Localizing F(ST) outliers on a QTL map reveals evidence for large genomic regions of reduced gene exchange during speciation-with-gene-flow.

Populations that maintain phenotypic divergence in sympatry typically show a mosaic pattern of genomic divergence, requiring a corresponding mosaic of genomic isolation (reduced gene flow). However, mechanisms that could produce the genomic isolation required for divergence-with-gene-flow have barely been explored, apart from the traditional localized effects of selection and reduced recombination near centromeres or inversions. By localizing F(ST) outliers from a genome scan of wild pea aphid host races on a Quantitative Trait Locus (QTL) map of key traits, we test the hypothesis that between-population recombination and gene exchange are reduced over large 'divergence hitchhiking' (DH) regions. As expected under divergence hitchhiking, our map confirms that QTL and divergent markers cluster together in multiple large genomic regions. Under divergence hitchhiking, the nonoutlier markers within these regions should show signs of reduced gene exchange relative to nonoutlier markers in genomic regions where ongoing gene flow is expected. We use this predicted difference among nonoutliers to perform a critical test of divergence hitchhiking. Results show that nonoutlier markers within clusters of F(ST) outliers and QTL resolve the genetic population structure of the two host races nearly as well as the outliers themselves, while nonoutliers outside DH regions reveal no population structure, as expected if they experience more gene flow. These results provide clear evidence for divergence hitchhiking, a mechanism that may dramatically facilitate the process of speciation-with-gene-flow. They also show the power of integrating genome scans with genetic analyses of the phenotypic traits involved in local adaptation and population divergence.

Population genetic structure and secondary symbionts in host-associated populations of the pea aphid complex.

Polyphagous insect herbivores experience different selection pressures on their various host plant species. How this affects population divergence and speciation may be influenced by the bacterial endosymbionts that many harbor. Here, we study the population structure and symbiont community of the pea aphid (Acyrthosiphon pisum), which feeds on a range of legume species and is known to form genetically differentiated host-adapted populations. Aphids were collected from eight legume genera in England and Germany. Extensive host plant associated differentiation was observed with this collection of pea aphids comprising nine genetic clusters, each of which could be associated with a specific food plant. Compared to host plant, geography contributed little to genetic differentiation. The genetic clusters were differentiated to varying degrees, but this did not correlate with their degree of divergence in host use. We surveyed the pea aphid clones for the presence of six facultative (secondary) bacterial endosymbionts and found they were nonrandomly distributed across the aphid genetic clusters and this distribution was similar in the two countries. Aphid clones on average carried 1.4 species of secondary symbiont with those associated with Lathyrus having significantly fewer. The results are interpreted in the light of the evolution of specialization and ecological speciation.

Divergence hitchhiking and the spread of genomic isolation during ecological speciation-with-gene-flow.

In allopatric populations, geographical separation simultaneously isolates the entire genome, allowing genetic divergence to accumulate virtually anywhere in the genome. In sympatric populations, however, the strong divergent selection required to overcome migration produces a genetic mosaic of divergent and non-divergent genomic regions. In some recent genome scans, each divergent genomic region has been interpreted as an independent incidence of migration/selection balance, such that the reduction of gene exchange is restricted to a few kilobases around each divergently selected gene. I propose an alternative mechanism, 'divergence hitchhiking' (DH), in which divergent selection can reduce gene exchange for several megabases around a gene under strong divergent selection. Not all genes/markers within a DH region are divergently selected, yet the entire region is protected to some degree from gene exchange, permitting genetic divergence from mechanisms other than divergent selection to accumulate secondarily. After contrasting DH and multilocus migration/selection balance (MM/SB), I outline a model in which genomic isolation at a given genomic location is jointly determined by DH and genome-wide effects of the progressive reduction in realized migration, then illustrate DH using data from several pairs of incipient species in the wild.

Natural selection in action during speciation.

