A continuum of genetic divergence from sympatric host races to species in the pea aphid complex.

1. Host‐specialised races of plant‐feeding insects are particularly informative models in the study of ecological speciation, that is, the evolution of reproductive isolation through divergent natural selection. However, within the enormous diversity of phytophagous insects, the mechanisms of ecological divergence have been elucidated in few host race systems. 2. Here we review the literature covering speciation through host‐plant specialisation in a well‐studied model, the pea aphid complex, Acyrthosiphon pisum , which encompasses numerous biotypes that parasitise different legume host species worldwide. 3. Published results are consistent with ecologically promoted reproductive isolation. Divergent host‐induced selection is pronounced across biotypes, and reflects genetic trade‐offs preventing the optimal use of multiple host plants. While these genetic trade‐offs may partly explain the unfitness of hybrids between biotypes, hybridisation occurring on plants is also limited by genetically‐based host preference, and by selection against migrants that chose unfavourable hosts. The continuum of genetic divergence displayed by 11 races and species of the pea aphid complex suggests that host races constitute an intermediate step in the speciation process, and that host specialisation may indeed lead to complete speciation. Uncertainties remain on the contribution of non‐ecological reproductive barriers to biotype divergence and on the physiological and molecular bases of host specialisation.

The pea aphid, Acyrthosiphon pisum Harris, constitutes a complex of sympatric populations that are specialised to distinct species of Fabaceae. So far, 12 such populations have been characterised genetically as genetic clusters associated with one or few legumes species. These clusters form a continuum of genetic divergence linking host‐associated races, which show moderate hybridization, to nascent species. They are also known to be associated with different species of endosymbiotic bacteria, which have environmentally dependent effects on aphid fitness. Here, we report on the genetic characterisation of new host‐associated populations in this species complex. We sampled pea aphids in eastern France on Genista tinctoria , G. sagittalis , Onobrychis viciifolia , and Hippocrepis comosa . Bayesian clustering methods based on genotypes obtained at 25 microsatellite loci showed that each sampled plant species hosted a specific pea aphid population. Comparison with previously characterised biotypes showed that the population associated with Hippocrepis comosa was no different from that on Securigera varia . Migrant aphids from other host plants and hybrids were found at various frequencies on the newly sampled plant species. They were particularly frequent on Onobrychis viciifolia , occasional between the two Genista ‐associated populations, and rare on Hippocrepis comosa . PCR‐based screening of bacterial species revealed new associations between aphid biotypes and facultative endosymbionts, chiefly the combination of Serratia symbiotica and Hamiltonella defensa within most individuals collected on Genista . The newly identified biotypes, which add up to a total of 15 within the pea aphid complex, offer new material to study the mechanisms and genetic bases of host‐specialisation and ecological speciation in this model aphid.

The enormous biodiversity of herbivorous insects may arise from ecological speciation via continuous host-plant switches. Whether such switches are successful depends on the trade-off between different selection pressures that act on herbivores. Decreased herbivore performance due to suboptimal nutrition might be compensated for by a reduced natural enemy pressure. As a consequence, an "enemy-free space" on a certain plant might facilitate host-plant switches and maintain biotypes. To test this hypothesis, we used the pea aphid (Acyrthosiphon pisum) complex, which consists of at least 11 genetically distinct host races that are native to specific legume host plants but can all develop on the universal host plant Vicia faba. Three A. pisum host races native to Trifolium pratense, Pisum sativum, and Medicago sativa were investigated in experiments on their respective host plants and on the universal host plant V. faba. We found that hoverflies preferred to oviposit on P. sativum and the universal host V. faba. Since feeding by hoverfly larvae suppressed aphid population growth on these host plants, the native hosts M. sativa and T. pratense provided enemy-free space for the respective A. pisum races. Mobile predators, such as ants and ladybird beetles, preferred Pisum race aphids on V. faba over P. sativum. Thus, all three of the native host plants studied supply enemy-free space for A. pisum compared to the universal host V. faba. Reducing encounters between aphid races on V. faba would reduce gene flow among them and could contribute to maintaining the host races.

