Differences in traits across seasons are known to occur in various organisms [21][22]. This phenomenon is suggested to be evidence supporting rapid evolution. However, comparing phenotypes in each season alone is not sufficient to prove rapid evolution among seasons. The effects of plasticity and the season-specific migration of populations with different genotypes can also explain the observed seasonal changes. One of the most important steps to detect rapid evolution in a natural population is improving the method for measuring phenotypic values in each season. The present study first demonstrated that the FST values between the spring and autumn periods of D. lutescens were very low; the two periods exhibited little genetic differentiation, and therefore, season-specific migration is unlikely to occur in the population that we observed. Nevertheless, we detected differences in heat tolerance and the wing to thorax ratio between the two collection time periods, while we did not find significant evidence for seasonal variation in cold tolerance and body size. Removing environmental effects, which are potential factors affecting phenotypic values, allows for strong confirmation of the presence of genetic changes across seasons. Thus, our results provide strong evidence indicating that seasonal environmental heterogeneity induces rapid evolution at a phenotypic level. Note that flies collected in the spring period were kept in our laboratory for a much longer time than flies collected in the autumn period. At this time, unfortunately, we cannot reject the possibility that a difference in breeding length in the laboratory affects the phenotypic values through inbreeding depression, stochastic drift, or adaptation to laboratory conditions [23]. To mitigate these effects, we should rear our lines for a longer time before phenotyping or reverse the order of seasonal sampling.
A higher heat tolerance after summer (i.e., autumn period) suggests that selection associated with seasonal climate drives the adaptive evolutionary response. The prevalence of individuals with higher heat tolerance might increase in the population during summer and decrease during winter. Many previous studies have focused on families of heat shock proteins (Hsps) as some of the most important genetic candidates for high-temperature responses, although Hsps also have a variety of deleterious consequences, such as low fecundity and retarded development [24][25]. In addition, other genes may contribute to the variation in the thermal response, including the Turandot [26] and Methuselah genes [27]. Further research including more genomic analysis is required to verify candidate genes contributing to the seasonal evolutionary response to heat tolerance.
Insects often show latitudinal variation in flight morphology, such as wing size and the ratio of wing size to body size, which directly affects foraging, mating, dispersal, and thus reproductive success. Along the environmental gradient on a continental scale, the wing size relative to body size of Drosophila spp. is known to be larger in cold regions than in warm regions. Larger wings are advantageous in the cold because ectotherms generate less energy per wingbeat [28]. However, the opposite pattern was observed for the wing-to-thorax ratio in the context of seasonal environmental changes; a larger ratio of wing-to-thorax length was observed in flies from the autumn period, which had just experienced summer. Such an opposite pattern could be explained by seasonal variation in population density. Previous theoretical studies demonstrated that a dispersal strategy could evolve in a density-dependent manner [29]. The wing to thorax ratio could reflect the ability to access resources; that is, a larger wing to thorax ratio could increase dispersal ability [30]. Since the population density of Drosophila spp. could be higher during a warm season than during a cold season, genotypes expressing a higher dispersal ability (i.e., larger wing to thorax ratio) may be favoured during summer, when densities and competition increase.