Divergent diapause life history timing drives both allochronic speciation and reticulate hybridization in an adaptive radiation of Rhagoletis flies.

Divergent adaptation to new ecological opportunities can be an important factor initiating speciation. However, as niches are filled during adaptive radiations, trait divergence driving reproductive isolation between sister taxa may also result in trait convergence with more distantly related taxa, increasing the potential for reticulated gene flow across the radiation. Here, we demonstrate such a scenario in a recent adaptive radiation of Rhagoletis fruit flies, specialized on different host plants. Throughout this radiation, shifts to novel hosts are associated with changes in diapause life history timing, which act as "magic traits" generating allochronic reproductive isolation and facilitating speciation-with-gene-flow. Evidence from laboratory rearing experiments measuring adult emergence timing and genome-wide DNA-sequencing surveys supported allochronic speciation between summer-fruiting Vaccinium spp.-infesting Rhagoletis mendax and its hypothesized and undescribed sister taxon infesting autumn-fruiting sparkleberries. The sparkleberry fly and R. mendax were shown to be genetically discrete sister taxa, exhibiting no detectable gene flow and allochronically isolated by a 2-month average difference in emergence time corresponding to host availability. At sympatric sites across the southern USA, the later fruiting phenology of sparkleberries overlaps with that of flowering dogwood, the host of another more distantly related and undescribed Rhagoletis taxon. Laboratory emergence data confirmed broadly overlapping life history timing and genomic evidence supported on-going gene flow between sparkleberry and flowering dogwood flies. Thus, divergent phenological adaptation can drive the initiation of reproductive isolation, while also enhancing genetic exchange across broader adaptive radiations, potentially serving as a source of novel genotypic variation and accentuating further diversification.

Genomically correlated trait combinations and antagonistic selection contributing to counterintuitive genetic patterns of adaptive diapause divergence in Rhagoletis flies.

Adaptation to novel environments can result in unanticipated genomic responses to selection. Here, we illustrate how multifarious, correlational selection helps explain a counterintuitive pattern of genetic divergence between the recently derived apple- and ancestral hawthorn-infesting host races of Rhagoletis pomonella (Diptera: Tephritidae). The apple host race terminates diapause and emerges as adults earlier in the season than the hawthorn host race, to coincide with the earlier fruiting phenology of their apple hosts. However, alleles at many loci associated with later emergence paradoxically occur at higher frequencies in sympatric populations of the apple compared to the hawthorn race. We present genomic evidence that historical selection over geographically varying environmental gradients across North America generated genetic correlations between two life history traits, diapause intensity and diapause termination, in the hawthorn host race. Moreover, the loci associated with these life history traits are concentrated in genomic regions in high linkage disequilibrium (LD). These genetic correlations are antagonistic to contemporary selection on local apple host race populations that favours increased initial diapause depth and earlier, not later, diapause termination. Thus, the paradox of apple flies appears due, in part, to pleiotropy or linkage of alleles associated with later adult emergence and increased initial diapause intensity, the latter trait strongly selected for by the earlier phenology of apples. Our results demonstrate how understanding of multivariate trait combinations and the correlative nature of selective forces acting on them can improve predictions concerning adaptive evolution and help explain seemingly counterintuitive patterns of genetic diversity in nature.

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.

Human-mediated disturbance in multitrophic interactions results in outbreak levels of North America's most venomous caterpillar.

Anthropogenic environmental change is predicted to disrupt multitrophic interactions, which may have drastic consequences for population-level processes. Here, we investigate how a large-scale human-mediated disturbance affects the abundance of North America's most venomous caterpillar species, Megalopyge opercularis. Specifically, we used a natural experiment where netting was deployed to cover the entire canopies of a subset of mature southern live oak trees (Quercus virginiana) to exclude urban pest birds (grackles and pigeons), throughout an 8.1 km2 area encompassing a medical centre in Houston, Texas. We used this experimental exclusion to test the following hypothesis: release from avian predators increases caterpillar abundance to outbreak levels, which increases the risk to human health. Results from a multi-year survey show that caterpillar abundance increased, on average, more than 7300% on netted versus non-netted trees. Thus, increases in caterpillar abundance due to anthropogenic enemy release increase human exposure to this venomous pest, and should be considered a health threat in the area. This study emphasizes the unforeseen consequences of ecological disturbance for species interactions and highlights the importance of considering ecology in urban planning.

