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Previous work by Demuth and Wade (2007a,b) discovered widespread adult hybrid incompatibility among populations of the red flour beetle, T. castaneum: 3% to 100% of adult interpopulation hybrids exhibit negative phenotypes such as deformities of the limbs, antennae, mouthparts, and wings. Quantitative genetic analyses revealed that these had a complex genetic architecture with maternal genetic effects and Genotype-by-Environment interactions. Some interpopulation crosses exhibited Haldane's Rule (Demuth and Wade 2007b), wherein the heterogametic male hybrids exhibited more frequent deformities than their homogametic hybrid sisters. The manifestation of this classic interspecific pattern in the interpopulation hybrids suggests that its origin lies early in the speciation process. In the Bateson-Dobzhansky-Muller (BDM) speciation model, genes with a positive fitness effect in the sympatric background but a negative effect in a hybrid background cause reproductive incompatibilities. Thus, the variation observed within the T. castaneum species for interpopulation compatibility represents the “polymorphic prelude to BDM incompatibilities” (Cutter 2012), affording an opportunity to dissect the genetic causes of incompatibility early in the speciation process. These data also support the view that hybrid incompatibility is an indirect by-product of adaptation to local environments, with some population pairs more divergent, and therefore more incompatible, than others.
Drury et al. (2011) mapped an extreme interpopulation incompatibility, the complete absence of F1 adults, to a region in linkage disequilibrium with the T. castaneum ecdysone receptor (EcR) homolog. EcR is a major regulatory switch, with multiple alternative splice forms; it is known to control larval molts, time of pupation, and eclosion (Li and Bender 2000). Although such extreme incompatibilities, with their alternative phenotypes, are easier to map, they represent the final stage in an underlying continuous process when hybrid inviability evolves as an indirect by-product of adaptation to local environments (Rice 1987). However, other population pairs manifesting partial incompatibility have received much less genetic attention.
Larval development and the transition from pupae to adult is a complex trait, especially in the holometabolous insects. It has long been speculated that small differences in developmental timing, known as heterochrony, might play an important role in speciation (McMillan et al. 1992; Mabee et al. 2000). The central idea is that a small difference in the timing of critical events during development may lead to larger, corresponding changes in adult morphology and thus contribute to rapid speciation (e.g., Alberch et al. 1979; Alberch 1980; Kamiya 1992). Our findings with regard to speciation and heterochrony are somewhat different. The adult and larval morphologies in our populations of T. castaneum are indistinguishable. However, the underlying genetic mechanisms for the timing developmental events during maturation and their sensitivity to density and temperature have become sufficiently different among populations to cause partial or complete developmental failure of the immatures.
Interactions between the maternal and zygotic genomes are critical to the control of events in early development and are known to diversify rapidly in fruit flies (Cruickshank and Wade 2008). Demuth and Wade (2007a,b) in quantitative genetic studies of adult hybrid deformities in T. castaneum found evidence of an important maternal genetic component. Here, we have extended those studies to the timing of hybrid larval development. Surprisingly, populations with similar patterns of larval development may produce larval hybrids whose development is different from that of either parent. This pattern suggests that local selection affects larval development and that small differences in timing can become the basis for reproductive isolation.
In contrast to studies of interpopulation adult hybrids (Demuth and Wade 2007a,b; Drury and Wade 2011; Drury et al. 2011), the earlier developmental stages, larvae and pupae, have not been characterized in interpopulation crosses. Because small errors early in development can be compounded during ontogeny (Arthur 2004), it is likely that the majority of hybrid adult developmental deficiencies result from earlier developmental deficiencies of the immatures. We investigated hybrid larvae from a factorial combination of crosses between four populations to determine the frequency of larval developmental deficiencies and their relationship to adult hybrid deformities. Earlier detection and better classification of hybrid abnormalities, in all developmental stages, can speed the characterization, differentiation, and mapping of nascent incompatibilities.
