The role of divergent host use and geography in the evolution of habitat isolation and sexual isolation among sister species of Belonocnema gall wasps.

Ecology and geography can play important roles in the evolution of reproductive isolation across the speciation continuum, but few studies address both at the later stages of speciation. This notable gap in knowledge arises from the fact that traditional ecological speciation studies have predominantly focused on the role of ecology in initiating the speciation process, while many studies exploring the effect of geography (e.g., reinforcement) concentrate on species pairs that lack divergent ecological characteristics. We simultaneously examine the strength of habitat isolation and sexual isolation among three closely related species of Belonocnema gall-forming wasps on two species of live oaks, Quercus virginiana and Q. geminata, that experience divergent selection from their host plants and variable rates of migration due to their geographic context. We find that the strength of both habitat isolation and sexual isolation is lowest among allopatric species pairs with the same host plant association, followed by allopatric species with different host plant associations, and highest between sympatric species with different host-plant associations. This pattern suggests that divergent selection due to different host use interacts with geography in the evolution of habitat isolation and sexual isolation during the later stages of speciation of Belonocnema wasps.

A test of trade-offs in dispersal and reproduction within and between a sister species pair of specialist insect herbivores.

Understanding the drivers of trade-offs among traits is vital for comprehending the evolution and maintenance of trait variation. Theoretical frameworks propose that evolutionary mechanisms governing trade-offs frequently exhibit a scale-dependent nature. However, empirical tests of whether trade-offs exhibited across various biological scales (i.e. individuals, populations, species, genera, etc.) remains scarce. In this study, we explore trade-off between dispersal and reproductive effort among sympatric sister species of wasps in the genus Belonocnema (Hymenoptera: Cynipini: Cynipidae) that form galls on live oaks: B. fossoria, which specializes on Quercus geminata, and B. treatae, which specializes on Q. virginiana. Specifically, our results suggest that B. fossoria has evolved reduced flight capability and smaller wings, but a larger abdomen and greater total reproductive effort than B. treatae, which has larger wings and is a stronger flier, but has a smaller abdomen and reduced total reproductive effort. These traits and the relationships among them remain unchanged when B. fossoria and B. treatae are transplanted and reared onto the alternative host plant, suggesting that trait divergence is genetically based as opposed to being a plastic response to the different rearing environments. However, when looking within species, we found no evidence of intraspecific trade-offs between wing length and reproductive traits within either B. fossoria or B. treatae. Overall, our results indicate that observed trade-offs in life history traits between the two gall former species are likely a result of independent adaptations in response to different environments as opposed to the amplified expression of within species intrinsic tradeoffs.

Context-Dependent Reproductive Isolation: Host Plant Variability Drives Fitness of Hybrid Herbivores.

AbstractThe role of divergent selection between alternative environments in promoting reproductive isolation (RI) between lineages is well recognized. However, most studies view each divergent environment as homogenous, thereby overlooking the potential role within-environment variation plays in RI between differentiating lineages. Here, we test the importance of microenvironmental variation in RI by using individual trees of two host plants, each harboring locally adapted populations of the cynipid wasp Belonocnema treatae. We compared the fitness surrogate (survival) of offspring from hybrid crosses with resident crosses across individual trees on each of two primary host plants, Quercus virginiana and Q. geminata. We found evidence of weak hybrid inviability between host-associated lineages of B. treatae despite strong genomic differentiation. However, averaging across environments masked great variation in hybrid fitness on individual trees, where hybrids performed worse than, equal to, or better than residents. Thus, considering the environmental context of hybridization is critical to improving the predictability of divergence under variable selection.

Host plant associations and geography interact to shape diversification in a specialist insect herbivore.

