Disparate genetic variants associated with distinct components of cowpea resistance to the seed beetle Callosobruchus maculatus.

KEY MESSAGE: Polygenic genome-wide association mapping identified two regions of the cowpea genome associated with different components of resistance to its major post-harvest pest, the seed beetle Callosobruchus maculatus. Cowpea (Vigna unguiculata) is an important grain and fodder crop in arid and semi-arid regions of Africa, Asia, and South America, where the cowpea seed beetle, Callosobruchus maculatus, is a serious post-harvest pest. Development of cultivars resistant to C. maculatus population growth in storage could increase grain yield and quality and reduce reliance on insecticides. Here, we use a MAGIC (multi-parent, advanced-generation intercross) population of cowpea consisting of 305 recombinant inbred lines (RILs) to identify genetic variants associated with resistance to seed beetles. Because inferences regarding the genetic basis of resistance may depend on the source of the pest or the assay protocol, we used two divergent geographic populations of C. maculatus and two complementary assays to measure several aspects of resistance. Using polygenic genome-wide association mapping models, we found that the cowpea RILs harbor substantial additive-genetic variation for most resistance measures. Variation in several components of resistance, including larval development time and survival, was largely explained by one or several linked loci on chromosome 5. A second region on chromosome 8 explained increased seed resistance via the induction of early-exiting larvae. Neither of these regions contained genes previously associated with resistance to insects that infest grain legumes. We found some evidence of gene-gene interactions affecting resistance, but epistasis did not contribute substantially to resistance variation in this mapping population. The combination of mostly high heritabilities and a relatively consistent and simple genetic architecture increases the feasibility of breeding for enhanced resistance to C. maculatus.

Measuring the effect of environmental stress on inbreeding depression alone obscures the relative importance of inbreeding-stress interactions on overall fitness in Callosobruchus maculatus.

Environmental stress can have a profound effect on inbreeding depression. Quantifying this effect is of particular importance in threatened populations, which are often simultaneously subject to both inbreeding and environmental stress. But while the prevalence of inbreeding-stress interactions is well known, the importance and broader applicability of such interactions in conservation are not clearly understood. We used seed beetles, Callosobruchus maculatus, as a model system to quantify how environmental stressors (here host quality and temperature stress) interact with inbreeding as measured by changes in the magnitude of inbreeding depression, δ, as well as the relative importance of inbreeding-stress interactions to overall fitness. We found that while both environmental stressors caused substantial inbreeding-stress interactions as measured by change in δ, the relative importance of these interactions to overall survival was modest. This suggests that assessing inbreeding-stress interactions within the framework of δ alone may give an inaccurate representation of the relevance of interactions to population persistence. Furthermore, we found that the effect of environmental stress on fitness, but not inbreeding depression, varied strongly among populations. These results suggest that the outcomes of inbreeding-stress interactions are not easily generalized, an important consideration in conservation settings.

Colonization of Marginal Host Plants by Seed Beetles (Coleoptera: Chrysomelidae): Effects of Geographic Source and Genetic Admixture.

The ability to adapt to a novel host plant may vary among insect populations with different genetic histories, and colonization of a marginal host may be facilitated by genetic admixture of disparate populations. We assembled populations of the seed beetle, Callosobruchus maculatus (F.), from four continents, and compared their ability to infest two hosts, lentil and pea. We also formed two cross-continent hybrids (Africa × N.A. and Africa × S.A.). In pre-selection assays, survival was only ~3% in lentil and ~40% in pea. For three replicate populations per line, colonization success on lentil was measured as cumulative exit holes after 75-175 d. On pea, we estimated the change in larval survival after five generations of selection. Females in all lines laid few eggs on lentil, and survival of F1 larvae was uniformly <5%. Subsequently, however, the lines diverged considerably in population growth. Performance on lentil was highest in the Africa × N.A. hybrid, which produced far more adults (mean > 11,000) than either parental line. At the other extreme, Asian populations on lentil appeared to have gone extinct. The Africa × N.A. line also exhibited the highest survival on pea, and again performed better than either parent line. However, no line displayed a rapid increase in survival on pea, as is sometimes observed on lentil. Our results demonstrate that geographic populations can vary substantially in their responses to the same novel resource. In addition, genetic admixtures (potentially caused by long-distance transport of infested seeds) may facilitate colonization of an initially poor host.

Combining Experimental Evolution and Genomics to Understand How Seed Beetles Adapt to a Marginal Host Plant.

