Diversification linked to larval host plant in the butterfly Eumedonia eumedon.

It is widely accepted that the relationship between phytophagous insects and their host plants influences insect diversification. However, studies addressed at documenting host-associated genetic differentiation (HAD) and the mechanisms that drive reproductive isolation in host-associated lineages (or host races) are still scarce relative to insect diversity. To uncover further evidence on the HAD processes in Lepidoptera, we investigated the genetic structure of the geranium argus butterfly (Eumedonia eumedon) and tested for isolation by ecology (IBE) vs. isolation by distance (IBD). Genomic data revealed an array of host races (three of them in the same mountain range, the Cantabrian Mountains, northern Iberia) at apparently distinct levels of reproductive isolation. We found a pattern of IBE mediated by HAD at both local and European scales, in which genetic differentiation between populations and individuals correlated significantly with the taxonomic relatedness of the host plants. IBD was significant only when considered at the wider European scale. We hypothesize that, locally, HAD between Geranium-feeding populations was caused (at least partially) by allochrony, that is via adaptation of adult flight time to the flowering period of each host plant species. Nevertheless, the potential reproductive isolation between populations using Erodium and populations using Geranium cannot be explained by allochrony or IBD, and other mechanisms are expected to be at play.

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.

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'.

Population genomics supports clonal reproduction and multiple independent gains and losses of parasitic abilities in the most devastating nematode pest.

The root-knot nematodes are the most devastating worms to worldwide agriculture with Meloidogyne incognita being the most widely distributed and damaging species. This parasitic and ecological success seems surprising given its supposed obligatory clonal reproduction. Clonal reproduction has been suspected based on cytological observations but, so far, never confirmed by population genomics data. As a species, M. incognita is highly polyphagous with thousands of host plants. However, different M. incognita isolates present distinct and overlapping patterns of host compatibilities. Historically, four "host races" had been defined as a function of ranges of compatible and incompatible plants. In this study, we used population genomics to assess whether (a) reproduction is actually clonal in this species, (b) the host races follow an underlying phylogenetic signal or, rather represent multiple independent transitions, and (c) how genome variations associate with other important biological traits such as the affected crops and geographical distribution. We sequenced the genomes of 11 M. incognita isolates across Brazil that covered the four host races in replicates. By aligning the genomic reads of these isolates to the M. incognita reference genome assembly, we identified point variations. Analysis of linkage disequilibrium and 4-gametes test showed no evidence for recombination, corroborating the clonal reproduction of M. incognita. The few point variations between the isolates showed no significant association with the host races, the geographical origin of the samples, or the crop on which they have been collected. Addition of isolates from other locations around the world confirmed this lack of underlying phylogenetic signal. This suggests multiple gains and losses of parasitic abilities and adaptations to different environments account for the broad host spectrum and wide geographical distribution of M. incognita and thus to its high economic impact. This surprising adaptability without sex poses both evolutionary and agro-economic challenges.

Untargeted Metabolomics Approach Reveals Differences in Host Plant Chemistry Before and After Infestation With Different Pea Aphid Host Races.

The pea aphid (Acyrthosiphon pisum), a phloem-sucking insect, has undergone a rapid radiation together with the domestication and anthropogenic range expansion of several of its legume host plants. This insect species is a complex of at least 15 genetically different host races that can all develop on the universal host plant Vicia faba. However, each host race is specialized on a particular plant species, such as Medicago sativa, Trifolium pratense, or Pisum sativum, which makes it an attractive model insect to study ecological speciation. Previous work revealed that pea aphid host plants produce a specific phytohormone profile depending on the host plant - host race combination. Native aphid races induce lower defense hormone levels in their host plant than non-native pea aphid races. Whether these changes in hormone levels also lead to changes in other metabolites is still unknown. We used a mass spectrometry-based untargeted metabolomic approach to identify plant chemical compounds that vary among different host plant-host race combinations and might therefore, be involved in pea aphid host race specialization. We found significant differences among the metabolic fingerprints of the four legume species studied prior to aphid infestation, which correlated with aphid performance. After infestation, the metabolic profiles of M. sativa and T. pratense plants infested with their respective native aphid host race were consistently different from profiles after infestation with non-native host races and from uninfested control plants. The metabolic profiles of P. sativum plants infested with their native aphid host race were also different from plants infested with non-native host races, but not different from uninfested control plants. The compounds responsible for these differences were putatively identified as flavonoids, saponins, non-proteinogenic amino acids and peptides among others. As members of these compound classes are known for their activity against insects and aphids in particular, they may be responsible for the differential performance of host races on native vs. non-native host plants. We conclude that the untargeted metabolomic approach is suitable to identify candidate compounds involved in the specificity of pea aphid - host plant interactions.

