Simulated climate warming causes asymmetric responses in insect life-history timing potentially disrupting a classic ecological speciation system.

Climate change may alter phenology within populations with cascading consequences for community interactions and on-going evolutionary processes. Here, we measured the response to climate warming in two sympatric, recently diverged (~170 years) populations of Rhagoletis pomonella flies specialized on different host fruits (hawthorn and apple) and their parasitoid wasp communities. We tested whether warmer temperatures affect dormancy regulation and its consequences for synchrony across trophic levels and temporal isolation between divergent populations. Under warmer temperatures, both fly populations developed earlier. However, warming significantly increased the proportion of maladaptive pre-winter development in apple, but not hawthorn, flies. Parasitoid phenology was less affected, potentially generating ecological asynchrony. Observed shifts in fly phenology under warming may decrease temporal isolation, potentially limiting on-going divergence. Our findings of complex sensitivity of life-history timing to changing temperatures predict that coming decades may see multifaceted ecological and evolutionary changes in temporal specialist communities.

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

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

Latitudinal gradient in species diversity provides high niche opportunities for a range-expanding phytophagous insect.

When species undergo poleward range expansions in response to anthropogenic change, they likely encounter less diverse communities in new locations. If low diversity communities provide weak biotic interactions, such as reduced competition or predation, range-expanding species may experience high niche opportunities. Here, we investigated if oak gall wasp communities follow a latitudinal diversity gradient (LDG) and if lower diversity communities provide weaker interactions at the poles for a range-expanding community member, Neuroterus saltatorius. We performed systematic surveys of gall wasps on a dominant oak, Quercus garryana, throughout most of its range, from northern California to Vancouver Island, British Columbia. On 540 trees at 18 sites, we identified 23 oak gall wasp morphotypes in three guilds (leaf detachable, leaf integral, and stem galls). We performed regressions between oak gall wasp diversity, latitude, and other abiotic (e.g. temperature) and habitat (e.g. oak patch size) factors to reveal if gall wasp communities followed an LDG. To uncover patterns in local interactions, we first performed partial correlations of gall wasp morphotype occurrences on trees within regions). We then performed regressions between abundances of co-occurring gall wasps on trees to reveal if interactions are putatively competitive or antagonistic. Q. garryana-gall wasp communities followed an LDG, with lower diversity at higher latitudes, particularly with a loss of detachable leaf gall morphotypes. Detachable leaf gall wasps, including the range-expanding species, co-occurred most on trees, with weak co-occurrences on trees in the northern expanded region. Abundances of N. saltatorius and detachable and integral leaf galls co-occurring on trees were negatively related, suggesting antagonistic interactions. Overall, we found that LDGs create communities with weaker associations at the poles that might facilitate ecological release in a range-expanding community member. Given the ubiquity of LDGs in nature, poleward range-expanding species are likely moving into low diversity communities. Yet, understanding if latitudinal diversity pattern provides weak biotic interactions for range-expanding species is not well explored. Our large-scale study documenting diversity in a related community of phytophagous insects that co-occur on a host plant reveals that LDGs create high niche opportunities for a range-expanding community member. Biogeographical patterns in diversity and species interactions are likely important mechanisms contributing to altered biotic interactions under range-expansions.

