melpomene males courting both species equally while H. timareta males strongly preferred conspecifics. Experiments with hybrid males suggested an important genetic component for such asymmetry. Behavioral observations support a key role for short‐distance cues in determining male choice in H. timareta. Scents extracts from wings and genitalia revealed interspecific divergence in chemical signatures, and hybrid female scent composition was significantly associated with courtship intensity by H. timareta males, providing candidate chemical mating cues involved in sexual isolation.     

Ongoing speciation within the Anastrepha fraterculus cryptic species complex: the case of the Andean morphotype

The Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is currently composed of seven taxonomically recognized morphotypes. Both, pre‐ and post‐zygotic isolation has been documented among four of these morphotypes, revealing that in fact they appear to be distinct biological entities. In order to progress in the full delimitation of species within the complex, we examined reproductive isolation between a Colombian population of the Andean morphotype and populations belonging to four other morphotypes spanning from Mexico to Argentina. Flies from the Andean morphotype exhibited strong pre‐zygotic mating isolation through temporal partitioning of mating activity. Post‐zygotic isolation was observed for crosses of males of all morphotypes and Andean morphotype females, yet most of the F1 hybrid ♂ × F1 hybrid ♀ self‐crosses showed normal levels of fertility, a finding suggesting a nuclear–cytoplasmic interaction according to previous studies. Overall, the Andean morphotype within the complex also appears to be a distinct biological entity. We discuss the implications of these findings for the understanding of speciation mechanisms in the Neotropical genus Anastrepha.     

Male courtship preferences demonstrate discrimination against allopatric colour morphs in a cichlid fish

Whether premating isolation is achieved by male‐specific, female‐specific or sex‐independent assortative preferences often depends on the underlying evolutionary processes. Here we test mate preferences of males presented with females of different allopatric colour variants of the cichlid fish Tropheus sp., a Lake Tanganyika endemic with rich geographical colour pattern variation, in which the strength of sexual isolation varies between populations. We conducted two‐way mate choice experiments to compare behaviour of males of a red‐bodied morph (population Moliro) towards females from their own population with behaviour towards females from four allopatric populations at different stages of phylogenetic and phenotypic divergence. Males courted same‐population females significantly more intensely than females of other populations, and reduced their heteromorphic courtship efforts both with increasing genetic and increasing phenotypic distinctness of the females. In particular, females of a closely related red‐bodied population received significantly more courtship than either genetically distinct, similarly coloured females (‘Kirschfleck’ morph) or genetically related, differently coloured females (‘yellow‐blotch’ morph), both of which were courted similarly. Genetically and phenotypically distinct females (Tropheus polli) were not courted at all. Consistent with previous female‐choice experiments, female courtship activity also decreased with increasing genetic distance from the males’ population. Given successful experimental and natural introgression between colour morphs and the pervasive allopatry of related variants, we consider it unlikely that assortative preferences of both sexes were driven by direct selection during periods of secondary contact or, in turn, drove colour pattern differentiation in allopatry. Rather, we suggest that sexual isolation evolved as by‐product of allopatric divergence.     

Absence of social facilitation of courtship in the wolf spider, Schizocosa ocreata (Hentz) (Araneae: Lycosidae)

Males of many animal species are reproductively limited by the difficulty and time costs of finding mates. Males of such species should be selected to take advantage of any cues that might reveal the location of prospective mates. Cues to female location are not restricted to those produced by females, but might also include the highly apparent courtship displays of males that have already found a female. By “eavesdropping” on these courting rivals, initiating sexual displays when courting rivals are detected (i.e., social facilitation of displays); males might effectively exploit the mate-searching efforts of their rivals. We tested the possibility that male Schizocosa ocreata wolf spiders exhibit social facilitation of courtship behaviors using a combination of live behavioral trials and video playback with single stimulus presentations. When exposed to visual cues from another male, male S. ocreata can discern the presence of another individual whether that individual is courting or not. However, we found no evidence of social facilitation of courtship or chemoexploratory behaviors in response to seismic or visual cues presented in isolation or combined. While complex, multimodal, male courtship signals are important in mate choice by female S. ocreata, males do not appear to use these cues to socially facilitate their own courtship.     

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.