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.

Intrinsic pre-zygotic reproductive isolation of distantly related pea aphid host races.

Human activities may weaken or destroy reproductive isolation between young taxa, leading to their fusion with consequences for population and community ecology. Pea aphid host races are adapted to different legume taxa, providing a degree of pre-mating isolation mediated by habitat choice. Yet, all races can feed and reproduce on the broad bean (Vicia faba), a major crop which represents a 'universal host plant', which can promote hybridization between races. Here, we ask if pea aphid host races have reproductive barriers which prevent or reduce gene flow when they co-occur on the universal host plant. We observed mating behaviour, female survival, number of eggs and egg fertilization rates for three types of crosses: among individuals of the same host race, between closely related host races and between distantly related host races. We did not find significant differences in mating behaviour and female survival among the three types of crosses. However, we observed a drastic reduction in the number of eggs laid, and in the number of fertilized eggs, in distant crosses. We conclude that widespread broad bean cultivation in agriculture may predispose closely related-but not distantly related-host races to hybridize, disrupting reproductive isolation between incipient species.

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.

Modulation of Legume Defense Signaling Pathways by Native and Non-native Pea Aphid Clones.

The pea aphid (Acyrthosiphon pisum) is a complex of at least 15 genetically different host races that are native to specific legume plants, but can all develop on the universal host plant Vicia faba. Despite much research, it is still unclear why pea aphid host races (biotypes) are able to colonize their native hosts while other host races are not. All aphids penetrate the plant and salivate into plant cells when they test plant suitability. Thus plants might react differently to the various pea aphid host races. To find out whether legume species vary in their defense responses to different pea aphid host races, we measured the amounts of salicylic acid (SA), the jasmonic acid-isoleucine conjugate (JA-Ile), other jasmonate precursors and derivatives, and abscisic acid (ABA) in four different species (Medicago sativa, Trifolium pratense, Pisum sativum, V. faba) after infestation by native and non-native pea aphid clones of various host races. Additionally, we assessed the performance of the clones on the four plant species. On M. sativa and T. pratense, non-native clones that were barely able to survive or reproduce, triggered a strong SA and JA-Ile response, whereas infestation with native clones led to lower levels of both phytohormones. On P. sativum, non-native clones, which survived or reproduced to a certain extent, induced fluctuating SA and JA-Ile levels, whereas the native clone triggered only a weak SA and JA-Ile response. On the universal host V. faba all aphid clones triggered only low SA levels initially, but induced clone-specific patterns of SA and JA-Ile later on. The levels of the active JA-Ile conjugate and of the other JA-pathway metabolites measured showed in many cases similar patterns, suggesting that the reduction in JA signaling was due to an effect upstream of OPDA. ABA levels were downregulated in all aphid clone-plant combinations and were therefore probably not decisive factors for aphid-plant compatibility. Our results suggest that A. pisum clones manipulate plant-defense signaling to their own advantage, and perform better on their native hosts due to their ability to modulate the SA- and JA-defense signaling pathways.

Evidence for ecological speciation via a host shift in the holly leaf miner, Phytomyza glabricola (Diptera: Agromyzidae).

Evolutionary radiations have been well documented in plants and insects, and natural selection may often underly these radiations. If radiations are adaptive, the diversity of species could be due to ecological speciation in these lineages. Agromyzid flies exhibit patterns of repeated host-associated radiations. We investigated whether host-associated population divergence and evidence of divergent selection exist in the leaf miner Phytomyza glabricola on its sympatric host plants, the holly species, Ilex coriacea and I. glabra. Using AFLPs and nuclear sequence data, we found substantial genetic divergence between host-associated populations of these flies throughout their geographic range. Genome scans using the AFLP data identified 13 loci under divergent selection, consistent with processes of ecological speciation. EF-1α data suggest that I. glabra is the original host of P. glabricola and that I. coriacea is the novel host, but the AFLP data are ambiguous with regard to directionality of the host shift.