Genes that affect adaptive traits have been identified, but our knowledge of the genetic basis of adaptation in a more general sense (across multiple traits) remains limited. We combined population-genomic analyses of evolve-and-resequence experiments, genome-wide association mapping of performance traits, and analyses of gene expression to fill this knowledge gap and shed light on the genomics of adaptation to a marginal host (lentil) by the seed beetle Callosobruchus maculatus. Using population-genomic approaches, we detected modest parallelism in allele frequency change across replicate lines during adaptation to lentil. Mapping populations derived from each lentil-adapted line revealed a polygenic basis for two host-specific performance traits (weight and development time), which had low to modest heritabilities. We found less evidence of parallelism in genotype-phenotype associations across these lines than in allele frequency changes during the experiments. Differential gene expression caused by differences in recent evolutionary history exceeded that caused by immediate rearing host. Together, the three genomic datasets suggest that genes affecting traits other than weight and development time are likely to be the main causes of parallel evolution and that detoxification genes (especially cytochrome P450s and beta-glucosidase) could be especially important for colonization of lentil by C. maculatus.

Components of Cowpea Resistance to the Seed Beetle Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae).

Cowpea, Vigna unguiculata (L.) Walp., serves as a major source of dietary protein in many tropical and subtropical regions around the world. To identify loci associated with agronomically desirable traits, eight elite cowpea cultivars were systematically inter-crossed for eight generations to yield 305 recombinant inbred lines. Here, we investigated whether these founder parents also possess resistance to the seed beetle Callosobruchus maculatus (F.), a highly destructive post-harvest pest. We estimated larval survival in seeds, egg-to-adult development time, adult mass at emergence, and seed acceptance for oviposition. Survival varied significantly among cowpea cultivars, but the pattern was complicated by an unexpected source of mortality; on three cultivars, mature larvae in a substantial fraction of seeds (20-36%) exited seeds prematurely, and consequently failed to molt into viable adults. Even if such seeds were eliminated from the analysis, survival in the remaining seeds varied from 49 to 92% across the eight parents. Development time and body mass also differed among hosts, with particularly slow larval development on three closely related cultivars. Egg-laying females readily accepted all cultivars except one with a moderately rugose seed coat. Overall, suitability ranks of the eight cultivars depended on beetle trait; a cultivar that received the most eggs (IT82E-18) also conferred low survival. However, one cultivar (IT93K-503-1) was a relatively poor host for all traits. Given the magnitude of variation among parental cultivars, future assays of genotyped recombinant progeny can identify genomic regions and candidate genes associated with resistance to seed beetles.

Dynamics of genomic change during evolutionary rescue in the seed beetle Callosobruchus maculatus.

Rapid adaptation can prevent extinction when populations are exposed to extremely marginal or stressful environments. Factors that affect the likelihood of evolutionary rescue from extinction have been identified, but much less is known about the evolutionary dynamics (e.g., rates and patterns of allele frequency change) and genomic basis of successful rescue, particularly in multicellular organisms. We conducted an evolve-and-resequence experiment to investigate the dynamics of evolutionary rescue at the genetic level in the cowpea seed beetle, Callosobruchus maculatus, when it is experimentally shifted to a stressful host plant, lentil. Low survival (~1%) at the onset of the experiment caused population decline. But adaptive evolution quickly rescued the population, with survival rates climbing to 69% by the F5 generation and 90% by the F10 generation. Population genomic data showed that rescue likely was caused by rapid evolutionary change at multiple loci, with many alleles fixing or nearly fixing within five generations of selection on lentil. Selection on these loci was only moderately consistent in time, but parallel evolutionary changes were evident in sublines formed after the lentil line had passed through a bottleneck. By comparing estimates of selection and genomic change on lentil across five independent C. maculatus lines (the new lentil-adapted line, three long-established lines and one case of failed evolutionary rescue), we found that adaptation on lentil occurred via somewhat idiosyncratic evolutionary changes. Overall, our results suggest that evolutionary rescue in this system can be caused by very strong selection on multiple loci driving rapid and pronounced genomic change.

Variable Responses to Novel Hosts by Populations of the Seed Beetle Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae).