A major goal in evolutionary biology is to uncover the genetic basis of adaptation. Divergent selection exerted on ecological traits may result in adaptive population differentiation and reproductive isolation and affect differentially the level of genetic divergence along the genome. Genome-wide scan of large sets of individuals from multiple populations is a powerful approach to identify loci or genomic regions under ecologically divergent selection. Here, we focused on the pea aphid, a species complex of divergent host races, to explore the organization of the genomic divergence associated with host plant adaptation and ecological speciation. We analysed 390 microsatellite markers located at variable distances from predicted genes in replicate samples of sympatric populations of the pea aphid collected on alfalfa, red clover and pea, which correspond to three common host-adapted races reported in this species complex. Using a method that accounts for the hierarchical structure of our data set, we found a set of 11 outlier loci that show higher genetic differentiation between host races than expected under the null hypothesis of neutral evolution. Two of the outliers are close to olfactory receptor genes and three other nearby genes encoding salivary proteins. The remaining outliers are located in regions with genes of unknown functions, or which functions are unlikely to be involved in interactions with the host plant. This study reveals genetic signatures of divergent selection across the genome and provides an inventory of candidate genes responsible for plant specialization in the pea aphid, thereby setting the stage for future functional studies.

Identifying the genomic bases of adaptation to novel environments is a long-term objective in evolutionary biology. Because genetic differentiation is expected to increase between locally adapted populations at the genes targeted by selection, scanning the genome for elevated levels of differentiation is a first step towards deciphering the genomic architecture underlying adaptive divergence. The pea aphid Acyrthosiphon pisum is a model of choice to address this question, as it forms a large complex of plant-specialized races and cryptic species, resulting from recent adaptive radiation. Here, we characterized genomewide polymorphisms in three pea aphid races specialized on alfalfa, clover and pea crops, respectively, which we sequenced in pools (poolseq). Using a model-based approach that explicitly accounts for selection, we identified 392 genomic hotspots of differentiation spanning 47.3Mb and 2,484 genes (respectively, 9.12% of the genome size and 8.10% of its genes). Most of these highly differentiated regions were located on the autosomes, and overall differentiation was weaker on the X chromosome. Within these hotspots, high levels of absolute divergence between races suggest that these regions experienced less gene flow than the rest of the genome, most likely by contributing to reproductive isolation. Moreover, population-specific analyses showed evidence of selection in every host race, depending on the hotspot considered. These hotspots were significantly enriched for candidate gene categories that control host-plant selection and use. These genes encode 48 salivary proteins, 14 gustatory receptors, 10 odorant receptors, five P450 cytochromes and one chemosensory protein, which represent promising candidates for the genetic basis of host-plant specialization and ecological isolation in the pea aphid complex. Altogether, our findings open new research directions towards functional studies, for validating the role of these genes on adaptive phenotypes.

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.

We investigated genetic diversity in West European populations of the fungal pathogen Microbotryum violaceum in sympatric, parapatric and allopatric populations of the host species Silene latifolia and S. dioica, using four polymorphic microsatellite loci. In allopatric host populations, the fungus was highly differentiated by host species, exhibiting high values of F(ST) and R(ST), and revealed clear and distinct host races. In sympatric and parapatric populations we found significant population differentiation as well, except for one sympatric population in which the two host species grew truly intermingled. The mean number of alleles per locus for isolates from each of the host species was significantly higher in sympatric/parapatric than in allopatric populations. This suggests that either gene flow between host races in sympatry, or in case of less neutral loci, selection in a more heterogeneous host environment can increase the level of genetic variation in each of the demes. The observed pattern of host-related genetic differentiation among these geographically spread populations suggest a long-term divergence between these host races. In sympatric host populations, both host races presumably come in secondary contact, and host-specific alleles are exchanged depending on the amount of fungal gene flow.