Heritability of Wing Size and Shape of the Rice and Corn Strains of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) represents a pest of economic importance in all Western Hemisphere. This polyphagous species has diverged into two populations that have been mainly recognized with various mitochondrial and nuclear molecular markers and named "the rice" and "the corn" strains. In Colombia, both strains have evolved prezygotic and postzygotic isolation. They differ in tolerance to Bacillus thuringiensis (Cry1Ac and Cry1Ab endotoxins) and the insecticides lambda-cyhalothrin and methomyl. In 2014, a wing morphometric analysis made in 159 individuals from a colony showed that both strains significantly differ in wing shape. The species also exhibits sexual dimorphism in the rice strain as in females wing size is larger than in males. Here, we continued this work with another wing morphometric approach in laboratory-reared strains to calculate wing size and shape heritabilities using a full-sib design and in wild populations to determine if this method distinguishes these strains. Our results show that male heritabilities of both traits were higher than female ones. Wild populations were significantly different in wing shape and size. These results suggest that wing morphometrics can be used as an alternative method to molecular markers to differentiate adults from laboratory-reared populations and wild populations of this pest, particularly in males of this species. Finally, Q ST values obtained for wing size and shape further demonstrated that both strains are genetically differentiated in nature.

Evidence of superclones in Australian cotton aphid Aphis gossypii Glover (Aphididae: Hemiptera).

BACKGROUND: Aphis gossypii is an important pest of cotton that has developed resistance to many chemicals used for its control. Any lack of understanding of its genetic structure, resistance status and host plant specialisation hampers effective management. RSULTS: Eight microsatellite markers were genotyped for a collection of Australian A. gossypii field isolates from 55 plant species from major Australian cotton-producing regions. The aphid's pirimicarb resistance status linked to the ACE1 (acetylcholinesterase) S431F mutation was determined by PCR-RFLP. Overall, the genetic diversity was low and there were only 13 multilocus genotype (MLG) groups found in a total of 936 aphids, suggesting asexual reproduction. Three MLGs (Aust-01, Aust-02 and Aust-04) represented 78% of all aphids tested. MLGs Aust-01 (41%) and Aust-02 (18%) were linked to the ACE1 S431F mutation and found on cotton and a range of hosts. Aust-04 (19%) hosted mainly on cotton (but also Asteraceae and Malvaceae) was predominantly susceptible to pirimicarb. Given their abundance and widespread occurrence, these three clones were considered to be superclones. CONCLUSION: The study demonstrated that any strategy to control A. gossypii and manage pirimicarb resistance should target A. gossypii strains of all MLG types residing on any plant species and not just cotton

Analysis of the genetic diversity in Metopolophium dirhodum (Walker) (Hemiptera, Aphididae) by RAPD markers

The emergence of host-races within aphids may constitute an obstacle to pest management by means of plant resistance. There are examples of host-races within cereals aphids, but their occurrence in Rose Grain Aphid, Metopolophium dirhodum (Walker, 1849), has not been reported yet. In this work, RAPD markers were used to assess effects of the hosts and geographic distance on the genetic diversity of M. dirhodum lineages. Twenty-three clones were collected on oats and wheat in twelve localitites of southern Brazil. From twenty-seven primers tested, only four primers showed polymorphisms. Fourteen different genotypes were revealed by cluster analysis. Five genotypes were collected only on wheat; seven only on oats and two were collected in both hosts. Genetic and geographical distances among all clonal lineages were not correlated. Analysis of molecular variance showed that some molecular markers are not randomly distributed among clonal lineages collected on oats and on wheat. These results suggest the existence of host-races within M. dirhodum, which should be further investigated using a combination of ecological and genetic data.

A emergência de raças hospedeiro-especialistas em afídeos pode constituir um obstáculo ao manejo de pragas por meio de plantas resistentes. Existem exemplos de raças hospedeiro-especialistas em afídeos de cereais, embora a ocorrência de raça hospedeiro-especialista no pulgão-verde-pálido-do-trigo Metopolophium dirhodum (Walker, 1849) (Hemiptera, Aphididae) não tenha sido relatada ainda. Marcadores RAPD foram utilizados para avaliar os efeitos da distância geográfica e do hospedeiro sobre a diversidade genética de linhas clonais de M. dirhodum. Vinte e três clones foram coletados em aveia e trigo em doze localidades do sul do Brasil. De vinte e sete iniciadores usados para a análise, apenas quatro iniciadores mostraram polimorfismos. A análise de agrupamento por similaridade genética revelou haver quatorze genótipos, cinco dos quais coletados exclusivamente em trigo, sete exclusivamente em aveia e dois em ambos hospedeiros. Não houve correlação entre as similaridades genéticas e a distância geográfica. A análise da variância molecular demonstrou que alguns marcadores RAPD não se distribuem aleatoriamente entre as linhagens clonais coletadas em aveia e em trigo. Estes resultados sugerem a existência de raças hospedeiro-especialistas em M. dirhodum no Brasil, hipótese esta que deve ser investigada combinando-se dados ecológicos e genéticos.