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.

Sequential divergence and the multiplicative origin of community diversity.

Phenotypic and genetic variation in one species can influence the composition of interacting organisms within communities and across ecosystems. As a result, the divergence of one species may not be an isolated process, as the origin of one taxon could create new niche opportunities for other species to exploit, leading to the genesis of many new taxa in a process termed "sequential divergence." Here, we test for such a multiplicative effect of sequential divergence in a community of host-specific parasitoid wasps, Diachasma alloeum, Utetes canaliculatus, and Diachasmimorpha mellea (Hymenoptera: Braconidae), that attack Rhagoletis pomonella fruit flies (Diptera: Tephritidae). Flies in the R. pomonella species complex radiated by sympatrically shifting and ecologically adapting to new host plants, the most recent example being the apple-infesting host race of R. pomonella formed via a host plant shift from hawthorn-infesting flies within the last 160 y. Using population genetics, field-based behavioral observations, host fruit odor discrimination assays, and analyses of life history timing, we show that the same host-related ecological selection pressures that differentially adapt and reproductively isolate Rhagoletis to their respective host plants (host-associated differences in the timing of adult eclosion, host fruit odor preference and avoidance behaviors, and mating site fidelity) cascade through the ecosystem and induce host-associated genetic divergence for each of the three members of the parasitoid community. Thus, divergent selection at lower trophic levels can potentially multiplicatively and rapidly amplify biodiversity at higher levels on an ecological time scale, which may sequentially contribute to the rich diversity of life.

A test of genomic modularity among life-history adaptations promoting speciation with gene flow.

Speciation with gene flow may require adaptive divergence of multiple traits to generate strong ecologically based reproductive isolation. Extensive negative pleiotropy or physical linkage of genes in the wrong phase affecting these diverging traits may therefore hinder speciation, while genetic independence or "modularity" among phenotypic traits may reduce constraints and facilitate divergence. Here, we test whether the genetics underlying two components of diapause life history, initial diapause intensity and diapause termination timing, constrain differentiation between sympatric hawthorn and apple-infesting host races of the fly Rhagoletis pomonella through analysis of 10,256 SNPs measured via genotyping-by-sequencing (GBS). Loci genetically associated with diapause termination timing were mainly observed for SNPs mapping to chromosomes 1-3 in the genome, most notably for SNPs displaying higher levels of linkage disequilibrium (LD), likely due to inversions. In contrast, selection on initial diapause intensity affected loci on all five major chromosomes of the genome, specifically those showing low levels of LD. This lack of overlap in genetically associated loci suggests that the two diapause phenotypes are largely modular. On chromosome 2, however, intermediate level LD loci and a subgroup of high LD loci displayed significant negative relationships between initial diapause intensity and diapause termination time. These gene regions on chromosome 2 therefore affected both traits, while most regions were largely independent. Moreover, loci associated with both measured traits also tended to exhibit highly divergent allele frequencies between the host races. Thus, the presence of nonoverlapping genetic modules likely facilitates simultaneous, adaptive divergence for the measured life-history components.

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.

Identification of Host Fruit Volatiles from Snowberry (Symphoricarpos albus), Attractive to Rhagoletis zephyria Flies from the Western United States.

A mixture of behaviorally active volatiles was identified from the fruit of snowberry, Symphoricarpos albus laevigatus, for Rhagoletis zephyria flies reared from snowberry fruit. A nine-component blend containing 3-methylbutan-1-ol (3%), dimethyl trisulfide (1%), 1-octen-3-ol (40%), myrcene (8%), nonanal (9%), linalool (13%), (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT, 6%), decanal (15%), and ß-caryophyllene (5%) was identified that gave consistent electroantennogram activity and was behaviorally active in flight tunnel tests. In other flight tunnel assays, snowberry flies from two sites in Washington state, USA, displayed significantly greater levels of upwind oriented flight to sources with the snowberry volatile blend compared with previously identified volatile blends from domestic apple (Malus domestica) and downy hawthorn (Crataegus mollis) fruit from the eastern USA, and domestic apple, black hawthorn (C. douglasii) and ornamental hawthorn (C. monogyna) from Washington state. Selected subtraction assays showed that whereas removal of DMNT or 1-octen-3-ol significantly reduced the level of upwind flight, removal of myrcene and ß-caryophyllene, or dimethyl trisulfide alone did not significantly affect the proportion of upwind flights. Our findings add to previous studies showing that populations of Rhagoletis flies infesting different host fruit are attracted to unique mixtures of volatile compounds specific to their respective host plants. Taken together, the results support the hypothesis that differences among flies in their behavioral responses to host fruit odors represent key adaptations involved in sympatric host plant shifts, contributing to host specific mating and generating prezygotic reproductive isolation among members of the R. pomonella sibling species complex.

