The impact of different reproductive barriers on species or population isolation may vary in different stages of speciation depending on evolutionary forces acting within species and through species’ interactions. Genetic incompatibilities between interacting species are expected to reinforce prezygotic barriers in sympatric populations and lead to cascade reinforcement between conspecific populations living within and outside the areas of sympatry. We tested these predictions and studied whether and how the strength and target of reinforcement between Drosophila montana and Drosophila flavomontana vary between sympatric populations with different histories and species abundances. All barriers between D. montana females and D. flavomontana males were nearly complete, while in the reciprocal cross strong postzygotic isolation was accompanied by prezygotic barriers whose strength varied according to population composition. Sexual isolation between D. flavomontana females and D. montana males was increased in long‐established sympatric populations, where D. flavomontana is abundant, while postmating prezygotic (PMPZ) barriers were stronger in populations where this species is a new invader and still rare and where female discrimination against heterospecific males was lower. Strengthening of sexual and PMPZ barriers in this cross also induced cascade reinforcement of respective barriers between D. flavomontana populations, which is a classic signature of reinforcement process.

The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are interrelated is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity.

Reproductive diapause is a primary mechanism used by arthropods to synchronize their life cycle with seasonal changes in temperate regions. Our study species, Drosophila montana, represents the northern insect species where flies enter reproductive diapause under short day conditions and where the precise timing of diapause is crucial for both survival and offspring production. We have studied clinal variation in the critical day length for female diapause induction (CDL) and their overall susceptibility to enter diapause (diapause incidence), as well as the temperature sensitivity of these traits. The study was performed using multiple strains from four latitudinal clines of the species -short clines in Finland and Alaska and long clines in the Rocky Mountains and the western coast of North America -and from one population in Kamchatka, Russia. CDL showed strong latitudinal clines on both continents, decreasing by one hour per five degrees decline in latitude, on average. CDL also decreased in all populations along with an increase in fly rearing temperature postponing the diapause to later calendar time, the effects of temperature being stronger in southern than in northern population. Female diapause incidence was close to 100% under short day/low temperature conditions in all populations, but decreased below 50% even under short days in 19°C in the southern North American western coast populations and in 22°C in most populations. Comparing a diversity of climatic data for the studied populations showed that while CDL is under a tight photoperiodic regulation linked with latitude, its length depends also on climatic factors determining the growing season length. Overall, the study deepens our understanding of how spatial and environmental parameters affect the seasonal timing of an important biological event, reproductive diapause and helps to estimate the evolutionary potential of insect populations to survive in changing climatic conditions.

Current advances in genetic analysis are opening up our knowledge of the genetics of species differences, but challenges remain, particularly for out‐bred natural populations. We constructed a microsatellite‐based linkage map for two out‐bred lines of Drosophila montana derived from divergent populations by taking advantage of the Drosophila virilis genome and available cytological maps of both species. Although the placement of markers was quite consistent with cytological predictions, the map indicated large heterogeneity in recombination rates along chromosomes. We also performed a quantitative trait locus (QTL) analysis on a courtship song character (carrier frequency), which differs between populations and is subject to strong sexual selection. Linkage mapping yielded two significant QTLs, which explained 3% and 14% of the variation in carrier frequency, respectively. Interestingly, as in other recent studies of traits which can influence speciation, the strongest QTL mapped to a genomic region partly covered by an inversion polymorphism.

The circadian clock plays an important role in adaptation in time and space by synchronizing changes in physiological, developmental, and behavioral traits of organisms with daily and seasonal changes in their environment. We have studied some features of the circadian activity and clock organization in a northern Drosophila species, Drosophila montana, at both the phenotypic and the neuronal levels. In the first part of the study, we monitored the entrained and free-running locomotor activity rhythms of females in different light-dark and temperature regimes. These studies showed that D. montana flies completely lack the morning activity component typical to more southern Drosophila species in an entrained environment and that they are able to maintain their free-running locomotor activity rhythm better in constant light than in constant darkness. In the second part of the study, we traced the expression of the PDF neuropeptide and the CRY protein in the neurons of the brain in D. montana adults and found differences in the number and location of PDF- and CRY-expressing neurons compared with those described in Drosophila melanogaster. These differences could account, at least in part, for the lack of morning activity and the reduced circadian rhythmicity of D. montana flies in constant darkness, both of which are likely to be adaptive features during the long and dark winters occurring in nature.