Disentangling the processes underlying geographic and environmental patterns of biodiversity challenges biologists as such patterns emerge from eco-evolutionary processes confounded by spatial autocorrelation among sample units. The herbivorous insect, Belonocnema treatae (Hymenoptera: Cynipidae), exhibits regional specialization on three plant species whose geographic distributions range from sympatry through allopatry across the southern United States. Using range-wide sampling spanning the geographic ranges of the three host plants and genotyping-by-sequencing of 1,217 individuals, we tested whether this insect herbivore exhibited host plant-associated genomic differentiation while controlling for spatial autocorrelation among the 58 sample sites. Population genomic structure based on 40,699 SNPs was evaluated using the hierarchical Bayesian model entropy to assign individuals to genetic clusters and estimate admixture proportions. To control for spatial autocorrelation, distance-based Moran's eigenvector mapping was used to construct regression variables summarizing spatial structure inherent among sample sites. Distance-based redundancy analysis (dbRDA) incorporating the spatial variables was then applied to partition host plant-associated differentiation (HAD) from spatial autocorrelation. By combining entropy and dbRDA to analyse SNP data, we unveiled a complex mosaic of highly structured differentiation within and among gall-former populations finding evidence that geography, HAD and spatial autocorrelation all play significant roles in explaining patterns of genomic differentiation in B. treatae. While dbRDA confirmed host association as a significant predictor of patterns of genomic variation, spatial autocorrelation among sites explained the largest proportion of variation. Our results demonstrate the value of combining dbRDA with hierarchical structural analyses to partition spatial/environmental patterns of genomic variation.

Temporal isolation between sympatric host plants cascades across multiple trophic levels of host-associated insects.

Phenological differences between host plants can promote temporal isolation among host-associated populations of insects with life cycles tightly coupled to plant phenology. Divergence in the timing of spring budbreak between two sympatric sister oak species has been shown to promote temporal isolation between host plants and their host-associated populations of a cynipid gall wasp. Here, we examined the generality of this mechanism by testing the hypothesis of cascading temporal isolation for five additional gall-formers and three natural enemy species associated with these same oak species. The timing of adult emergence from galls differed significantly between host-associated populations for all nine species and parallels the direction of the phenological differences between host plants. Differences in emergence timing can reduce gene flow between host-associated populations by diminishing mating opportunities and/or reducing the fitness of immigrants due to differences in the availability of ephemeral resources. Our study suggests that cascading temporal isolation could be a powerful 'biodiversity generator' across multiple trophic levels in tightly coupled plant-insect systems.

Diversity and distribution of Wolbachia in relation to geography, host plant affiliation and life cycle of a heterogonic gall wasp.

BACKGROUND: The maternally inherited endosymbiont Wolbachia is widespread in arthropods and nematodes and can play an important role in the ecology and evolution of its host through reproductive manipulation. Here, we survey Wolbachia in Belonocnema treatae, a widely distributed North American cynipid gall forming wasp that exhibits regional host specialization on three species of oaks and alternation of sexually and asexually reproducing generations. We investigated whether patterns of Wolbachia infection and diversity in B. treatae are associated with the insect's geographic distribution, host plant association, life cycle, and mitochondrial evolutionary history. RESULTS: Screening of 463 individuals from 23 populations including sexual and asexual generations from all three host plants across the southern U.S. showed an average infection rate of 56% with three common Wolbachia strains: wTre1-3 and an additional rare variant wTre4. Phylogenetic analysis based on wsp showed that these strains are unrelated and likely independently inherited. We found no difference in Wolbachia infection frequency among host plant associated populations or between the asexual and sexual generations, or between males and females of the sexual generation. Partially incomplete Wolbachia transmission rates might explain the occurrence of uninfected individuals. A parallel analysis of the mitochondrial cytochrome oxidase I gene in B. treatae showed high mtDNA haplotype diversity in both infected and uninfected populations suggesting an ancestral infection by Wolbachia as well as a clear split between eastern and western B. treatae mtDNA clades with a sequence divergence of > 6%. The strain wTre1 was present almost exclusively in the western clade while wTre2 and wTre3 occur almost exclusively in eastern populations. In contrast, the same strains co-occur as double-infections in Georgia and triple-infections in two populations in central Florida. CONCLUSIONS: The diversity of Wolbachia across geographically and genetically distinct populations of B. treatae and the co-occurrence of the same strains within three populations highlights the complex infection dynamics in this system. Moreover, the association of distinct Wolbachia strains with mitochondrial haplotypes of its host in populations infected by different Wolbachia strains suggests a potential role of the endosymbiont in reproductive isolation in B. treatae.

Independent life history evolution between generations of bivoltine species: a case study of cyclical parthenogenesis.