Specialized Feeding Behavior Influences Both Ecological Specialization and Assortative Mating in Sympatric Host Races of Pea Aphids.

Not only is ecological specialization a defining feature of much of Earth's biological diversity, the evolution of specialization may also play a central role in generating diversity by facilitating speciation. To understand how ecological specialization evolves, we must know the particular characters that cause organisms to be specialized. For example, most theories of specialization in herbivorous insects emphasize physiological trade-offs in response to toxic plant chemicals. However, even in herbivores, it is likely that other characters are also involved in resource specialization. Knowing the causes of ecological specialization is also crucial for linking specialization to speciation. When the same character(s) that cause specialization also influence assortative mating, speciation may occur particularly rapidly because specialization and reproductive isolation become coupled in a positive feedback that speeds the evolution of both. Indeed, a central hypothesis in the study of ecological speciation is that specialization in recently diverged taxa may often be due to characters that also produce assortative mating. We test this hypothesis by evaluating the causes of ecological specialization among host-associated populations of an herbivorous insect, the pea aphid (Acyrthosiphon pisum). These populations are highly specialized on different host plants (alfalfa or clover; "alternate hosts"), and the races are partially reproductively isolated. Here, we identify key characters responsible for host plant specialization. Our results suggest that the major proximal determinant of host specialization is the behavioral acceptance of a plant rather than the toxicity of the food source. Pea aphids rapidly assess alfalfa and clover and reject the alternate host based on chemical cues that are perceived before the initiation of feeding. This rapid behavioral rejection of the alternate host by a given race has two consequences. First, unrestrained aphids quickly leave the alternate host and search for other plants. Because pea aphids mate on their host plants, divergence in host acceptance among ecologically specialized races leads to congregation on the favored host. This results in de facto assortative mating when sexual forms are produced in late summer. Second, specialized aphids that are held on the alternate host will not feed in a 7.2-h trial, even in the face of starvation. Thus, a complex trait, behavioral acceptance of a plant as host, influences both reproductive isolation (through host-associated assortative mating) and ecological specialization (because of low nutritional uptake on the alternate host). This dual influence of feeding behavior on both assortative mating and resource specialization is central to the maintenance of these divergent races, and it may also have been involved in their origin.

A FIELD TEST FOR HOST-PLANT DEPENDENT SELECTION ON LARVAE OF THE APPLE MAGGOT FLY, RHAGOLETIS POMONELLA.

Host-plant dependent fitness trade-offs refer to traits that enhance the performance of an insect on one plant species to its detriment on others. Such trade-offs are central to models of sympatric speciation via host shifts, but have proven difficult to empirically demonstrate. Here, we test for host-plant dependent selection on larvae of apple (Malus pumila L.)- and hawthorn (Crataegus mollis L. spp.)-infesting races of Rhagoletis pomonella (Walsh). Samples of larvae were reared in the field and under protective conditions in a garage. Our rationale was that the garage should slow rates of fruit rot relative to the field, relaxing selection pressures associated with declining fruit quality. Four findings emerged from the study. (1) Larvae suffered higher mortality in fruits in the field than the garage. (2) The increase in mortality was greater for larvae in haws. (3) Larvae possessing the alleles Me 100, Acon-2 95, and Mpi 37, three allozymes displaying host-related differentiation in R. pomonella that map to linkage group II in the fly, left fruits earlier than other genotypes. (4) Allele frequencies for Me 100, Acon-2 95, and Mpi 37 were significantly higher in both apple and haw larvae surviving the field versus the garage treatment. Our results suggested that field conditions favored larvae that rapidly developed and left rotting fruits. Since these individuals tended to possess the alleles Me 100, Acon-2 95, and Mpi 37, frequencies of these allozymes were higher in the field. Selection on larvae was directional for Me 100, Acon-2 95, and Mpi 37 (or linked genes) in both host races. We previously showed that these same alleles can be disfavored in the pupal stage, especially in the apple race, where they correlate with premature diapause termination. Fitness trade-offs in Rhagoletis may therefore be due as much to differences in the relative strengths of directional selection pressures acting on different life stages as to disruptive selection affecting any one particular stage. The necessity to consider details of the entire life-cycle highlights one of the many challenges posed to documenting fitness trade-offs for phytophagous insects.