The role of natural selection in speciation, first described by Darwin, has finally been widely accepted. Yet, the nature and time course of the genetic changes that result in speciation remain mysterious. To date, genetic analyses of speciation have focused almost exclusively on retrospective analyses of reproductive isolation between species or subspecies and on hybrid sterility or inviability rather than on ecologically based barriers to gene flow. However, if we are to fully understand the origin of species, we must analyze the process from additional vantage points. By studying the genetic causes of partial reproductive isolation between specialized ecological races, early barriers to gene flow can be identified before they become confounded with other species differences. This population-level approach can reveal patterns that become invisible over time, such as the mosaic nature of the genome early in speciation. Under divergent selection in sympatry, the genomes of incipient species become temporary genetic mosaics in which ecologically important genomic regions resist gene exchange, even as gene flow continues over most of the genome. Analysis of such mosaic genomes suggests that surprisingly large genomic regions around divergently selected quantitative trait loci can be protected from interrace recombination by "divergence hitchhiking." Here, I describe the formation of the genetic mosaic during early ecological speciation, consider the establishment, effects, and transitory nature of divergence hitchhiking around key ecologically important genes, and describe a 2-stage model for genetic divergence during ecological speciation with gene flow.

The genetic mosaic suggests a new role for hitchhiking in ecological speciation.

Early in ecological speciation, the genomically localized effects of divergent selection cause heterogeneity among loci in divergence between incipient species. We call this pattern of genomic variability in divergence the 'genetic mosaic of speciation'. Previous studies have used F(ST) outliers as a way to identify divergently selected genomic regions, but the nature of the relationship between outlier loci and quantitative trait loci (QTL) involved in reproductive isolation has not yet been quantified. Here, we show that F(ST) outliers between a pair of incipient species are significantly clustered around QTL for traits that cause ecologically based reproductive isolation. Around these key QTL, extensive 'divergence hitchhiking' occurs because reduced inter-race mating and negative selection decrease the opportunity for recombination between chromosomes bearing different locally adapted QTL alleles. Divergence hitchhiking is likely to greatly increase the opportunity for speciation in populations that are sympatric, regardless of whether initial divergence was sympatric or allopatric. Early in ecological speciation, analyses of population structure, gene flow or phylogeography based on different random or arbitrarily chosen neutral markers should be expected to conflict--only markers in divergently selected genomic regions will reveal the evolutionary history of adaptive divergence and ecologically based reproductive isolation. Species retain mosaic genomes for a very long time, and gene exchange in hybrid zones can vary dramatically among loci. However, in hybridizing species, the genomic regions that affect ecologically based reproductive isolation are difficult to distinguish from regions that have diverged for other reasons.

Population differentiation and genetic variation in performance on eight hosts in the pea aphid complex.

Phytophagous insects frequently use multiple host-plant species leading to the evolution of specialized host-adapted populations and sometimes eventually to speciation. Some insects are confronted with a large number of host-plant species, which may provide complex routes of gene flow between host-adapted populations. The pea aphid (Acyrthosiphon pisum) attacks a broad range of plants in the Fabaceae and it is known that populations on Trifolium pratense and Medicago sativa can be highly specialized at exploiting these species. To find out whether adaptation to a broad range of co-occurring hosts has occurred, we tested the performance of pea aphid clones collected from eight host-plant genera on all of these plants in a reciprocal transfer experiment. We provide evidence for pervasive host-plant specialization. The high performance of all aphid clones on Vicia faba suggests that this host plant could be a site of gene flow between different populations that could limit further host-associated divergence. The genetic variance in host-plant usage was partitioned into within- and among-population components, which represent different levels of host adaptation. Little evidence of within-population trade-offs in performance on different plant species was found.

Population differentiation and genetic variation in host choice among pea aphids from eight host plant genera.

