LOCAL ADAPTATION IN TWO SUBSPECIES OF AN ANNUAL PLANT: IMPLICATIONS FOR MIGRATION AND GENE FLOW

Plant populations often adapt to local environmental conditions. Here we demonstrate local adaptation in two subspecies of the California native annual Gilia capitata using standard reciprocal transplant techniques in two sites (coastal and inland) over three consecutive years. Subspecies performance in each site was measured in four ways: probability of seedling emergence, early vegetative size (length of longest leaf), probability of flowering, and total number of inflorescences produced per plant. Analysis of three of the four variables demonstrated local adaptation through site-by-subspecies interactions in which natives outperformed immigrants. The disparity between natives and immigrants in their probability of emergence and probability of flowering was greater at the coastal site than at the inland site. Treated in isolation, these two fitness components suggest that migration from the coast to the inland site may be less restricted by selection than migration in the opposite direction. Two measurements of individual size (leaf length and number of inflorescences), suggest (though not strongly) that immigrants may be subject to weaker selection at the coastal site than at the inland site. A standard cohort life table is used to compare replacement rates (R₀) for each subspecies at each site. Comparisons of R₀s suggest that immigrants are under a severe demographic disadvantage at the coastal site, but only a small disadvantage at the inland site. The results point out the importance of integrating over several fitness components when documenting the magnitude of local adaptation.     

FREQUENCY-DEPENDENT SEED PRODUCTION AND HYBRIDIZATION RATES: IMPLICATIONS FOR GENE FLOW BETWEEN LOCALLY ADAPTED PLANT POPULATIONS

Gene flow between genetically distinct plant populations can have significant evolutionary consequences. It can increase genetic diversity, create novel gene combinations, and transfer adaptations from one population to another. This study addresses the roles of frequency-dependent selection and mating system in gene exchange between two subspecies of Gilia capitata (Polemoniaceae). Long-distance migrants are likely to be rare in new habitats, and the importance of immigrant frequency to fitness, gene exchange, and ultimately introgression, has not been explored. To test for the importance of frequency in migration, a field experiment was conducted in which artificial populations (arrays) composed of different mixtures of the two subspecies were placed in the home habitats of both. Female function (seed production) and a portion of male function (hybridization rate) were compared for the two subspecies to assess the potential for gene exchange and introgression between them. Individual fitness (through both hybridization and seed production) for the inland subspecies varied with its frequency as an immigrant at the coastal site. Rare immigrants produced fewer seeds and fathered fewer hybrid offspring. In contrast, both forms of reproductive function were frequency independent for the coastal subspecies when it was an immigrant at the inland site. Seed production was high and insensitive to frequency, and immigrants from the coast never successfully fertilized the inland subspecies' seeds. To control for the effects of frequency-dependent pollinator behavior in the field, hand crosses were performed in the greenhouse using a range of pollen mixtures. The greenhouse experiment demonstrated that cross-fertilization is possible in only one direction, that cross-pollination in the other direction is only partially successful, and that pollen from the coastal subspecies has a strong negative effect on seed production by the inland subspecies. Experimental pollen supplementation in the field verified both the unilateral incompatibility and the negative effect of coastal pollen on inland plant seed production observed in the greenhouse. Contrasts between field array and greenhouse results suggest that pollinator behavior and other ecological factors act to exaggerate reproductive barriers between the two subspecies. In this system, immigrant frequency interacts with reproductive biology and pollinator ecology to enhance gene flow between the populations in one direction, while restricting gene establishment and introgression in the other direction.     

GENETIC INTERACTIONS AND NATURAL SELECTION IN LOUISIANA IRIS HYBRIDS

Hybridization between divergent lineages has long been assumed to give rise to unfavorable interactions between the parental genomes. These deleterious genetic interactions are further assumed to result in the production of hybrid offspring with decreased levels of viability and/or fertility. To test this assumption, we investigated the role of both nuclear and cytonuclear epistatic interactions in determining the frequencies of F₂ genotypes produced in crosses between two species of Louisiana iris, Iris fulva and I. brevicaulis. Overall, these crosses revealed a significant deficit of intermediate hybrid genotypes accompanied by an excess of parental-like genotypes, suggesting that genetic interactions may promote postmating reproductive isolation between these species. However, analyses of single and multilocus segregation patterns revealed a variety of negative and positive interactions between the genomes of the parental taxa at the nuclear and cytonuclear levels. Taken together, these results indicate that the traditional view that interactions between divergent genomes are always deleterious is an oversimplification. Rather, it seems likely that crosses between divergent lineages can lead to the production of both fit and unfit hybrid genotypes.     