Successive generations of bi- and multivoltine species encounter differing biotic and abiotic environments intra-annually. The question of whether selection can independently adjust the relationship between body size and components of reproductive effort within successive generations in response to generation-specific environmental variation is applicable to a diversity of taxa. Herein, we develop a conceptual framework that illustrates increasingly independent life history adjustments between successive generations of taxa exhibiting complex life cycles. We apply this framework to the reproductive biology of the gall-forming insect, Belonocnema treatae (Hymenoptera: Cynipidae). This bivoltine species expresses cyclical parthenogenesis in which alternating sexual and asexual generations develop in different seasons and different environments. We tested the hypotheses that ecological divergence between the alternate generations is accompanied by generational differences in body size, egg size, and egg number and by changes in the relationships between body size and these components of reproductive effort. Increased potential reproductive effort of sexual generation B. treatae is attained by increased body size and egg number (with no trade-off between egg number and egg size) and by a significant increase in the slope of the relationship between body size and potential fecundity. These generation-specific relationships, interpreted in the context of the model framework, suggest that within each generation selection has independently molded the relationships relating body size to potential fecundity and potential reproductive effort in B. treatae. The conceptual framework is broadly applicable to comparisons involving the alternating generations of bi- and multivoltine species.

Divergent host-plant use promotes reproductive isolation among cynipid gall wasp populations.

Ecological speciation occurs when reproductive isolation evolves as a consequence of divergent natural selection among environments. A direct prediction of this process is that ecologically divergent pairs of populations will exhibit greater reproductive isolation than ecologically similar pairs of populations. By comparing allopatric populations of the cynipid gall wasp Belonocnema treatae infesting Quercus virginiana and Quercus geminata, we tested the role that divergent host use plays in generating ecological divergence and sexual isolation. We found differences in body size and gall structure associated with divergent host use, but no difference in neutral genetic divergence between populations on the same or different host plant. We observed significant assortative mating between populations from alternative host plants but not between allopatric populations on the same host plant. Thus, we provide evidence that divergent host use promotes speciation among gall wasp populations.

Speciation in Nearctic oak gall wasps is frequently correlated with changes in host plant, host organ, or both.

Quantifying the frequency of shifts to new host plants within diverse clades of specialist herbivorous insects is critically important to understand whether and how host shifts contribute to the origin of species. Oak gall wasps (Hymenoptera: Cynipidae: Cynipini) comprise a tribe of ∼1000 species of phytophagous insects that induce gall formation on various organs of trees in the family Fagacae-primarily the oaks (genus Quercus; ∼435 sp.). The association of oak gall wasps with oaks is ancient (∼50 my), and most oak species are galled by one or more gall wasp species. Despite the diversity of both gall wasp species and their plant associations, previous phylogenetic work has not identified the strong signal of host plant shifting among oak gall wasps that has been found in other phytophagous insect systems. However, most emphasis has been on the Western Palearctic and not the Nearctic where both oaks and oak gall wasps are considerably more species rich. We collected 86 species of Nearctic oak gall wasps from most of the major clades of Nearctic oaks and sequenced >1000 Ultraconserved Elements (UCEs) and flanking sequences to infer wasp phylogenies. We assessed the relationships of Nearctic gall wasps to one another and, by leveraging previously published UCE data, to the Palearctic fauna. We then used phylogenies to infer historical patterns of shifts among host tree species and tree organs. Our results indicate that oak gall wasps have moved between the Palearctic and Nearctic at least four times, that some Palearctic wasp clades have their proximate origin in the Nearctic, and that gall wasps have shifted within and between oak tree sections, subsections, and organs considerably more often than previous data have suggested. Given that host shifts have been demonstrated to drive reproductive isolation between host-associated populations in other phytophagous insects, our analyses of Nearctic gall wasps suggest that host shifts are key drivers of speciation in this clade, especially in hotspots of oak diversity. Although formal assessment of this hypothesis requires further study, two putatively oligophagous gall wasp species in our dataset show signals of host-associated genetic differentiation unconfounded by geographic distance, suggestive of barriers to gene flow associated with the use of alternative host plants.

The Arthropod Associates of 155 North American Cynipid Oak Galls.