Cuando las especies se expanden hacia los polos en respuesta al cambio antropogénico, es probable que las encuentren comunidades menos diversas en sus nuevos lugares. Si las comunidades de baja diversidad proporcionan interacciones bióticas débiles, como competencia reducida o depredación, las especies que se expanden pueden tener 'oportunidades de alto nicho'. Aquí, investigamos si las comunidades de avispas de las agallas muestran un gradiente de diversidad latitudinal (LDG) y si las comunidades de menor diversidad proporcionan interacciones antagónicas más débiles en los polos para las comunidades que se expanden. Específicamente, investigamos estas relaciones en la comunidad de Neuroterus saltatorius. Realizamos estudios sistemáticos de las avispas de las agallas en un roble dominante, Quercus garryana en la mayor parte de su área de distribución, desde el norte de California hasta la isla de Vancouver, en la Columbia Británica. Sobre 540 árboles en 18 sitios y 6 regiones, nos identificamos 23 mofotipos de avispas de las agallas en tres gremios (hoja desmontable, hoja integral y agallas del tallo). Ejecutamos regresiones entre la diversidad de avispas de las agallas, la latitud, y otros factores abióticos (p. ej., temperatura) y de hábitat (p. ej., tamaño del parche de roble) para revelar si las comunidades de avispas de las agallas siguieron un LDG. Para descubrir tendencias en las interacciones locales, primero ejecutamos correlaciones parciales de ocurrencias de morfotipos de avispas de las agallas en árboles dentro de las regiones. Luego ejecutamos regresiones entre las coocurrencias de las abundancias deavispas de las agallas en los árboles para revelar si las interacciones son supuestamente competitivas o antagónicas. Las comunidades de avispas Q. garryana-gall, siguieron un LDG, con menor diversidad en latitudes más altas, particularmente con una pérdida de morfotipos de agallas de hoja desmontable. Las avispas de las agallas de las hojas desprendibles, incluidas a las especies que se expanden su hábitat, se encontraron en sobre todos los árboles, con coocurrencias débiles en los árboles de la región expandida del norte. Las coocurrencias de las las abundancias de N. saltatorius,las agallas foliares desmontables, e agallas integrales en los árboles se relacionaron negativamente, lo que sugiere interacciones antagónicas. En general, encontramos que los LDG crean comunidades con asociaciones más débiles en los polos que podrían facilitar la liberación ecológica en un miembro de la comunidad que se expanden. Debido de la ubicuidad de los LDG en la naturaleza, es probable que las especies que se expanden hacia los polos se estén trasladando a comunidades de baja diversidad. Sin embargo, no se explora bien la comprensión de si las tendenciasde diversidad latitudinal proporcionan interacciones bióticas débiles para las especies que se expanden. Nuestro estudio degran escala documenta la diversidad en una comunidad relacionada de insectos fitófagos que coexisten en una planta hospedante. Como resultado, revela que los LDG pueden crear oportunidades de grandes nichos para un miembro de la comunidad que se expande. Los patrones biogeográficos en la diversidad y las interacciones entre especies son probablemente mecanismos importantes que contribuyen a las interacciones bióticas alteradas bajo la expansión del hábitat.

The Build-Up of Population Genetic Divergence along the Speciation Continuum during a Recent Adaptive Radiation of Rhagoletis Flies.

New species form through the evolution of genetic barriers to gene flow between previously interbreeding populations. The understanding of how speciation proceeds is hampered by our inability to follow cases of incipient speciation through time. Comparative approaches examining different diverging taxa may offer limited inferences, unless they fulfill criteria that make the comparisons relevant. Here, we test for those criteria in a recent adaptive radiation of the Rhagoletis pomonella species group (RPSG) hypothesized to have diverged in sympatry via adaptation to different host fruits. We use a large-scale population genetic survey of 1568 flies across 33 populations to: (1) detect on-going hybridization, (2) determine whether the RPSG is derived from the same proximate ancestor, and (3) examine patterns of clustering and differentiation among sympatric populations. We find that divergence of each in-group RPSG taxon is occurring under current gene flow, that the derived members are nested within the large pool of genetic variation present in hawthorn-infesting populations of R. pomonella, and that sympatric population pairs differ markedly in their degree of genotypic clustering and differentiation across loci. We conclude that the RPSG provides a particularly robust opportunity to make direct comparisons to test hypotheses about how ecological speciation proceeds despite on-going gene flow.

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.

Rescaling Biology: Increasing Integration Across Biological Scales and Subdisciplines to Enhance Understanding and Prediction.