MULTIVARIATE ANALYSIS OF SELECTION IN EXPERIMENTAL POPULATIONS DERIVED FROM HYBRIDIZATION OF TWO ECOTYPES OF THE ANNUAL PLANT DIODIA TERES W. (RUBIACEAE)

Morphologically variable F₂ genotypes derived from hybridization of coastal and inland ecotypes of the annual plant Diodia teres were used to identify selection on morphological traits in the natural habitat of each ecotype. These ecotypes occur in very different habitats, and have evolved pronounced morphological differentiation. Selection analysis can suggest whether present patterns of selection can explain morphological differences between ecotypes. F₂ genotypes were characterized morphologically, clonally replicated, and transplanted into the habitat of each ecotype. Selection was measured on six morphological traits. Directional and stabilizing selection occurred on many traits; direction and strength of selection varied sharply at different stages of growth, as revealed by a path-analysis approach that divided selection into a set of independent components. Directional selection favored traits of the native population at the coastal habitat, but less so at the inland habitat. Selection was of sufficient strength to create the observed morphological differences between ecotypes in 25–100 generations, given constant selection and sufficient genetic variation. In effects on fitness, most traits were neither independent nor consistently interactive with other traits. Rather, many traits entered into strong but evanescent interactions affecting particular components of fitness. Observed interactions did not support the hypothesis that the morphology of each ecotype was functionally integrated to a high degree.     

NARROW HYBRID ZONE BETWEEN TWO SUBSPECIES OF BIG SAGEBRUSH (ARTEMISIA TRIDENTATA: ASTERACEAE). IV. RECIPROCAL TRANSPLANT EXPERIMENTS

Does endogenous or exogenous selection stabilize the big sagebrush (Artemisia tridentata) hybrid zone? After two years of study, our reciprocal transplant experiments showed significant genotype by environment interactions for a number of fitness components, including germination, growth, and reproduction. Hybrids were the most fit within the hybrid garden. In the parental gardens, the native parental taxon was more fit than either the alien parental or hybrids. These results are consistent with the bounded hybrid superiority model, which assumes exogenous selection, but are clearly at odds with the dynamic equilibrium model, which assumes endogenous selection and universal hybrid unfitness.     

ECOLOGICAL SEGREGATION IN A SMALL MAMMAL HYBRID ZONE: HABITAT‐SPECIFIC MATING OPPORTUNITIES AND SELECTION AGAINST HYBRIDS RESTRICT GENE FLOW ON A FINE SPATIAL SCALE

The degree to which closely related species interbreed is determined by a complex interaction of ecological, behavioral, and genetic factors. We examine the degree of interbreeding between two woodrat species, Neotoma bryanti and N. lepida, at a sharp ecological transition. We identify the ecological association of each genotypic class, assess the opportunity for mating between these groups, and test whether they have similar patterns of year‐to‐year persistence on our study site. We find that 13% of individuals have a hybrid signature but that the two parental populations and backcrosses are highly segregated by habitat type and use. Also, we find that adult hybrids are comparable to parental types in terms of year‐to‐year persistence on our site but that, among juveniles, significantly fewer hybrids reach adulthood on site compared to their purebred counterparts. Our analyses show that this hybrid zone is maintained by occasional nonassortative mating coupled with hybrid fertility, but that these factors are balanced by lower apparent survival of juvenile hybrids and habitat‐based preference or selection that limits heterospecific mating while promoting backcrossing to habitat‐specific genotypes. This system presents a novel example of the role that sharp resource gradients play in reproductive isolation and the potential for genetic introgression.     

Ecological selection maintains cytonuclear incompatibilities in hybridizing sunflowers

Despite the recent renaissance in studies of ecological speciation, the connection between ecological selection and the evolution of reproductive isolation remains tenuous. We tested whether habitat adaptation of cytoplasmic genomes contributes to the maintenance of reproductive barriers in hybridizing sunflower species, Helianthus annuus and Helianthus petiolaris. We transplanted genotypes of the parental species, reciprocal F1 hybrids and all eight possible backcross combinations of nuclear and cytoplasmic genomes into the contrasting xeric and mesic habitats of the parental species. Analysis of survivorship across two growing seasons revealed that the parental species' cytoplasms were strongly locally adapted and that cytonuclear interactions (CNIs) significantly affected the fitness and architecture of hybrid plants. A significant fraction of the CNIs have transgenerational effects, perhaps due to divergence in imprinting patterns. Our results suggest a common means by which ecological selection may contribute to speciation and have significant implications for the persistence of hybridizing species.     