Experimental evidence of genome-wide impact of ecological selection during early stages of speciation-with-gene-flow.

Theory predicts that speciation-with-gene-flow is more likely when the consequences of selection for population divergence transitions from mainly direct effects of selection acting on individual genes to a collective property of all selected genes in the genome. Thus, understanding the direct impacts of ecologically based selection, as well as the indirect effects due to correlations among loci, is critical to understanding speciation. Here, we measure the genome-wide impacts of host-associated selection between hawthorn and apple host races of Rhagoletis pomonella (Diptera: Tephritidae), a model for contemporary speciation-with-gene-flow. Allele frequency shifts of 32 455 SNPs induced in a selection experiment based on host phenology were genome wide and highly concordant with genetic divergence between co-occurring apple and hawthorn flies in nature. This striking genome-wide similarity between experimental and natural populations of R. pomonella underscores the importance of ecological selection at early stages of divergence and calls for further integration of studies of eco-evolutionary dynamics and genome divergence.

Ecological adaptation and reproductive isolation in sympatry: genetic and phenotypic evidence for native host races of Rhagoletis pomonella.

Ecological speciation with gene flow may be an important mode of diversification for phytophagous insects. The recent shift of Rhagoletis pomonella from its native host downy hawthorn (Crataegus mollis) to introduced apple (Malus domestica) in the northeastern United States is a classic example of sympatric host race formation. Here, we test whether R. pomonella has similarly formed host races on four native Crataegus species in the southern United States: western mayhaw (C. opaca), blueberry hawthorn (C. brachyacantha), southern red hawthorn (C. mollis var. texana) and green hawthorn (C. viridis). These four southern hosts differ from each other in their fruiting phenology and in the volatile compounds emitted from the surface of their fruits. These two traits form the basis of ecological reproductive isolation between downy hawthorn and apple flies in the north. We report evidence from microsatellite population surveys and eclosion studies supporting the existence of genetically differentiated and partially reproductively isolated host races of southern hawthorn flies. The results provide an example of host shifting and ecological divergence involving native plants and imply that speciation with gene flow may be commonly initiated in Rhagoletis when ecological opportunity presents itself.

Cold tolerance and diapause within and across trophic levels: Endoparasitic wasps and their fly host have similar phenotypes.

Low temperatures associated with winter can limit the survival of organisms, especially ectotherms whose body temperature is similar to their environment. However, there is a gap in understanding how overwintering may vary among groups of species that interact closely, such as multiple parasitoid species that attack the same host insect. Here, we investigate cold tolerance and diapause phenotypes in three endoparasitoid wasps of the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae): Utetes canaliculatus, Diachasma alloeum, and Diachasmimorpha mellea (Hymenoptera: Braconidae). Using a combination of respirometry and eclosion tracking, we found that all three wasp species exhibited the same three diapause duration phenotypes as the fly host. Weak (short duration) diapause was rare, with <5 % of all three wasp species prematurely terminating diapause at 21 °C. Most D.mellea (93 %) entered a more intense (longer duration) diapause that did not terminate within 100 d at this warm temperature. The majority of U.canaliculatus (92 %) and D. alloeum (72 %) averted diapause (non-diapause) at 21 °C. There was limited interspecific variation in acute cold tolerance among the three wasp species: wasps and flies had similarly high survival (>87 %) following exposure to extreme low temperatures (-20 °C) as long as their body fluids did not freeze. The three wasp species also displayed little interspecific variation in survival following prolonged exposure to mild chilling of 8 or more weeks at 4 °C. Our study thus documents a remarkable conservation of cold tolerance and diapause phenotypes within and across trophic levels.

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.

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.