The identities of most arthropod associates of cynipid-induced oak galls in the western Palearctic are generally known. However, a comprehensive accounting of associates has been performed for only a small number of the galls induced by the estimated 700 species of cynipid gall wasps in the Nearctic. This gap in knowledge stymies many potential studies of diversity, coevolution, and community ecology, for which oak gall systems are otherwise ideal models. We report rearing records of insects and other arthropods from more than 527,306 individual galls representing 201 different oak gall types collected from 32 oak tree species in North America. Of the 201 gall types collected, 155 produced one or more arthropods. A total of 151,075 arthropods were found in association with these 155 gall types, and of these 61,044 (40.4%) were gall wasps while 90,031 (59.6%) were other arthropods. We identified all arthropods to superfamily, family, or, where possible, to genus. We provide raw numbers and summaries of collections, alongside notes on natural history, ecology, and previously published associations for each taxon. For eight common gall-associated genera (Synergus, Ceroptres, Euceroptres, Ormyrus, Torymus, Eurytoma, Sycophila, and Euderus), we also connect rearing records to gall wasp phylogeny, geography, and ecology -including host tree and gall location (host organ), and their co-occurrence with other insect genera. Though the diversity of gall wasps and the large size of these communities is such that many Nearctic oak gall-associated insects still remain undescribed, this large collection and identification effort should facilitate the testing of new and varied ecological and evolutionary hypotheses in Nearctic oak galls.

Asymmetric, but opposing reductions in immigrant viability and fecundity promote reproductive isolation among host-associated populations of an insect herbivore.

Immigrant inviability can contribute to reproductive isolation (RI) during ecological speciation by reducing the survival of immigrants in non-native environments. However, studies that assess the fitness consequence of immigrants moving from native to non-native environments typically fail to explore the potential role of concomitant reductions in immigrant fecundity despite recent evidence suggesting its prominent role during local adaptation. Here, we evaluate the directionality and magnitude of both immigrant viability and fecundity to RI in a host-specific gall-forming wasp, Belonocnema treatae. Using reciprocal transplant experiments replicated across sites, we measure immigrant viability and fecundity by comparing differences in the incidence of gall formation (viability) and predicted the number of eggs per female (fecundity) between residents and immigrants in each of two host-plant environments. Reduced immigrant viability was found in one host environment while reduced immigrant fecundity was found in the other. Such habitat-dependent barriers resulted in asymmetric RI between populations. By surveying recent literature on local adaptation, we find that asymmetry in immigrant viability and fecundity are widespread across disparate taxa, which highlights the need to combine estimates of both common and overlooked barriers in cases of potential bi-directional gene flow to create a more comprehensive view of the evolution of RI.

Developmental plasticity and reduced susceptibility to natural enemies following host plant defoliation in a specialized herbivore.

Host-specific phytophagous insects that are short lived and reliant on ephemeral plant tissues provide an excellent system in which to investigate the consequences of disruption in the timing of resource availability on consumer populations and their subsequent interactions with higher tropic levels. The specialist herbivore, Belonocnema treatae (Hymenoptera: Cynipidae) induces galls on only newly flushed leaves of live oak, Quercus fusiformis. In central Texas (USA) episodic defoliation of the host creates variation in the timing of resource availability and results in heterogeneous populations of B. treatae that initiate development at different times. We manipulated the timing of leaf flush in live oak via artificial defoliation to test the hypothesis that a 6- to 8-week delay in the availability of resources alters the timing of this gall former's life cycle events, performance and survivorship on its host, and susceptibility to natural enemies. B. treatae exhibits plasticity in development time, as the interval from egg to emergence was significantly reduced when gallers oviposited into the delayed leaf flush. As a consequence, the phenologies of gall maturation and adult emergence remain synchronized in spite of variation in the timing of resource availability. Per capita gall production and gall-former performance are not significantly affected by the timing of resource availability. The timing of resource availability and natural enemies interact, however, to produce strong effects on survivorship: when exposed to natural enemies, B. treatae developing in galls initiated by delayed oviposition exhibited an order-of-magnitude increase in survivorship. Developmental plasticity allows this gall former to circumvent disruptions in resource availability, maintain synchrony of life cycle events, and results in reduced vulnerability to natural enemies following defoliation of the host plant.

Cascading reproductive isolation: Plant phenology drives temporal isolation among populations of a host-specific herbivore.