The organization of the living world covers a vast range of spatiotemporal scales, from molecules to the biosphere, seconds to centuries. Biologists working within specialized subdisciplines tend to focus on different ranges of scales. Therefore, developing frameworks that enable testing questions and predictions of scaling requires sufficient understanding of complex processes across biological subdisciplines and spatiotemporal scales. Frameworks that enable scaling across subdisciplines would ideally allow us to test hypotheses about the degree to which explicit integration across spatiotemporal scales is needed for predicting the outcome of biological processes. For instance, how does genomic variation within populations allow us to explain community structure? How do the dynamics of cellular metabolism translate to our understanding of whole-ecosystem metabolism? Do patterns and processes operate seamlessly across biological scales, or are there fundamental laws of biological scaling that limit our ability to make predictions from one scale to another? Similarly, can sub-organismal structures and processes be sufficiently understood in isolation of potential feedbacks from the population, community, or ecosystem levels? And can we infer the sub-organismal processes from data on the population, community, or ecosystem scale? Concerted efforts to develop more cross-disciplinary frameworks will open doors to a more fully integrated field of biology. In this paper, we discuss how we might integrate across scales, specifically by (1) identifying scales and boundaries, (2) determining analogous units and processes across scales, (3) developing frameworks to unite multiple scales, and (4) extending frameworks to new empirical systems.

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.

Genome-wide variation and transcriptional changes in diverse developmental processes underlie the rapid evolution of seasonal adaptation.

Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. We tested the hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster Our transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, our data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella We further discuss patterns that suggest hourglass-like developmental divergence early and late in diapause development and an important role for hub genes in the evolution of transcriptional divergence.

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'.

A rapidly evolved shift in life-history timing during ecological speciation is driven by the transition between developmental phases.

For insect species in temperate environments, seasonal timing is often governed by the regulation of diapause, a complex developmental programme that allows insects to weather unfavourable conditions and synchronize their life cycles with available resources. Diapause development consists of a series of distinct phases including initiation, maintenance, termination and post-diapause development. The evolution of insect seasonal timing depends in part on how these phases of diapause development and post-diapause development interact to affect variation in phenology. Here, we dissect the physiological basis of a recently evolved phenological shift in Rhagoletis pomonella (Diptera: Tephritidae), a model system for ecological divergence. A recently derived population of R. pomonella shifted from specializing on native hawthorn fruit to earlier fruiting introduced apples, resulting in a 3-4 week shift in adult emergence timing. We tracked metabolic rates of individual flies across post-winter development to test which phases of development may act either independently or in combination to contribute to this recently evolved divergence in timing. Apple and hawthorn flies differed in a number of facets of their post-winter developmental trajectories. However, divergent adaptation in adult emergence phenology in these flies was due almost entirely to the end of the pupal diapause maintenance phase, with post-diapause development having a very small effect. The relatively simple underpinnings of variation in adult emergence phenology suggest that further adaptation to seasonal change in these flies for this trait might be largely due to the timing of diapause termination unhindered by strong covariance among different components of post-diapause development.

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.

Geographic and Ecological Dimensions of Host Plant-Associated Genetic Differentiation and Speciation in the Rhagoletis cingulata (Diptera: Tephritidae) Sibling Species Group.

Ascertaining the causes of adaptive radiation is central to understanding how new species arise and come to vary with their resources. The ecological theory posits adaptive radiation via divergent natural selection associated with novel resource use; an alternative suggests character displacement following speciation in allopatry and then secondary contact of reproductively isolated but ecologically similar species. Discriminating between hypotheses, therefore, requires the establishment of a key role for ecological diversification in initiating speciation versus a secondary role in facilitating co-existence. Here, we characterize patterns of genetic variation and postzygotic reproductive isolation for tephritid fruit flies in the Rhagoletis cingulata sibling species group to assess the significance of ecology, geography, and non-adaptive processes for their divergence. Our results support the ecological theory: no evidence for intrinsic postzygotic reproductive isolation was found between two populations of allopatric species, while nuclear-encoded microsatellites implied strong ecologically based reproductive isolation among sympatric species infesting different host plants. Analysis of mitochondrial DNA suggested, however, that cytoplasmic-related reproductive isolation may also exist between two geographically isolated populations within R cingulata. Thus, ecology associated with sympatric host shifts and cytoplasmic effects possibly associated with an endosymbiont may be the key initial drivers of the radiation of the R. cingulata group.