Rapid and repeatable host plant shifts drive reproductive isolation following a recent human-mediated introduction of the apple maggot fly, Rhagoletis pomonella.

Ecological speciation via host-shifting is often invoked as a mechanism for insect diversification, but the relative importance of this process is poorly understood. The shift of Rhagoletis pomonella in the 1850s from the native downy hawthorn, Crataegus mollis, to introduced apple, Malus pumila, is a classic example of sympatric host race formation, a hypothesized early stage of ecological speciation. The accidental human-mediated introduction of R. pomonella into the Pacific Northwest (PNW) in the late 1970s allows us to investigate how novel ecological opportunities may trigger divergent adaptation and host race formation on a rapid timescale. Since the introduction, the fly has spread in the PNW, where in addition to apple, it now infests native black hawthorn, Crataegus douglasii, and introduced ornamental hawthorn, Crataegus monogyna. We use this "natural experiment" to test for genetic differentiation among apple, black, and ornamental hawthorn flies co-occurring at three sympatric sites. We report evidence that populations of all three host-associations are genetically differentiated at the local level, indicating that partial reproductive isolation has evolved in this novel habitat. Our results suggest that conditions suitable for initiating host-associated divergence may be common in nature, allowing for the rapid evolution of new host races when ecological opportunity arises.

Can the genomics of ecological speciation be predicted across the divergence continuum from host races to species? A case study in Rhagoletis.

Studies assessing the predictability of evolution typically focus on short-term adaptation within populations or the repeatability of change among lineages. A missing consideration in speciation research is to determine whether natural selection predictably transforms standing genetic variation within populations into differences between species. Here, we test whether and how host-related selection on diapause timing associates with genome-wide differentiation during ecological speciation by comparing ancestral hawthorn and newly formed apple-infesting host races of Rhagoletis pomonella to their sibling species Rhagoletis mendax that attacks blueberries. The associations of 57 857 single nucleotide polymorphisms in a diapause genome-wide-association study (GWAS) on the hawthorn race strongly predicted the direction and magnitude of genomic divergence among the three fly populations at a field site in Fennville, MI, USA. The apple race and R. mendax show parallel changes in the frequencies of putative inversions on three chromosomes associated with the earlier fruiting times of apples and blueberries compared to hawthorns. A diapause GWAS on R. mendax revealed compensatory changes throughout the genome accounting for the earlier eclosion of blueberry, but not apple flies. Thus, a degree of predictability, although not complete, exists in the genomics of diapause across the ecological speciation continuum in Rhagoletis. The generality of this result is placed in the context of other similar systems. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Evidence for spatial clines and mixed geographic modes of speciation for North American cherry-infesting Rhagoletis (Diptera: Tephritidae) flies.

An important criterion for understanding speciation is the geographic context of population divergence. Three major modes of allopatric, parapatric, and sympatric speciation define the extent of spatial overlap and gene flow between diverging populations. However, mixed modes of speciation are also possible, whereby populations experience periods of allopatry, parapatry, and/or sympatry at different times as they diverge. Here, we report clinal patterns of variation for 21 nuclear-encoded microsatellites and a wing spot phenotype for cherry-infesting Rhagoletis (Diptera: Tephritidae) across North America consistent with these flies having initially diverged in parapatry followed by a period of allopatric differentiation in the early Holocene. However, mitochondrial DNA (mtDNA) displays a different pattern; cherry flies at the ends of the clines in the eastern USA and Pacific Northwest share identical haplotypes, while centrally located populations in the southwestern USA and Mexico possess a different haplotype. We hypothesize that the mitochondrial difference could be due to lineage sorting but more likely reflects a selective sweep of a favorable mtDNA variant or the spread of an endosymbiont. The estimated divergence time for mtDNA suggests possible past allopatry, secondary contact, and subsequent isolation between USA and Mexican fly populations initiated before the Wisconsin glaciation. Thus, the current genetics of cherry flies may involve different mixed modes of divergence occurring in different portions of the fly's range. We discuss the need for additional DNA sequencing and quantification of prezygotic and postzygotic reproductive isolation to verify the multiple mixed-mode hypothesis for cherry flies and draw parallels from other systems to assess the generality that speciation may commonly involve complex biogeographies of varying combinations of allopatric, parapatric, and sympatric divergence.