All organisms exist within a complex network of interacting species, thus evolutionary change may have reciprocal effects on multiple taxa. Here, we demonstrate "cascading reproductive isolation," whereby ecological differences that reduce gene flow between populations at one trophic level affect reproductive isolation (RI) among interacting species at the next trophic level. Using a combination of field, laboratory and common-garden studies and long-term herbaria records, we estimate and evaluate the relative contribution of temporal RI to overall prezygotic RI between populations of Belonocnema treatae, a specialist gall-forming wasp adapted to sister species of live oak (Quercus virginiana and Q. geminata). We link strong temporal RI between host-associated insect populations to differences between host plant budbreak phenology. Budbreak initiates flowering and the production of new leaves, which are an ephemeral resource critical to insect reproduction. As flowering time is implicated in RI between plant species, budbreak acts as a "multitrophic multi-effect trait," whereby differences in budbreak phenology contribute to RI in plants and insects. These sister oak species share a diverse community of host-specific gall-formers and insect natural enemies similarly dependent on ephemeral plant tissues. Thus, our results set the stage for testing for parallelism in a role of plant phenology in driving temporal cascading RI across multiple species and trophic levels.

Cynipid gall wasps.

Egan et al. introduce the reader to gall wasps, including a description of their life cycle and complex ecological interactions with host plants and natural enemies.

Host plant quality and local adaptation determine the distribution of a gall-forming herbivore.

Herein we report results of transplant experiments that link variation in host plant quality to herbivore fitness at the local scale (among adjacent plants) with the process of local (demic) adaptation at the landscape scale to explain the observed distribution of the specialist gall former Belonocnema treatae (Hymenoptera: Cynipidae) within populations of its host plant, Quercus fusiformis. Field surveys show that leaf gall densities vary by orders of magnitude among adjacent trees and that high-gall-density trees are both rare (< 5%) and patchily distributed. B. treatae from each of five high-gall-density trees were reared on (1) the four nearest low-gall-density trees, (2) the four alternative high-gall-density trees, and (3) their natal trees (control). Each treatment (source X rearing site) was replicated three times. Nine components of performance that sequentially contribute to fitness were evaluated with over 21000 galls censused across the 25 experimental trees. When reared on their natal trees and compared with low-gall-density neighbors, transplanted gall formers had higher gall initiation success (P < 0.05), produced more (P < 0.001) and larger galls (P < 0.001), and produced a higher proportion of galls that exceeded the threshold size for natural enemy avoidance (P < 0.05). Comparison of gall-former performance on natal vs. alternative high-gall-density trees demonstrated significant (P < 0.001) differences in six performance measures with five differing in the direction predicted by the hypothesis of local adaptation. Overall, these linked experiments document direct and indirect effects of host plant variation on gall-former performance and demonstrate convincingly that (1) high-gall-density trees equate to high-quality trees that are surrounded by trees of relatively lower quality to the herbivore and (2) gall-former populations have become locally adapted to individual trees.

Chromosomes of Belonocnematreatae Mayr, 1881 (Hymenoptera, Cynipidae).

Chromosomes of the asexual and sexual generation of the gall wasp Belonocnematreatae Mayr, 1881 (Cynipidae) were analyzed. Females of both generations have 2n = 20, whereas males of the sexual generation have n = 10. Cyclical deuterotoky is therefore confirmed in this species. All chromosomes are acrocentric and form a continuous gradation in size. This karyotype structure is probably ancestral for many gall wasps and perhaps for the family Cynipidae in general. Chromosome no. 7 carries a characteristic achromatic gap that appears to represent a nucleolus organizing region.

Host plant-mediated variation in overwintering site quality: implications for the size and composition of populations of Acanthoscelides alboscutellatus (Coleoptera: Bruchidae).