We made all possible reciprocal crosses among four populations from India, South America, Canada, and Georgia (U.S.A.). Eggs from each cross were reared at a controlled, constant density, as well as at a natural density achieved by 24 hours of egg laying. Eggs at each density were also raised at two temperatures, 26 and 34°C, the rearing temperatures of the hybrid adults characterized by Demuth and Wade (2007a,b). At 4-day intervals, we censused each set of developing eggs or larvae, photographed them through a stereomicroscope, and measured larval length from the stored digital images. We used these measurements to estimate a series of developmental parameters, including growth rates, asymptotic larval size, and size at adulthood as functions of temperature and life stage (egg, larval instar, pupa, adult). We also report larval to adult viabilities. We compared the developmental trajectories from egg to adult of interpopulation hybrids with those of the pure-bred parent populations to determine the extent of hybrid developmental dysfunction and its stage specificity.
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We find that hybrid incompatibility manifests early in development as changes in the duration of larval instars and as diminished success in the transition between instars relative to the pure-bred parent populations. Parent populations with similar developmental profiles may nevertheless produce hybrids with disrupted development. The degree and the timing of expression of the larval hybrid inviability are conditional upon the populations crossed, the direction of the cross, and the environment in which the hybrids are raised. Our findings suggest that local adaptation tends to affect the timing of events in early development, which, in turn, plays a significant role in generating hybrid incompatibilities between populations.
We find that the variation among crosses and treatments for growth parameters is not correlated with eventual incompatibility. Crosses whose hybrids prove inviable at the pupal or adult stage tend to have larval growth rates, egg-to-larval viabilities, and asymptotic sizes equal to or exceeding those of the parent populations. The degree of pupal or adult inviability is not correlated with the parameters of larval development. Not including the combinations previously shown to be larval inviable, the larval portion of the beetle's lifespan is unaffected by the novel genomic environment produced by hybridization. While hybrids in a portion of crosses never reach reproductive maturity, we find that the inviabilities are clustered at discrete time points, particularly, at the transitions between larval stages or between the pupal and adult stage. This suggests that the coordinated expression of genes involved in these transitional periods of development is the underlying cause of hybrid incompatibility in this species.
As shown previously (Drury et al. 2011), Indian and Colombian hybrid offspring cease development at the third larval instar, but do not die. They live as third instar larvae for a period as long as the normal adult lifespan. Interestingly, between hatching and the onset of the third instar, they appear to develop normally; when reared at low density, they have the 11th highest k and, reared at low density, the 22nd highest k (out of the total 28 crosses reared at 34°C). This suggests that discrete stage-dependent developmental errors are being caused by differences between populations in a developmental switch or timing mechanism.
A phenotypically distinct hybrid incompatibility occurs in crosses between the Canadian and Indian populations. Here, larval development proceeds normally to pupation before the hybrid inviability is manifested; the nature of the inviability depends up on the direction of the hybrid cross and the environment in which the hybrids develop. When Canadian sires are mated to Indian dams and the resulting hybrids are reared at 26°C, there is almost complete failure to complete pupation. In contrast, when reared at 34°C, the hybrid offspring progress successfully through the pupal stage, to become free-living adults, all of which have malformed elytra. The reciprocal cross produces the same hybrid phenotypes but they differ in abundance. For the Indian sire-by-Colombian dam offspring reared at 26°C, 60% fail to eclose from pupation; while the other 40% successfully eclose to adults, but incur the elytral deformity. When raised at the higher temperature, the degree of incompatibility diminishes further: 40% of the hybrid adults have malformed elytra, while the other 60% are normal adults. Thus, higher temperature partially rescues hybrid inviability. Those beetles surviving to adulthood, with or without deformed elytra, are fertile and lay viable eggs.
Overall, we find that interpopulation hybrids manifest a variety of hybrid incompatibilities, which vary in degree from partial to complete reciprocal inviability. Furthermore, the expression of hybrid inviability occurs at different but highly specific developmental transitions, depending upon the populations crossed, the direction of the cross, and the temperature at which the hybrid larvae are reared. The among-population variation in hybrid inviability and the population variation in its sensitivity to temperature reveal that pairs of populations can be incompatible in different ways. This suggests that local adaptation affects developmental timing with the result that the developmental incompatibilities between populations have different underlying genetic causes. Although all the developmental differences among hybrids affect the successful progression from one developmental stage to the next, the timing varies with cross, cross direction, and temperature. Until more incompatibilities are mapped to the gene level (e.g., Drury and Wade 2011), it is not clear whether populations are beginning to differentiate in a small number of regulatory genes controlling an iterated set of molts or in a much larger number of genes in a complex regulatory network turned off and on at each larval molt.