Cosmopolitan pests can consist of geographic populations that differ in their current host ranges or in their ability to colonize a novel host. We compared the responses of cowpea-adapted, seed-beetle populations (Callosobruchus maculatus [F.] (Coleoptera: Chrysomelidae: Bruchinae)) from Africa, North America, and South America to four novel legumes: chickpea, lentil, mung bean, and pea. We also qualitatively compared these results to those obtained earlier for an Asian population. For each host, we measured larval survival to adult emergence and used both no-choice and choice tests to estimate host acceptance. The pattern of larval survival was similar among populations: high or moderately high survival on cowpea, mung bean, and chickpea, intermediate survival on pea, and very low survival on lentil. One exception was unusually high survival of African larvae on pea, and there was modest variation among populations for survival on lentil. The African population was also an outlier with respect to host acceptance; under no-choice conditions, African females showed a much greater propensity to accept the two least preferred hosts, chickpea and lentil. However, greater acceptance of these hosts by African females was not evident in choice tests. Inferences about population differences in host acceptance can thus strongly depend on experimental protocol. Future selection experiments can be used to determine whether the observed population differences in initial performance will affect the probability of producing self-sustaining populations on a marginal crop host.

Genomic evidence that resource-based trade-offs limit host-range expansion in a seed beetle.

Trade-offs have often been invoked to explain the evolution of ecological specialization. Phytophagous insects have been especially well studied, but there has been little evidence that resource-based trade-offs contribute to the evolution of host specialization in this group. Here, we combine experimental evolution and partial genome resequencing of replicate seed beetle selection lines to test the trade-off hypothesis and measure the repeatability of evolution. Bayesian estimates of selection coefficients suggest that rapid adaptation to a poor host (lentil) was mediated by standing genetic variation at multiple genetic loci and involved many of the same variants in replicate lines. Sublines that were then switched back to the ancestral host (mung bean) showed a more gradual and variable (less repeatable) loss of adaptation to lentil. We were able to obtain estimates of variance effective population sizes from genome-wide differences in allele frequencies within and between lines. These estimates were relatively large, which suggests that the contribution of genetic drift to the loss of adaptation following reversion was small. Instead, we find that some alleles that were favored on lentil were selected against during reversion on mung bean, consistent with the genetic trade-off hypothesis.

Adaptation to a novel host by a seed beetle (Coleoptera: Chrysomelidae: Bruchinae): effect of source population.

Geographic populations of a widespread species can differ in their ability to adapt to a novel environment because they possess different amounts of the requisite genetic variation. We compared responses to the same novel host in ecologically and genetically divergent populations of the seed beetle Callosobruchus maculatus (F.). Populations from Africa and Asia had been derived from and maintained on different legume hosts. In preselection assays, both populations exhibited lower survival, slower development, and smaller size on a third host (adzuki bean), and the difference in performance between the ancestral and novel hosts was especially high for the African population. Replicate lines of each population were switched to adzuki bean or maintained on the ancestral host, and beetle performance was measured on both hosts after 12 generations. Survival on adzuki bean increased substantially in the adzuki-bean lines of the African population, but improved only slightly in the Asian lines. Similarly, only the African adzuki-bean lines exhibited significantly faster development on adzuki bean. Improved performance on adzuki bean did not simultaneously reduce performance on the ancestral host. Together with previous studies, these results confirm that populations of C. maculatus often possess sufficient standing genetic variation for rapid adaptation to a novel host, but the magnitude of the response may depend on the source population. Although international trade in grain legumes can expand beetle host ranges and produce unusual biotypes, the consistent absence of strong genetic trade-offs in larval performance or adult oviposition across hosts makes it unlikely that this insect would form distinct host races.

Genetic architecture underlying convergent evolution of egg-laying behavior in a seed-feeding beetle.

Independent populations subjected to similar environments often exhibit convergent evolution. An unresolved question is the frequency with which such convergence reflects parallel genetic mechanisms. We examined the convergent evolution of egg-laying behavior in the seed-feeding beetle Callosobruchus maculatus. Females avoid ovipositing on seeds bearing conspecific eggs, but the degree of host discrimination varies among geographic populations. In a previous experiment, replicate lines switched from a small host to a large one evolved reduced discrimination after 40 generations. We used line crosses to determine the genetic architecture underlying this rapid response. The most parsimonious genetic models included dominance and/or epistasis for all crosses. The genetic architecture underlying reduced discrimination in two lines was not significantly different from the architecture underlying differences between geographic populations, but the architecture underlying the divergence of a third line differed from all others. We conclude that convergence of this complex trait may in some cases involve parallel genetic mechanisms.