GENE FLOW AND SELECTION ON PHENOTYPIC PLASTICITY IN AN ISLAND SYSTEM OF RANA TEMPORARIA

Gene flow is often considered to be one of the main factors that constrains local adaptation in a heterogeneous environment. However, gene flow may also lead to the evolution of phenotypic plasticity. We investigated the effect of gene flow on local adaptation and phenotypic plasticity in development time in island populations of the common frog Rana temporaria which breed in pools that differ in drying regimes. This was done by investigating associations between traits (measured in a common garden experiment) and selective factors (pool drying regimes and gene flow from other populations inhabiting different environments) by regression analyses and by comparing pairwise F_ST values (obtained from microsatellite analyses) with pairwise Q_ST values. We found that the degree of phenotypic plasticity was positively correlated with gene flow from other populations inhabiting different environments (among‐island environmental heterogeneity), as well as with local environmental heterogeneity within each population. Furthermore, local adaptation, manifested in the correlation between development time and the degree of pool drying on the islands, appears to have been caused by divergent selection pressures. The local adaptation in development time and phenotypic plasticity is quite remarkable, because the populations are young (less than 300 generations) and substantial gene flow is present among islands.     

Experimental studies of adaptation in Clarkia xantiana. III. Phenotypic selection across a subspecies border

Sister taxa with distinct phenotypes often occupy contrasting environments in parapatric ranges, yet we generally do not know whether trait divergence reflects spatially varying selection. We conducted a reciprocal transplant experiment to test whether selection favors “native phenotypes” in two subspecies of Clarkia xantiana (Onagraceae), an annual plant in California. For four quantitative traits that differ between subspecies, we estimated phenotypic selection in subspecies’ exclusive ranges and their contact zone in two consecutive years. We predicted that in the arid, pollinator‐scarce eastern region, selection favors phenotypes of the native subspecies parviflora: small leaves, slow leaf growth, early flowering, and diminutive flowers. In the wetter, pollinator‐rich, western range of subspecies xantiana, we expected selection for opposite phenotypes. We investigated pollinator contributions to selection by comparing naturally pollinated and pollen‐supplemented individuals. For reproductive traits and for subspecies xantiana, selection generally matched expectations. The contact zone sometimes showed distinctive selection, and in ssp. parviflora selection sometimes favored nonnative phenotypes. Pollinators influenced selection on flowering time but not on flower size. Little temporal variation in selection occurred, possibly because of plastic trait responses across years. Though there were exceptions and some causes of selection remain obscure, phenotypic differentiation between subspecies appears to reflect spatially variable selection.     

Local adaptation and cogradient selection in the alpine plant, Poa hiemata, along a narrow altitudinal gradient

Alpine environments are particularly susceptible to environmental changes associated with global warming but there is potential for alpine plants to adapt to warming if local adaptation occurs and gene flow allows genotypes adapted to low altitudes to colonize higher altitude sites. Here we examine the adaptive potential of a common alpine grass, Poa hiemata, within the restricted alpine habitat of Australian mountains, across a narrow altitudinal gradient replicated in three areas. Grasses at high altitude sites had shorter leaf lengths and larger circumferences than those at lower sites. Transplant experiments with clonal material and plants grown from seed indicated that these differences were partly genetic, with environmental and genetic factors both contributing to the differences between altitudes. Differences in altitudinal forms were also evident in a common garden experiment. Plants showed a home-site advantage in terms of survival. A fitness analysis indicated that at high altitude sites, selection favored plants with short leaves and larger circumferences, whereas these traits were selected in the opposite direction at the low altitude sites. These findings indicate cogradient selection and potential for both plastic and genotypic shifts in response to climate change in P. hiemata.     