Habitat choice plays a critical role in the processes of host range evolution, specialization, and ecological speciation. Pea aphid, Acyrthosiphon pisum, populations from alfalfa and red clover in eastern North America are known to be genetically differentiated and show genetic preferences for the appropriate host plant. This species feeds on many more hosts, and here we report a study of the genetic variation in host plant preference within and between pea aphid populations collected from eight genera of host plants in southeastern England. Most host-associated populations show a strong, genetically based preference for the host plant from which they were collected. Only in one case (populations from Vicia and Trifolium) was there little difference in the plant preference spectrum between populations. All populations showed a significant secondary preference for the plant on which all the aphid lines were reared: broad bean, Vicia faba, previously suggested to be a "universal host" for pea aphids. Of the total genetic variance in host preference within our sample, 61% could be attributed to preference for the collection host plant and a further 9% to systematic differences in secondary preferences with the residual representing within-population genetic variation between clones. We discuss how a combination of host plant preference and mating on the host plant may promote local adaptation and possibly ecological speciation, and whether a widely accepted host could oppose speciation by mediating gene flow between different populations.

Back to the future: genetic correlations, adaptation and speciation.

Genetic correlations can affect the course of phenotypic evolution. Although genetic correlations among traits are a common feature of quantitative genetic analyses, they have played a very minor role in recent linkage-map based analyses of the genetic architecture of quantitative traits. Here, we use our work on host-associated races in pea aphids to illustrate how quantitative trait locus (QTL) mapping can be used to test specific hypotheses about how genetic correlations may facilitate ecological specialization and speciation.

The ecological genetics of speciation.

Ecological interactions and the natural selection they cause play a prominent causal role in biological diversification and speciation. As a discipline, ecological genetics integrates the two components of adaptive evolution (natural selection and genetic variability) to study the mechanisms of evolution. Ecological genetics is a fruitful approach to the study of how reproductive isolation can evolve under natural selection. The essence of this way of thinking and the ways in which it can be used to address persistent open questions in speciation are discussed.

The genetic architecture of ecological specialization: correlated gene effects on host use and habitat choice in pea aphids.

Genetic correlations among phenotypic characters result when two traits are influenced by the same genes or sets of genes. By reducing the degree to which traits in two environments can evolve independently (e.g., Lande 1979; Via and Lande 1985), such correlations are likely to play a central role in both the evolution of ecological specialization and in its link to speciation. For example, negative genetic correlations between fitness traits in different environments (i.e., genetic trade-offs) are thought to influence the evolution of specialization, while positive genetic correlations between performance and characters influencing assortative mating can accelerate the evolution of reproductive isolation between ecologically specialized populations. We first discuss how the genetic architecture of a suite of traits may affect the evolutionary role of genetic correlations among them and review how the mechanisms of correlations can be analyzed using quantitative trait locus (QTL) mapping. We then consider the implications of such data for understanding the evolution of specialization and its link to speciation. We illustrate this approach with a QTL analysis of key characters in two races of pea aphids that are highly specialized on different host plants and partially reproductively isolated. Our results suggest that antagonism among QTL effects on performance in the two environments leads to a genetic trade-off in this system. We also found evidence for parallel QTL effects on host-plant acceptance and fecundity on the accepted host, which could produce assortative mating. These results suggest that the genetic architecture of traits associated with host use may have played a central role in the evolution of specialization and reproductive isolation in pea aphids.

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.

EVOLUTION OF AN APHID-PARASITOID INTERACTION: VARIATION IN RESISTANCE TO PARASITISM AMONG APHID POPULATIONS SPECIALIZED ON DIFFERENT PLANTS.