Host races of phytophagous insects are sympatric populations that have different host preferences and between which gene flow is restricted because of the difference in host preference. Sympatric, host-associated sibling species are sympatric populations that use different host plants and that do not interbreed because of the presence of isolating mechanisms not related to host preference (Jaenike, 1981; see Mayr, 1970, and Bush, 1969, for slightly different definitions of these terms). Although the existence of host races and host-associated sibling species of phytophagous insects has been suspected for over 50 years (e.g., Thorpe, 1930), the evolutionary mechanism of host race formation remains controversial. In particular, there is disagreement about the importance of sympatric divergence in generating host races and species. White (1978) has argued that sympatric speciation must be invoked to explain, in part, the great diversity of specialized herbivorous insects, while Bush (1974, 1975) has forcefully advocated the operation of sympatric divergence in the creation of sympatric host races of tephritid flies. By contrast, Futuyma and Mayer (1980; see also Futuyma, 1983a) have argued that there is no reliable experimental evidence to support these claims and that models of sympatric speciation are based on assumptions that are probably not met by most phytophagous insects. Several authors have developed formal models of sympatric divergence and speciation (Maynard Smith, 1966; Dickinson and Antonovics, 1973; Caisse and Antonovics, 1978; Pimm, 1979; Felsenstein, 1981). Although these models do not account for the evolution of differences in host plant preference associated with purported sympatric speciation in phytophagous insects, Bush and Diehl (1983) and Rausher (1984) have suggested how modification of these models could remedy this problem. A common assumption of all these models is that fitness on one host is negatively correlated with fitness on a second. This type of negative correlation is manifested in these models in the assumption that genetic variation exists at loci that exhibit a genotype x host plant interaction, i.e., at loci at which some genotypes have high fitness on one host but low fitness on a second host while other genotypes have low fitness on the first but high fitness on the second host. This negative correlation seems to be necessary for sympatric speciation because it permits linkage disequilibrium to be established between loci influencing, say, viability on different hosts and loci influencing host preference. The resulting coadapted preference-viability gene complexes represent incipient host races or species. Moreover, the breakdown of these coadapted gene complexes by recombination provides the selection pressure that improves reproductive isolation, whether isolation is achieved via mating on the host (e.g., Bush, 1974, 1975) or by a separate assortative mating locus in linkage disequilibrium with loci affecting preference and fitness (e.g., Felsenstein, 1981). Without a crossing interaction, one homozygote genotype would have maximal fitness on both host species. Consequently, unless reproductive isolation were achieved instantaneously by a mutation

The existence of a continuous array of sympatric biotypes - from polymorphisms, through ecological or host races with increasing reproductive isolation, to good species - can provide strong evidence for a continuous route to sympatric speciation via natural selection. Host races in plant-feeding insects, in particular, have often been used as evidence for the probability of sympatric speciation. Here, we provide verifiable criteria to distinguish host races from other biotypes: in brief, host races are genetically differentiated, sympatric populations of parasites that use different hosts and between which there is appreciable gene flow. We recognize host races as kinds of species that regularly exchange genes with other species at a rate of more than ca. 1% per generation, rather than as fundamentally distinct taxa. Host races provide a convenient, although admittedly somewhat arbitrary intermediate stage along the speciation continuum. They are a heuristic device to aid in evaluating the probability of speciation by natural selection, particularly in sympatry. Speciation is thereby envisaged as having two phases: (i) the evolution of host races from within polymorphic, panmictic populations; and (ii) further reduction of gene flow between host races until the diverging populations can become generally accepted as species. We apply this criterion to 21 putative host race systems. Of these, only three are unambiguously classified as host races, but a further eight are strong candidates that merely lack accurate information on rates of hybridization or gene flow. Thus, over one-half of the cases that we review are probably or certainly host races, under our definition. Our review of the data favours the idea of sympatric speciation via host shift for three major reasons: (i) the evolution of assortative mating as a pleiotropic by-product of adaptation to a new host seems likely, even in cases where mating occurs away from the host; (ii) stable genetic differences in half of the cases attest to the power of natural selection to maintain multilocus polymorphisms with substantial linkage disequilibrium, in spite of probable gene flow; and (iii) this linkage disequilibrium should permit additional host adaptation, leading to further reproductive isolation via pleiotropy, and also provides conditions suitable for adaptive evolution of mate choice (reinforcement) to cause still further reductions in gene flow. Current data are too sparse to rule out a cryptic discontinuity in the apparently stable sympatric route from host-associated polymorphism to host-associated species, but such a hiatus seems unlikely on present evidence. Finally, we discuss applications of an understanding of host races in conservation and in managing adaptation by pests to control strategies, including those involving biological control or transgenic parasite-resistant plants.