TWO HERBIVORES AND CONSTRAINTS ON SELECTION FOR RESISTANCE IN BRASSICA RAPA

Although most plants experience herbivory by several insect species, there has been little empirical work directed toward understanding plant responses to these simultaneous selection pressures. In an experiment in which herbivory by flea beetles (Phyllotreta cruciferae) and diamondback moths (Plutella xylostella) was manipulated in a factorial design, I found that selection for resistance to these herbivores is not independent in Brassica rapa. Specifically, the effect of flea beetle damage on B. rapa fitness depends on the amount of diamondback moth damage a plant experiences: damage by these herbivores has a nonadditive effect on plant fitness. When diamondbacks are abundant, plants that sustain high levels of damage by flea beetles are favored by natural selection, but when diamondbacks are rare, a low level of damage by flea beetles is favored. However, resistance to the later-feeding diamondback moth is not affected by the presence or absence of damage by early-feeding flea beetles. Thus, there are no plant-mediated ecological interactions between these herbivores that affect the outcome of selection for resistance. Because these herbivores do not independently affect plant fitness, neither is likely to develop a pairwise coevolutionary relationship with its host. Instead, coevolution is diffuse.     

Strong patterns of intraspecific variation and local adaptation in Great Basin plants revealed through a review of 75 years of experiments

Variation in natural selection across heterogeneous landscapes often produces (a) among‐population differences in phenotypic traits, (b) trait‐by‐environment associations, and (c) higher fitness of local populations. Using a broad literature review of common garden studies published between 1941 and 2017, we documented the commonness of these three signatures in plants native to North America's Great Basin, an area of extensive restoration and revegetation efforts, and asked which traits and environmental variables were involved. We also asked, independent of geographic distance, whether populations from more similar environments had more similar traits. From 327 experiments testing 121 taxa in 170 studies, we found 95.1% of 305 experiments reported among‐population differences, and 81.4% of 161 experiments reported trait‐by‐environment associations. Locals showed greater survival in 67% of 24 reciprocal experiments that reported survival, and higher fitness in 90% of 10 reciprocal experiments that reported reproductive output. A meta‐analysis on a subset of studies found that variation in eight commonly measured traits was associated with mean annual precipitation and mean annual temperature at the source location, with notably strong relationships for flowering phenology, leaf size, and survival, among others. Although the Great Basin is sometimes perceived as a region of homogeneous ecosystems, our results demonstrate widespread habitat‐related population differentiation and local adaptation. Locally sourced plants likely harbor adaptations at rates and magnitudes that are immediately relevant to restoration success, and our results suggest that certain key traits and environmental variables should be prioritized in future assessments of plants in this region.     

THE RESPONSE TO DIFFERING SELECTION ON PLANT PHYSIOLOGICAL TRAITS: EVIDENCE FOR LOCAL ADAPTATION

Understanding adaptive evolution to differing environments requires studies of genetic variances, of natural selection, and of the genetic differentiation between populations. Plant physiological traits such as leaf size and water-use efficiency (the ratio of carbon gained per water lost) have been suggested by physiological plant ecologists to be important in local adaptation to environments differing in water availability. In this study, I raised families of Cakile edentula var lacustris derived from a wet-site population and a dry-site population in a common greenhouse environment to determine the degree of genetic differentiation between the two populations and the genetic architecture of the traits. The dry-site population had significantly smaller leaf size and significantly greater water-use efficiency than the wet-site population. I used a retrospective selection analysis to compare long-term selection inferred from these results to measures of phenotypic selection from a field experiment. Both direct measures in the field and the retrospective selection gradients were consistent with the hypothesis that greater water-use efficiency and smaller leaves were adaptive in drier environments. Though the correlation between population means for water-use efficiency and leaf size was negative, the genetic correlation within populations between water-use efficiency and leaf size was positive and thus would be expected to constrain the evolutionary response to selection.     

NATURAL SELECTION ON FLORAL TRAITS IN TWO SPECIES OF LOBELIA WITH DIFFERENT POLLINATORS

Using both multivariate and univariate regression techniques, I measured selection acting through female reproductive success in two hermaphroditic species with precise pollen placement but different pollinators: hummingbird-pollinated Lobelia cardinalis and bumblebee-pollinated L. siphilitica. Six traits were analyzed in two populations of L. cardinalis and one population of L. siphilitica: flower number, mean number of flowers open per day, inflorescence height, number of days in flower, median-flower date and nectar-stigma distance. In another study it was found that female reproductive success in one population of L. cardinalis was much less pollen limited than in the other two populations, and it was therefore expected that selection of female reproductive traits in this population would be weaker. In the univariate analyses correlations caused nearly all traits to have significant directional selection coefficients. However, in the multivariate analyses no traits in L. siphilitica experienced directional or quadratic selection. Selection acted differently in the two L. cardinalis populations. The less pollen-limited population experienced positive directional selection on flower number and median-flower date, while in the other L. cardinalis population there was positive directional selection on flower number and nectar-stigma distance and both positive directional and positive quadratic selection on height. The functional significance of floral traits in these two species and the probable effect of increased sample sizes are discussed.     