The evolution of associations between herbivorous insects and their parasitoids is likely to be influenced by the relationship between the herbivore and its host plants. If populations of specialized herbivorous insects are structured by their host plants such that populations on different hosts are genetically differentiated, then the traits affecting insect-parasitoid interactions may exhibit an associated structure. The pea aphid (Acyrthosiphon pisum) is a herbivorous insect species comprised of genetically distinct groups that are specialized on different host plants (Via 1991a, 1994). Here, we examine how the genetic differentiation of pea aphid populations on different host plants affects their interaction with a parasitoid wasp, Aphidius ervi. We performed four experiments. (1) By exposing pea aphids from both alfalfa and clover to parasitoids from both crops, we demonstrate that pea aphid populations that are specialized on alfalfa are successfully parasitized less often than are populations specialized on clover. This difference in parasitism rate does not depend upon whether the wasps were collected from alfalfa or clover fields. (2) When we controlled for potential differences in aphid and parasitoid behavior between the two host plants and ensured that aphids were attacked, we found that pea aphids from alfalfa were still parasitized less often than pea aphids from clover. Thus, the difference in parasitism rates is not due to behavior of either aphids or wasps, but appears to be a physiologically based difference in resistance to parasitism. (3) Replicates of pea aphid clones reared on their own host plant and on a common host plant, fava bean, exhibited the same pattern of resistance as above. Thus, there do not appear to be nutritional or secondary chemical effects on the level of physiological resistance in the aphids due to feeding on clover or alfalfa, and therefore the difference in resistance on the two crops appears to be genetically based. (4) We assayed for genetic variation in resistance among individual pea aphid clones collected from clover fields and found no detectable genetic variation for resistance to parasitism within two populations sampled from clover. This is in contrast to Henter and Via's (1995) report of abundant genetic variation in resistance to this parasitoid within a pea aphid population on alfalfa. Low levels of genetic variation may be one factor that constrains the evolution of resistance to parasitism in the populations of pea aphids from clover, leading them to remain more susceptible than populations of the same species from alfalfa.

REPRODUCTIVE ISOLATION BETWEEN SYMPATRIC RACES OF PEA APHIDS. I. GENE FLOW RESTRICTION AND HABITAT CHOICE.

Determining the extent and causes of barriers to gene flow between genetically divergent populations or races of single species is an important complement to post facto analyses of the causes of reproductive isolation between recognized species. Sympatric populations of pea aphids (Acyrthosiphon pisum Harris, Homoptera: Aphididae) on alfalfa and red clover are highly genetically divergent and locally adapted. Here, hierarchical estimates of population structure based on Fst suggest that gene exchange between closely adjacent aphid populations on the two hosts is highly restricted relative to that among fields of the same host plant. Although these host-associated races are presently considered to be the same subspecies, they appear to be significantly reproductively isolated, suggesting incipient speciation. Habitat (host) choice was investigated as the first in a temporal series of factors that could reduce gene exchange between these sympatric populations. Field studies of winged colonists to newly planted fields of each host suggest pronounced habitat fidelity. This result was verified using replicated observations of the host choice behavior of different aphid genotypes for which the relative demographic performance on each host was known. These laboratory observations of behavior revealed a strong genetic correlation between habitat choice (or acceptance) and the relative performance in each habitat. Because mating occurs on the host plant, habitat choice in this system leads to assortative mating and is therefore a major cause of reproductive isolation between the sympatric pea aphid populations on alfalfa and clover. However, the extent of dispersal between hosts estimated from the field study of winged colonists (9-11%) is too great to be consistent with the genetic divergence estimated between the races. This suggests that barriers to gene flow other than host choice also exist, such as selection against migrants or hybrids in the parental environments, hybrid sterility, or hybrid breakdown.

SHORT-TERM EVOLUTION IN THE SIZE AND SHAPE OF PEA APHIDS.