ABSTRACTThe evolution of host‐associated sympatric populations in phytophagous insects (so called “host races”) connects adaptive divergence to barriers to gene flow. Pea aphid (Acyrthosiphon pisum) host races specialise on legume species, and because host plant choice leads to assortative mating, the genetic basis of host plant acceptance is key to understanding speciation. Aphids use smell and taste in their host plant selection. While chemosensory genes frequently emerge as “outliers” in genome scans, their link to plant acceptance behaviour remains unclear. We examined the genetic basis of host‐associated phenotypes using an F2 cross between two pea aphid host‐associated races (specialised on alfalfa—Medicago sativa‐ and pea—Pisum sativum), assaying behaviour on both host plants and conducting QTL and regional heritability analyses based on a high‐resolution linkage map. We identified five regions of moderate effect associated with acceptance of alfalfa, two with pea acceptance and two with survival on alfalfa. Two QTLs, one for alfalfa and one for pea acceptance, are located within a large rearranged region on chromosome 1, while other QTLs linked to alfalfa acceptance and survival are in the same region on chromosome 3—linking host plant choice to fitness. These findings highlight the polygenic basis of acceptance behaviour and the role of gene clustering and chromosomal rearrangements in promoting coupling among barrier loci. We identified 60 chemosensory genes within regions connected to acceptance, 24 of which were divergent among pea aphid races in previous genome scan or gene expression analyses. Evidence linking these genes to acceptance phenotypes supports their role in determining host plant specificity and as barrier loci contributing to pea aphid speciation.

Host‐associated races of phytophagous insects provide a model for understanding how adaptation to a new environment can lead to reproductive isolation and speciation, ultimately enabling us to connect barriers to gene flow to adaptive causes of divergence. The pea aphid (Acyrthosiphon pisum) comprises host races specializing on legume species and provides a unique system for examining the early stages of diversification along a gradient of genetic and associated adaptive divergence. As host choice produces assortative mating, understanding the underlying mechanisms of choice will contribute directly to understanding of speciation. As host choice in the pea aphid is likely mediated by smell and taste, we use capture sequencing and SNP genotyping to test for the role of chemosensory genes in the divergence between eight host plant species across the continuum of differentiation and sampled at multiple locations across western Europe. We show high differentiation of chemosensory loci relative to control loci in a broad set of pea aphid races and localities, using a model‐free approach based on principal component analysis. Olfactory and gustatory receptors form the majority of highly differentiated genes and include loci that were already identified as outliers in a previous study focusing on the three most closely related host races. Consistent indications that chemosensory genes may be good candidates for local adaptation and barriers to gene flow in the pea aphid open the way to further investigations aiming to understand their impact on gene flow and to determine their precise functions in response to host plant metabolites.

Interspecific interaction with host plants have important consequences for the host race formation of herbivorous insects. Plant secondary metabolites, particularly those that are involved in host races specializing on plants, warrant the theory of host specialization. Acyrthosiphon pisum comprises various host races that adapt to different Fabaceae plants, which provides an ideal system for determining the behavioral and physiological mechanisms underlying host-adaptive diversification. The current study evaluated the effects of host transfer on population fitness, feeding behavior and the transcriptome-wide gene expression of the two host races of A. pisum, one of which was originally from Medicago sativa and the other from Pisum sativum. The results showed that the Pisum host race of A. pisum had a lower population abundance and feeding efficiency than the Medicago host race in terms of a longer penetration time and shorter duration times of phloem ingestion when fed on M. sativa. In contrast, few differences were found in the population abundance and feeding behavior of A. pisum between the two host races when fed on P. sativum. Meanwhile, of the nine candidate phenolic compounds, only genistein was significantly affected by aphid infestation; higher levels of genistein were detected in M. sativa after feeding by the Pisum host race, but these levels were reduced relative to uninfested controls after feeding by the Medicago host race, which suggested that genistein may be involved in the specialization of the aphid host race on M. sativa. Further exogenous application of genistein in artificial diets showed that the increase in genistein reduced the survival rate of the Pisum host race but had little effect on that of the Medicago host race. The transcriptomic profiles indicated that the transcripts of six genes with functions related to detoxification were up-regulated in the Pisum host race relative to the Medicago host race of A. pisum. These results suggested that the inducible plant phenolics and associated metabolic process in aphids resulted in their differential adaptations to their Fabaceae host.