Standing geographic variation in eclosion time and the genomics of host race formation in Rhagoletis pomonella fruit flies.

Taxa harboring high levels of standing variation may be more likely to adapt to rapid environmental shifts and experience ecological speciation. Here, we characterize geographic and host-related differentiation for 10,241 single nucleotide polymorphisms in Rhagoletis pomonella fruit flies to infer whether standing genetic variation in adult eclosion time in the ancestral hawthorn (Crataegus spp.)-infesting host race, as opposed to new mutations, contributed substantially to its recent shift to earlier fruiting apple (Malus domestica). Allele frequency differences associated with early vs. late eclosion time within each host race were significantly related to geographic genetic variation and host race differentiation across four sites, arrayed from north to south along a 430-km transect, where the host races co-occur in sympatry in the Midwest United States. Host fruiting phenology is clinal, with both apple and hawthorn trees fruiting earlier in the North and later in the South. Thus, we expected alleles associated with earlier eclosion to be at higher frequencies in northern populations. This pattern was observed in the hawthorn race across all four populations; however, allele frequency patterns in the apple race were more complex. Despite the generally earlier eclosion timing of apple flies and corresponding apple fruiting phenology, alleles on chromosomes 2 and 3 associated with earlier emergence were paradoxically at lower frequency in the apple than hawthorn host race across all four sympatric sites. However, loci on chromosome 1 did show higher frequencies of early eclosion-associated alleles in the apple than hawthorn host race at the two southern sites, potentially accounting for their earlier eclosion phenotype. Thus, although extensive clinal genetic variation in the ancestral hawthorn race exists and contributed to the host shift to apple, further study is needed to resolve details of how this standing variation was selected to generate earlier eclosing apple fly populations in the North.

Identification of a New Blend of Host Fruit Volatiles from Red Downy Hawthorn, Crataegus mollis, Attractive to Rhagoletis pomonella Flies from the Northeastern United States.

A new blend of volatiles was identified for the fruit of downy red hawthorn, Crataegus mollis, that is attractive to Rhagoletis pomonella flies infesting this host in the northeastern USA. The new blend was as attractive as the previously identified mixture but is more complex in the number of odorants (six in the old versus ten in the new) and differs significantly in the ratio of three volatiles, 3-methylbutan-1-ol, butyl hexanoate, and dihydro-ß-ionone, that are common to both blends and exerted agonist or antagonist effects on behavior in a flight tunnel assay. However, behavioral results with the old and new northern hawthorn blends, as well as modified blends with substituted ratios of 3-methylbutan-1-ol, butyl hexanoate, dihydro-ß-ionone, indicated that the 'agonist' or 'antagonist' effects of these volatiles depended on the ratio, or balance of compounds within the blend. In addition, the new blend contains a number of esters identified from the headspace of domesticated apple, Malus domestica, that are attractive to apple-origin R. pomonella, and present in the five other blends from southern hawthorns, including the southern C. mollis var. texana blend, but are not part of the previously identified blend from northern C. mollis fruit. This finding supports the hypothesis that in addition to providing specificity to the odor blends of the northern and southern hawthorn populations, the presence of the significant amounts of ester compounds in the new northern hawthorn blend might have provided a source of standing variation that could help explain the shift in host preference by C. mollis-infesting flies to introduced apple in the mid-1800's.

Genomic Differentiation during Speciation-with-Gene-Flow: Comparing Geographic and Host-Related Variation in Divergent Life History Adaptation in Rhagoletis pomonella.