SPATIAL VARIATION IN SELECTIVE REGIMES: SEXUAL SELECTION IN THE WATER STRIDER,GERRIS ODONTOGASTER

Studies of phenotypic selection in natural populations are often concerned with simply detecting selection. In adopting a more mechanistic approach, this study compares the sexual selection regimes in natural populations of the water strider Gerris odontogaster with a priori predictions of selection, based on a number of previous field and laboratory studies of the behavioral mechanisms of selection. In this species, a general reluctance of females to mate allows for intersexual selection for ability to subdue reluctant females in males. Female reluctance to mate has been shown to decrease with increasing population density, suggesting that sexual selection should be weaker in high density populations. Three different populations with large differences in population density were studied. A number of traits including parasite load, body mass, body size and male abdominal process length were found to experience significant sexual selection. The investigated populations differed considerably with regard to the total strength of selection on the measured traits and the form of selection on single traits. In general, males in the population with the highest density experienced the weakest selection for grasping ability. This pattern is ascribed to density-related alterations of female mating behavior. Selection for male grasping ability, as reflected by selection on male abdominal process length, is reduced in high-density populations where reluctant females are more easily subdued. Further, the studied populations differed significantly in mean phenotype and phenotypic variance for male abdominal process length. It is suggested that interpopulational differences in selective regimes may generate local adaptations with respect to male abdominal process length, and that gene flow may contribute to the maintenance of the high genetic variation in this trait. It is further suggested that more empirical effort should be made in quantifying and understanding spatial and temporal variation in selection in natural populations, since this may provide information on the prevalence of local adaptations in metric traits and on the mechanisms of selection.     

RESPONSE TO NATURAL ENVIRONMENTAL HETEROGENEITY: MATERNAL EFFECTS AND SELECTION ON LIFE-HISTORY CHARACTERS AND PLASTICITIES IN MIMULUS GUTTATUS

Recent studies in plant populations have found that environmental heterogeneity and phenotypic selection vary at local spatial scales. In this study, I ask if there is evolutionary change in response to environmental heterogeneity and, if so, whether the response occurs for characters or character plasticities. I used vegetative clones of Mimulus guttatus to create replicate populations of 75 genotypes. These populations were planted into the natural habitat where they differed in mean growth, flowering phenology, and life span. This phenotypic variation was used to define selective environments. There was variation in fitness (flower production) among genotypes across all planting sites and in genotype response to the selective environment. Offspring from each site were grown in the greenhouse in two water treatments. Because each population initially had the same genetic composition, variation in the progeny between selective environments reveals either evolutionary change in response to environmental heterogeneity or environmental maternal effects. Plants from experimental sites that flowered earlier, had shorter life spans and were less productive, produced offspring that had more flowers, on average, and were less plastic in vegetative allocation than offspring of longer-lived plants from high-productivity areas. However, environmental maternal effects masked phenotypic differences in flower production. Therefore, although there was evidence of genetic differentiation in both life-history characters and their plasticities in response to small-scale environmental heterogeneity, environmental maternal effects may slow evolutionary change. Response to local-scale selective regimes suggests that environmental heterogeneity and local variation in phenotypic selection may act to maintain genetic variation.     

ADAPTIVE PHENOTYPIC PLASTICITY AND ITS DIFFERENCE BETWEEN UNIVOLTINE AND MULTIVOLTINE POPULATIONS IN A BRUCHID BEETLE,KYTORHINUS SHARPIANUS

The multivoltine bruchid Kytorhinus sharpianus shows seasonal phenotypic plasticity in adult longevity, the preoviposition period, and the number of eggs laid without feeding between the diapausing and nondiapausing generations. This study compared the norms of reaction in three life-history traits between the univoltine Aomori and multivoltine Mitsuma populations. The directions of response in the norms of reaction were similar in both populations, although their response curves differed between populations. This result indicated a potential for variation in seasonal phenotypic plasticity in the univoltine population. However, the variation in the norms of reaction was small in both populations, suggesting strong selection pressure on the plasticity in the multivoltine population. These results also suggest that the univoltine Aomori population may have originated from a multivoltine population.     