Field assessments were conducted to examine the interplay between host plant and predation in complex agricultural mosaic on pea aphid clover and alfalfa races. In one experiment, we examined the relative fitness on clover race (CR) and alfalfa race (AR) pea aphids on broad bean, red clover and alfalfa alone. But because clover is typically grown in a more complex agricultural mosaic with alfalfa and broad bean, a second experiment was conducted to assess the fitness consequences under predation in a more complex agricultural field setting that also included potential apparent competition with AR pea aphids. In a third experiment we tested for the effect of differential host race density on the fitness of the other host race mediated by a predator effect. CR pea aphids always had fitness losses when on broad bean (had lower fitness on broad bean relative to red clover) and fitness benefits when on red clover (higher fitness on red clover relative to broad bean), whether or not in apparent competition with alfalfa race aphids on bean and alfalfa. AR suffered fitness loss on both alfalfa and bean in apparent competition with CR on clover. Therefore we can conclude that the predation rate between host races was highly asymmetrical. The complexity of the agricultural mosaic thus can influence prey selection by predators on different host plants. These may have evolutionary consequences through context dependent fitness benefits on particular host plants.

1. Sympatric populations of insects adapted to different host plants are good model systems not only to study how they adapt to the chemistry of their food plant, but also to investigate whether morphological modifications evolved enabling them to live successfully on a certain plant species. 2. The pea aphid, Acyrthosiphon pisum ( H arris) encompasses at least 11 genetically distinct sympatric host races, each showing a preference for a certain legume species. The leaflet surfaces of these legumes differ considerably in their wax coverage. 3. It was investigated whether the attachment structures of three pea aphid genotypes from different host races are adapted to the different surface properties of their host plants and whether they show differences in their attachment ability on the respective host and non‐host plants. 4. The surface morphology of plants and aphid tarsi was examined using SEM (scanning electron microscopy). The ability of the aphids to walk on specific surfaces was tested using traction force measurements. 5. The presence of wax blooms on the leaflets lowers the aphids' attachment ability considerably and diminishes their subsequent attachment on ‘neutral’ surfaces like glass. The pea aphid host races differ in their ability to walk on certain surfaces. However, the genotype from the adapted aphid host race was not necessarily the one with the best walking performance on their host plant. All aphids, regardless of the original host plant, were most efficient on the neutral control surface glass. The general host plant Vicia faba was the plant with the most favourable surface for all aphid host races.

Generalist parasites regularly evolve host-specific races that each specialize on one particular host species. Many host-specific races originate from geographically structured populations where local adaptations to different host species drive the differentiation of distinct races. However, in sympatric populations where several host races coexist, gene flow could potentially disrupt such host-specific adaptations. Here, we analyse genetic differentiation among three sympatrically breeding host races of the brood-parasitic common cuckoo, Cuculus canorus. In this species, host-specific adaptations are assumed to be controlled by females only, possibly via the female-specific W-chromosome, thereby avoiding that gene flow via males disrupts local adaptations. Although males were more likely to have offspring in two different host species (43% versus 7%), they did not have significantly more descendants being raised outside their putative foster species than females (9% versus 2%). We found significant genetic differentiation for both biparentally inherited microsatellite DNA markers and maternally inherited mitochondrial DNA markers. To our knowledge, this is the first study that finds significant genetic differentiation in biparentally inherited markers among cuckoo host-specific races. Our results imply that males also may contribute to the evolution and maintenance of the different races, and hence that the genes responsible for egg phenotype may be found on autosomal chromosomes rather than the female-specific W-chromosome as previously assumed.