During the last decade, interest in speciation has been renewed as a major topic of research [1], [2]. Bean beetles, Callosobruchus maculatus, are herbivorous pest insects that are found in Africa and Asia. In this study, maternal effects could potentially influence preference and performance on the two hosts, but our results suggest that this is not the case. It would be more likely that mate choice is based on some other, nonvisual cue, such as smell for example. In all of these cases, the strains selected on different hosts showed signs of reproductive isolation. Replicates 1–9 were run on BE beans, and replicates 10–18 on mung beans. This requires linkage disequilibrium to build up between loci determining resource use and loci determining mating preference. <> We terminated the experiment after eight generations, and seven lines showed a significant excess of heterozygotes compared to Hardy-Weinberg expectations at that point (mean I  = −0.20±0.052; Figure 2e). Eggs are layed singly, and hatch into larvae (maggots) several days later. In a large population with no selection, random mating, no mutations and no gene flow, genotype frequencies can be calculated from gene frequencies [18]. Host preference and host performance was then compared between females of different colours. The mixture contained the resource that the particular morph type had been reared on during the selection process, and a newly introduced resource. Thus, our novel result is that a difference in fitness on the two alternative host plants is not necessary to induce assortative mating, provided that mating at the time of selection takes place on the food resource. After egg-laying, we separated the two resources and counted the eggs laid on each resource. As a measure of assortative mating, we used the heterozygote deficiency index, I  = 1- Hetobs/Hetexp [1], which is zero in the case of no assortative mating, and one if there is total reproductive isolation. In our study, we selected the lines in a way to create a close linkage between colour and host preference, and thus mate choice. Metrics details. Low effective population size could also lead to inbreeding and subsequent loss of fecundity and hatching success. A number of conditions need to be met for speciation via host-shift to occur, such as evolution of assortative mating and selection against hybrids, but if these are fulfilled, speciation can be rapid. In a more general sense, this implies that only a fraction of the host shifts will eventually result in a genetic differentiation between populations. [27]. The bruchid beetle Callosobruchus maculatus is known to be fairly plastic when it comes to host use. This suggests that partial reproductive isolation can arise as a consequence of adaptation/habituation to different types of food resources. We did this for all lines on each of the two resources. The species is a cosmopolitan pest of stored legumes (Fabaceae), particularly beans of the genus Vigna [16]. 20 virgin females and males from each morph type were selected randomly, resulting in 80 individuals on 80 grams of beans (40 g of M beans and 40 g of BE beans). This may represent a valid explanation for rapid breakdown of the partial reproductive isolation obtained in the two first generations and the return to random mating between the two distinct morphs. However, fecundity and hatching success were at the level found in the control lines, and hence no obvious inbreeding effects were observed. Populations were mixed in a pair wise fashion, such that line1 on M beans were mixed with line 1 on BE beans and so on. No major differences were found between the beetle strains. After ten generations of selection on the two different host beans BE and M, we introduced individuals from the BE lines to the alternative resource, M beans, for one generation. The larva then burrows into the bean and will form a pupa 21–30 days after the egg was deposited.


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