A major goal of evolutionary biology is to understand how variation within populations gets partitioned into differences between reproductively isolated species. Here, we examine the degree to which diapause life history timing, a critical adaptation promoting population divergence, explains geographic and host-related genetic variation in ancestral hawthorn and recently derived apple-infesting races of Rhagoletis pomonella. Our strategy involved combining experiments on two different aspects of diapause (initial diapause intensity and adult eclosion time) with a geographic survey of genomic variation across four sites where apple and hawthorn flies co-occur from north to south in the Midwestern USA. The results demonstrated that the majority of the genome showing significant geographic and host-related variation can be accounted for by initial diapause intensity and eclosion time. Local genomic differences between sympatric apple and hawthorn flies were subsumed within broader geographic clines; allele frequency differences within the races across the Midwest were two to three-fold greater than those between the races in sympatry. As a result, sympatric apple and hawthorn populations displayed more limited genomic clustering compared to geographic populations within the races. The findings suggest that with reduced gene flow and increased selection on diapause equivalent to that seen between geographic sites, the host races may be recognized as different genotypic entities in sympatry, and perhaps species, a hypothesis requiring future genomic analysis of related sibling species to R. pomonella to test. Our findings concerning the way selection and geography interplay could be of broad significance for many cases of earlier stages of divergence-with-gene flow, including (1) where only modest increases in geographic isolation and the strength of selection may greatly impact genetic coupling and (2) the dynamics of how spatial and temporal standing variation is extracted by selection to generate differences between new and discrete units of biodiversity.

Genetic Evidence for the Introduction of Rhagoletis pomonella (Diptera: Tephritidae) into the Northwestern United States.

The apple maggot fly, Rhagoletis pomonella Walsh (Diptera: Tephritidae), is a serious quarantine pest in the apple-growing regions of central Washington and Oregon. The fly is believed to have been introduced into the Pacific Northwest via the transport of larval-infested apples near Portland, Oregon, within the last 40 yr. However, R. pomonella also attacks native black hawthorn, Crataegus douglasii Lindley (Rosales: Rosaceae), and introduced ornamental hawthorn, Crataegus monogyna Jacquin, in the region. It is, therefore, possible that R. pomonella was not introduced but has always been present on black hawthorn. If true, then the fly may have independently shifted from hawthorn onto apple in the Pacific Northwest within the last 40 yr after apples were introduced. Here, we test the introduction hypothesis through a microsatellite genetic survey of 10 R. pomonella sites in Washington and 5 in the eastern United States, as well as a comparison to patterns of genetic variation between populations of Rhagoletis cingulata Loew and Rhagoletis indifferens Curran, two sister species of cherry-infesting flies known to be native to the eastern and western United States, respectively. We report results based on genetic distance networks, patterns of allelic variation, and estimated times of population divergence that are consistent with the introduction hypothesis for R. pomonella. The results have important implications for R. pomonella management, suggesting that black hawthorn-infesting flies near commercial apple-growing regions of central Washington may harbor sufficient variation to utilize apple as an alternate host, urging careful monitoring, and possible removal of hawthorn trees near orchards.

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.

Divergence of the diapause transcriptome in apple maggot flies: winter regulation and post-winter transcriptional repression.

The duration of dormancy regulates seasonal timing in many organisms and may be modulated by day length and temperature. Though photoperiodic modulation has been well studied, temperature modulation of dormancy has received less attention. Here, we leverage genetic variation in diapause in the apple maggot fly, Rhagoletis pomonella, to test whether gene expression during winter or following spring warming regulates diapause duration. We used RNAseq to compare transcript abundance during and after simulated winter between an apple-infesting population and a hawthorn-infesting population where the apple population ends pupal diapause earlier than the hawthorn-infesting population. Marked differences in transcription between the two populations during winter suggests that the 'early' apple population is developmentally advanced compared with the 'late' hawthorn population prior to spring warming, with transcripts participating in growth and developmental processes relatively up-regulated in apple pupae during the winter cold period. Thus, regulatory differences during winter ultimately drive phenological differences that manifest themselves in the following summer. Expression and polymorphism analysis identify candidate genes in the Wnt and insulin signaling pathways that contribute to population differences in seasonality. Both populations remained in diapause and displayed a pattern of up- and then down-regulation (or vice versa) of growth-related transcripts following warming, consistent with transcriptional repression. The ability to repress growth stimulated by permissive temperatures is likely critical to avoid mismatched phenology and excessive metabolic demand. Compared with diapause studies in other insects, our results suggest some overlap in candidate genes/pathways, though the timing and direction of changes in transcription are likely species specific.

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.