This study provides an example of how variation in the quality of overwintering sites provided by the host plant of an insect seed predator can influence both the probability of overwintering survival and the size and composition of postwintering populations. Thus, the concept of host plant quality is extended to include variation in the suitability of the overwintering site of temperate region insects that overwinter within, or in habitats created by, their host plant. Adult Acanthoscelides alboscutellatus (Horn) (Coleoptera: Bruchidae) overwinter inside the fruit of Ludwigia alternifolia (L.) (Onagraceae). In early winter, however, fruits begin to dehisce, i.e., one or more of the fruit's four sides and/or top are shed. Variation in the onset and extent of dehiscence creates a range of overwintering habitats that vary in exposure to ambient conditions. In this study the frequency of possible overwintering sites in natural populations of L. alternifolia was determined by monitoring the phenology of fruit dehiscence from October through May in two populations for four years and for a third population for three years. Winter survivorship of adult A. alboscutellatus was assessed experimentally in eight environments representative of the conditions created by variation in dehiscence. These environments were produced by crossing four levels of exposure (degree of dehiscence) with two locations of the overwintering site, i.e., above or on the ground surface. The onset, phenology, and overall frequency of fruit dehiscence varied markedly among populations and years. Exposure, location, and their interaction had strong effects on survival and accounted for 80% of the observed variation in winter survival. Survivorship was higher on than above the ground, and in both locations decreased with increasing exposure. Thus, variation in fruit dehiscence among L. alternifolia populations will influence the size of postwintering A. alboscutellatus populations by dictating the quality of overwintering sites. Adult beetles that over-winter inside indehiscent fruit experience selection for small body size, associated with high mortality, when they attempt to exit the fruit at eclosion. As a consequence, the frequency of fruit dehiscence at eclosion coupled with the relative survival rates of adults within indehiscent fruit will determine the body size composition of postwintering populations and hence the response to selection for small body size in this species.

Body size selection inAcanthoscelides alboscutellatus (Coleoptera: Bruchidae) : I. Entrapment within the fruit ofLudwigia alternifolia (Onagraceae).

Direct observations and analyses of selection occurring in natural populations are rare. The biology of the bruchid beetle,Acanthoscelides alboscutellatus, on its host plant,Ludwigia alternifolia, provides an anusual opportunity to study the process of selection on the morphology of an organism under field conditions.A. alboscutellatus larvae mature within the variably dehiscent fruit ofL. alternifolia. At eclosion, adults are confined within indehiscent fruit but are not confined within dehiscent fruit. Beetles can escape from indehiscent fruit only by forcing their bodies through the fruit's apical pore (a circular opening in the top of the fruit). Thus, during the eclosion stage of this beetle's life cycle the relationship between body size and differential fitness appears to be clearly defined.We examined entrapment ofA. alboscutellatus within indehiscentL. alternifolia fruit in a natural population. Only 8.8% of the beetles that attempted to escape were successful. Smaller beetles were trapped within a narrower range of pore diameters than were larger beetles; and trapped beetles had only limited abilities to enlarge fruit pore diameter. These data suggest (1) that escape from indehiscent fruit is regulated by the relationship between adult body diameter and fruit pore diameter and (2) that adult beetles may experience strong selection for small body diameter (size) within idehiscent fruit.

Limited dispersal and its effect on population structure in the milkweed beetle Tetraopes tetraophthalmus.

The movement patterns of adult milkweed beetles, Tetraopes tetraphthalmus, were monitored via a mark-recapture technique. Movement or dispersal patterns were studied in two natural populations, one in which the host plant, Asclepias syriaca, was nearly continuously distributed over a 250×90 m area and another where Asclepias was distributed in 17 small discrete patches. In both populations dispersal distances resulting from the flight patterns of the adult beetles were quite short, averaging less than 40 m from the point of first encounter 10 days after marking. Males were shown to be more vagile than females. The distribution of dispersal distances collected from one of the populations was fit to three statistical distributions cited in the literature as expected from dispersal by many small-scale movements or observed in other species. It was found that an equation describing an exponential decay gave the best statistical fit to the data collected here for milkweed beetles. The data is discussed in the context of the effects of the limited dispersal power of the beetles and the distribution of suitable habitat on the population structure of Tetraopes.

Parallel patterns of morphological and behavioral variation among host-associated populations of two gall wasp species.

A powerful approach to address the general factors contributing to ecological speciation is to compare distantly related taxa that inhabit the same selective environments. In this design, similarities among taxa can elucidate general mechanisms of the process whereas differences may uncover specific factors important to the process for individual taxa. Herein, we present evidence of parallel patterns of morphological and behavioral variation among host-associated populations of two species of cynipid gall wasps, Belonocnema treatae and Disholcaspis quercusvirens, that each exhibit a life cycle intimately tied to the same two host plant environments, Quercus geminata and Q. virginiana. Across both gall-former species we find consistent differences in body size and gall morphology associated with host plant use, as well as strong differences in host plant preference, a measure of habitat isolation among populations. These consistent differences among taxa highlight the important role of host plant use in promoting reproductive isolation and morphological variation among herbivorous insect populations-a prerequisite for ecological speciation.