GENERAL-PURPOSE GENOTYPES FOR HOST SPECIES UTILIZATION IN A NEMATODE PARASITE OF DROSOPHILA.

The nematode Howardula aoronymphium parasitizes several species of mushroom-feeding Drosophila. A survey of isofemale strains of H. aoronymphium and a 25-generation selection experiment revealed that this species does not comprise host races, and that it harbors little heritable variation for adaptation to specific hosts No tradeoffs in performance on the different host species were evident. General-purpose genotypes, which can utilize all host species, characterize H. aoronymphium. An important feature of the natural history of these nematodes-correlated epidemiology across host species-is postulated to be both a cause and a consequence of the evolution of general-purpose genotypes in this species.

THE EFFECTS OF WINTER LENGTH ON THE GENETICS OF APPLE AND HAWTHORN RACES OF RHAGOLETIS POMONELLA (DIPTERA: TEPHRITIDAE).

Host plant-associated fitness trade-offs are central to models of sympatric speciation proposed for certain phytophagous insects. But empirical evidence for such trade-offs is scant, which has called into question the likelihood of nonallopatric speciation. Here, we report on the second in a series of studies testing for host-related selection on pupal life-history characteristics of apple- (Malus pumila L.) and hawthorn- (Crataegus mollis L. spp.) infesting races of the Tephritid fruit fly, Rhagoletis pomonella (Walsh). In particular, we examine the effects of winter length on the genetics of these flies. We have previously found that the earlier fruiting phenology of apple trees exposes apple-fly pupae to longer periods of warm weather preceding winter than hawthorn-fly pupae. Because R. pomonella has a facultative diapause, we hypothesized that this selects for pupae with more recalcitrant pupal diapauses (or slower metabolic/development rates) in the apple-fly race. A study in which we experimentally manipulated the length of the prewintering period for hawthorn-origin pupae supported this prediction. If the period preceding winter is important for apple- and hawthorn-fly pupae, then so too should be the length (duration) of winter; the rationale for this prediction is that "fast developing" pupae that break diapause too early will deplete their energy reserves and disproportionately die during long winters. To test this possibility, we chilled apple- and hawthorn-origin pupae collected from a field site near Grant, Michigan, in a refrigerator at 4°C for time periods ranging from one week to two years. Our a priori expectation was that longer periods of cold storage would select against allozyme markers that were associated with faster rates of development in our earlier study. Since these electromorphs are typically found at higher frequencies in hawthorn flies, extending the overwintering period should favor "apple-fly alleles" in both races. The results from this "overwinter" experiment supported the diapause hypothesis. The anticipated genetic response was observed in both apple and hawthorn races, as allele frequencies became significantly more "apple-fly-like" in eclosing adults surviving longer chilling periods. This indicates that it is the combination of environmental conditions before and during winter that selects on the host races. Many tests for trade-offs fail to adequately consider the interplay between insect development, host plant phenology, and local climatic conditions. Our findings suggest that such oversight may help to explain the paucity of reported fitness trade-offs.

SYMPATRIC SPECIATION VIA HABITAT SPECIALIZATION DRIVEN BY DELETERIOUS MUTATIONS.

Theoretical studies have suggested that the evolution of habitat (host) races, regarded as a prelude to sympatric speciation, requires strong trade-offs in adaptation to different habitats: alleles that improve fitness in some habitats and have deleterious effects of similar magnitude in other habitats must be segregating in the population. I argue that such trade-offs are not necessary; the evolution of habitat races can also be driven by genetic variation due to loci that affect fitness in one habitat and are neutral or nearly so in others, that is, when performance in different habitats is genetically independent. One source of such genetic variation are deleterious mutations with habitat-specific fitness effects. I use deterministic two-locus and multilocus models to show that the presence of such mutations in the gene pool results in indirect selection favoring habitat fidelity or habitat preference over acceptance of both suitable habitats. This leads to the evolution of largely genetically isolated populations that use different habitats, from a single panmictic population of individuals accepting both habitats. This study suggests that the conditions favoring habitat race formation, and thus possibly sympatric speciation, are much less stringent than previously thought.