Phenotypic evolution in contemporary populations can generally be witnessed only when novel selective forces produce rapid evolution. Examples of conditions that have led to rapid evolution include drastic environmental change, invasion of a new predator, or a host-range expansion. In cyclical parthenogens, however, yearly cycles of phenotypic evolution may occur due to the loss of adaptation during recombination in the sexual phase (genetic slippage), permitting an opportunity to observe adaptive evolutionary change in contemporary populations that are not necessarily subject to new patterns of natural selection. In insect herbivores, comparative studies suggest that morphological features that aid individuals in remaining on the plant or exploiting it as a food source are likely targets for selection. Here, we estimated the genetic variability of morphological traits in a cyclical parthenogen, the pea aphid (Acyrthosiphon pisum), to determine the potential for their evolution and we tested the hypothesis that size and/or shape evolves by clonal selection during one season of parthenogenetic reproduction. Genetic variation in a set of morphological traits was estimated using laboratory-reared descendents of clones collected from a single alfalfa field in May 1988 and April 1989 (henceforth, the "early" collections). In both years, there was significant clonal heritability early in the season both for overall morphology and for several individual aspects of size and shape. Because the course of short-term evolutionary change in the multivariate phenotype is a function of patterns of genetic covariance among characters, genetic correlations between size and 12 shape variables were also estimated for these early collections. A comparison between the mean phenotype of each early collection and that of a corresponding "late" collection made from the same field seven to eight clonal generations later in the same years revealed qualitatively similar changes in the average multivariate morphological phenotypes between the time periods in both years, although the difference was only significant for the 1989 samples. The pattern of genetic correlations that we estimated early in the 1989 season between overall size and various shape variables suggests that the observed short-term evolutionary changes in shape could have been due to natural selection acting only to increase overall size. We tested this hypothesis by estimating selection on size using a separate data set in which both demographic and morphological variables were measured on individuals reared under field conditions. Highly significant regressions of individual relative fitness on size were found for two major fitness components. Thus, it is likely that the evolutionary change in morphology that we observed is attributable to natural selection, possibly acting primarily through body size. A shift back to smaller size between the late 1988 and early 1989 collections from the same field suggests that either a cost of recombination or opposing selective forces during overwintering may produce persistent yearly cycles of morphological evolution in this cyclically parthenogenetic species.

THE POTENTIAL FOR COEVOLUTION IN A HOST-PARASITOID SYSTEM. I. GENETIC VARIATION WITHIN AN APHID POPULATION IN SUSCEPTIBILITY TO A PARASITIC WASP.

For coevolution to occur, there must be genetic variation in each species for traits relevant to their interaction. Here, statistically significant variation in susceptibility to a parasitic wasp was found among pea-aphid clones collected from a single population. In a subset of clones that was tested further, wasps were found to oviposit in aphids from both resistant and susceptible lines, but eggs failed to develop in resistant hosts. Significant genetic variance in susceptibility provides evidence that this aphid population has the potential to evolve resistance in response to selection by one of its major natural enemies. Predictions of an expected response to selection based on the experimental measures of variation and field parasitism rates suggested that there should be a detectable change in susceptibility over the course of a season. However, an experimental comparison of mean susceptibility of clones collected early and late in the summer, a period of several generations, revealed no response to selection by the wasps. Aphids collected late in the season were as susceptible, on the average, as those collected early in the summer. Possible constraints on the response of the aphids to selection by this natural enemy are considered.

THE GENETIC STRUCTURE OF HOST PLANT ADAPTATION IN A SPATIAL PATCHWORK: DEMOGRAPHIC VARIABILITY AMONG RECIPROCALLY TRANSPLANTED PEA APHID CLONES.