QUANTITATIVE GENETICS OF BRYOZOAN PHENOTYPIC EVOLUTION. I. RATE TESTS FOR RANDOM CHANGE VERSUS SELECTION IN DIFFERENTIATION OF LIVING SPECIES

The possible roles of random genetic change and natural selection in bryozoan speciation were analyzed using quantitative genetic methods on breeding data for traits of skeletal morphology in two closely related species of the cheilostome Stylopoma. The hypothesis that morphologic differences between the species are caused entirely by mutation and genetic drift could not be rejected for reasonable rates of mutation maintained for as few as 10³ to 10⁴ generations. Divergence times this short or shorter are consistent with the abrupt appearances of many invertebrate species in the fossil record, commonly followed by millions of years of morphologic stasis. To produce these differences over 10³ generations or fewer, directional selection acting alone would require unrealistically high levels of minimum selective mortality throughout divergence. Thus, selection is unnecessary to explain the divergence of these species, except as a means of accelerating the effects of random genetic change on shorter time scales (directional selection), or decelerating them over longer ones (stabilizing selection). These results are consistent with a variety of models of phenotypic evolution involving random shifts between multiple adaptive peaks. Similar results were obtained by substituting trait heritabilities and genetic covariances reconstructed by partitioning within- and among-colony phenotypic variance in place of the values based on breeding data. Quantitative genetic analysis of speciation in fossil bryozoan lineages is thus justified.     

LOCAL ADAPTATION AND AGENTS OF SELECTION IN A MOBILE INSECT

The deme-formation hypothesis states that selection can produce adaptive genetic variation within and among phytophagous insect populations. We conducted three field experiments and tested this prediction by transferring eggs and measuring performance of a mobile leafmining insect, Stilbosis quadricustatella. In Experiment 1, we compared the rate of mine initiation of leafminers transferred to natal and novel sites. In Experiment 2, we compared mine-initiation rate of leafminers transferred to natal and novel host-plant species. In Experiment 3, we compared the mine-initiation rate, mine-completion rate, and sources of mortality of miners transferred to neighboring natal and novel Quercus geminata trees. In the first, second, and third experiments, leafminer larvae initiated significantly more mines at the natal site, on the natal plant species, and on the natal Q. geminata tree, evidence for adaptive differentiation. Furthermore, plant-mediated mortality was significantly lower among miners transferred to natal Q. geminata trees. This result supports a key assumption of the deme-formation hypothesis: insects adapt to the defensive phenotypes of individual trees. However, natural-enemy mortality was significantly higher among miners transferred to natal trees, essentially reversing the plant effect. Therefore, rates of successful mine completion were similar on natal (19%) and novel (17%) trees. This experiment suggests that host plants and natural enemies may represent opposing forces of selection. Leafminers adapted to individual trees may realize a selective advantage only when natural-enemy densities are low.     

HYBRIDIZATION,NATURAL SELECTION,AND EVOLUTION OF REPRODUCTIVE ISOLATION: A 25‐YEARS SURVEY OF AN ARTIFICIAL SYMPATRIC AREA BETWEEN TWO MOSQUITO SIBLING SPECIES OF THE Aedes mariae COMPLEX

Natural selection can act against maladaptive hybridization between co‐occurring divergent populations leading to evolution of reproductive isolation among them. A critical unanswered question about this process that provides a basis for the theory of speciation by reinforcement, is whether natural selection can cause hybridization rates to evolve to zero. Here, we investigated this issue in two sibling mosquitoes species, Aedes mariae and Aedes zammitii, that show postmating reproductive isolation (F1 males sterile) and partial premating isolation (different height of mating swarms) that could be reinforced by natural selection against hybridization. In 1986, we created an artificial sympatric area between the two species and sampled about 20,000 individuals over the following 25 years. Between 1986 and 2011, the composition of mating swarms and the hybridization rate between the two species were investigated across time in the sympatric area. Our results showed that A. mariae and A. zammitii have not completed reproductive isolation since their first contact in the artificial sympatric area. We have discussed the relative role of factors such as time of contact, gene flow, strength of natural selection, and biological mechanisms causing prezygotic isolation to explain the observed results.