RESOURCE-ASSOCIATED POPULATION SUBDIVISION IN A SYMBIOTIC CORAL-REEF SHRIMP.

The importance of sympatric speciation remains controversial. An empirical observation frequently offered in its support is the occurrence of sister taxa living in sympatry but using different resources. To examine the possibility of sympatric differentiation in producing such cases, I measured genetic, behavioral, and demographic differentiation between populations of the tropical sponge-dwelling shrimp Synalpheus brooksi occupying two alternate host species on three reefs in Caribbean Panama. This species belongs to an apparently monophyletic group of ≥ 30 species of mostly obligate, host-specific sponge-dwellers, many of which occur in sympatry. Demographic data demonstrated the potential for disruptive selection imposed by the two host species: shrimp demes from the sponge Agelas clathrodes were consistently denser, poorer in mature females, more heavily parasitized by branchial bopyrid isopods, and less parasitized by thoracic isopods, than conspecific shrimp from the sponge Spheciospongia vesparium. Laboratory assays demonstrated divergence in host preference: shrimp on all three reefs tended to choose their native sponge species more often than did conspecific shrimp from the other host. Because S. brooksi mates within the host, this habitat selection should foster assortative mating by host species. A hierarchical survey of protein-electrophoretic variation also supported host-mediated divergence, revealing the following: (1) shrimp from the two hosts are conspecific, as evidenced by absence of fixed allelic differences at any of nine allozyme loci scored; (2) strong genetic subdivision among populations of this philopatric shrimp on reefs separated by 1-3 km; and (3) significant host-associated genetic differentiation within two of the three reefs. Finally, intersexual aggression (a proxy for mating incompatibility) between shrimp from different host species was significantly elevated on the one reef where host-associated genetic differences were strongest, demonstrating concordance between genetic and behavioral estimates of divergence. Adjacent reefs appear to be semi-independent sites of host-associated differentiation, as evidenced by differences in the degree of host-associated behavioral and genetic differentiation, and in the specific loci involved, on different reefs. In philopatric organisms with highly subdivided populations, such as S. brooksi, resource-associated differentiation can occur independently in different populations, thus providing multiple "experiments" in differentiation and resulting in a mosaic pattern of polymorphism as reflected by neutral genetic markers. Several freshwater fishes, an amphipod, and a snail similarly show independent but remarkably convergent patterns of resource-associated divergence in different conspecific populations, often in the absence of obvious spatial barriers. In each case, substantial differentiation has occurred in the face of continuing gene flow.

Intra- and interspecific competition and host race formation in the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae).

Intra- and interspecific resource competition are potentially important factors affecting host plant use by phytophagous insects. In particular, escape from competitors could mediate a successful host shift by compensating for decreased feeding performance on a new plant. Here, we examine the question of host plant-dependent competition for apple (Malus pumila)- and hawthorn (Crataegus mollis)-infesting larvae of the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae) at a field site near Grant, Michigan, USA. Interspecific competition from tortricid (Cydia pomonella, Grapholita prunivora, and Grapholita packardi) and agonoxenid (subfamily Blastodacninae) caterpillars and a curculionid weevil (Conotrachelus crataegi) was much stronger for R. pomonella larvae infesting the ancestral host hawthorn than the derived host apple. Egg to pupal survivorship was estimated as 52.8% for fly larvae infesting hawthorn fruit without caterpillars and weevils compared to only 27.3% for larvae in harthorns with interspecific insects. Survivorship was essentially the same between fly larvae infesting apples in the presence (44.8%) or absence (42.6%) of interspecific insects. Intraspecific competition among maggots was also stronger in hawthorns than apples. The order or time that a larva exited a hawthorn fruit was a significant determinant of its pupal mass, with earlier emerging larvae being heavier than later emerging larvae. This was not the case for larvae in apples, as the order or time that a larva exited an apple fruit had relatively little influence on its pupal mass. Our findings suggest that decreased performance related to host plant chemistry/nutrition may restrict host range expansion and race formation in R. pomonella to those plants where biotic/ecological factors (i.e. escape from competitors and parasitoids) adequately balance the survivorship equation. This balance permits stable fly populations to persist on novel plants, setting the stage for the evolution of host specialization under certain mitigating conditions (e.g. when mating is host specific and host-associated fitness trade-offs exist).