Populations of insect herbivores that feed on several host plant species may experience different selective forces on each host. When the hosts cooccur in a local area, herbivore populations can provide useful models for the study of evolutionary mechanisms in patchy environments. A first step in such a study involves determination of the genetic structure of host adaptation in the region: how is genetic variation for host use structured within and between subpopulations of herbivores on each host? The structure of genetic variation for host use reveals patterns of local adaptation, probable selective consequences of migration between hosts, and the potential for further evolution. To estimate the population structure of host adaptation in a patchwork, 7-11 pea aphid clones were collected at the beginning of the summer from each of two alfalfa and two red clover fields within a very localized area (about 15-20 km2 ). Using a reciprocal transplant in the field, replicates of these 35 clones were allowed to develop individually on each of the two crops. A complete life table was made for each replicate. Individual fitness was calculated from the life tables as the expected rate of population increase; longevity, age at first reproduction, and total fecundity were also measured for each clonal replicate. Currently, experimental estimates of genetic variation in complete life tables are virtually nonexistent for natural populations, even for single environments (Charlesworth, 1987); field studies are even less common. Because clones from each of two source crops were tested reciprocally on both hosts, variation in relative genotypic fitness on alfalfa and clover could be partitioned among clones within source crops, between fields of the same crop, and between source crops (alfalfa or red clover), providing a view of population structure. Significant clonal variation in relative performance on alfalfa and red clover was found: clones tended to have higher fitness on the crop from which they had been collected (the "home" crop) than they did on the "away" crop, suggesting local adaptation in response to patchy patterns of selection. Clonal variability within collections from the two crops suggests the potential for changes in the genetic constitution of these aphid populations within established fields as a result of clonal selection during the summer season. Significantly negative genetic correlations across crops were found for fitness and its major components. The possibility that these negative cross-environment correlations could act as evolutionary constraints on adaptation to the patchwork is considered.

GENETIC COVARIANCE BETWEEN OVIPOSITION PREFERENCE AND LARVAL PERFORMANCE IN AN INSECT HERBIVORE.

An experimental study determined that females of the herbivorous fly species Liriomyza sativae (Diptera: Agromyzidae) preferentially oviposit on the plant species on which their female progeny attain the greatest pupal weight. A modified parent/offspring regression was used to quantify this relationship as an additive genetic covariance between host-plant preference and relative performance of female larvae on different plant species. The implications of a genetic covariance between preference and performance on the course of evolution in herbivores are discussed. Several females from one population refused to oviposit on one of the plant species; this population also suffered the only significant larval mortality on this plant. These results corroborate the avoidance of unsuitable host plants seen in the genetic analyses of individuals, but relative to the genetic data, such population-level data are of limited usefulness in the study of evolutionary mechanisms by which insect populations become adapted to their host plants.

GENOTYPE-ENVIRONMENT INTERACTION AND THE EVOLUTION OF PHENOTYPIC PLASTICITY.

Studies of spatial variation in the environment have primarily focused on how genetic variation can be maintained. Many one-locus genetic models have addressed this issue, but, for several reasons, these models are not directly applicable to quantitative (polygenic) traits. One reason is that for continuously varying characters, the evolution of the mean phenotype expressed in different environments (the norm of reaction) is also of interest. Our quantitative genetic models describe the evolution of phenotypic response to the environment, also known as phenotypic plasticity (Gause, 1947), and illustrate how the norm of reaction (Schmalhausen, 1949) can be shaped by selection. These models utilize the statistical relationship which exists between genotype-environment interaction and genetic correlation to describe evolution of the mean phenotype under soft and hard selection in coarse-grained environments. Just as genetic correlations among characters within a single environment can constrain the response to simultaneous selection, so can a genetic correlation between states of a character which are expressed in two environments. Unless the genetic correlation across environments is ± 1, polygenic variation is exhausted, or there is a cost to plasticity, panmictic populations under a bivariate fitness function will eventually attain the optimum mean phenotype for a given character in each environment. However, very high positive or negative correlations can substantially slow the rate of evolution and may produce temporary maladaptation in one environment before the optimum joint phenotype is finally attained. Evolutionary trajectories under hard and soft selection can differ: in hard selection, the environments with the highest initial mean fitness contribute most individuals to the mating pool. In both hard and soft selection, evolution toward the optimum in a rare environment is much slower than it is in a common one. A subdivided population model reveals that migration restriction can facilitate local adaptation. However, unless there is no migration or one of the special cases discussed for panmictic populations holds, no geographical variation in the norm of reaction will be maintained at equilibrium. Implications of these results for the interpretation of spatial patterns of phenotypic variation in natural populations are discussed.