Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females.

Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. Here we used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. We estimated CTemp for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50% in all temperatures. In continuous darkness, the diapause proportion was above 50% in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, we measured changes in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, our findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be less vulnerable to climate warming in high- than low-latitude populations.

Experimental introgression in Drosophila: Asymmetric postzygotic isolation associated with chromosomal inversions and an incompatibility locus on the X chromosome.

Interspecific gene flow (introgression) is an important source of new genetic variation, but selection against it can reinforce reproductive barriers between interbreeding species. We used an experimental approach to trace the role of chromosomal inversions and incompatibility genes in preventing introgression between two partly sympatric Drosophila virilis group species, D. flavomontana and D. montana. We backcrossed F1 hybrid females from a cross between D. flavomontana female and D. montana male with the males of the parental species for two generations and sequenced pools of parental strains and their reciprocal second generation backcross (BC2 mon and BC2 fla) females. Contrasting the observed amount of introgression (mean hybrid index, HI) in BC2 female pools along the genome to simulations under different scenarios allowed us to identify chromosomal regions of restricted and increased introgression. We found no deviation from the HI expected under a neutral null model for any chromosome for the BC2 mon pool, suggesting no evidence for genetic incompatibilities in backcrosses towards D. montana. In contrast, the BC2 fla pool showed high variation in the observed HI between different chromosomes, and massive reduction of introgression on the X chromosome (large X-effect). This observation is compatible with reduced recombination combined with at least one dominant incompatibility locus residing within the X inversion(s). Overall, our study suggests that genetic incompatibilities arising within chromosomal inversions can play an important role in speciation.

Divergence and introgression among the virilis group of Drosophila.

Speciation with gene flow is now widely regarded as common. However, the frequency of introgression between recently diverged species and the evolutionary consequences of gene flow are still poorly understood. The virilis group of Drosophila contains 12 species that are geographically widespread and show varying levels of prezygotic and postzygotic isolation. Here, we use de novo genome assemblies and whole-genome sequencing data to resolve phylogenetic relationships and describe patterns of introgression and divergence across the group. We suggest that the virilis group consists of three, rather than the traditional two, subgroups. Some genes undergoing rapid sequence divergence across the group were involved in chemical communication and desiccation tolerance, and may be related to the evolution of sexual isolation and adaptation. We found evidence of pervasive phylogenetic discordance caused by ancient introgression events between distant lineages within the group, and more recent gene flow between closely related species. When assessing patterns of genome-wide divergence in species pairs across the group, we found no consistent genomic evidence of a disproportionate role for the X chromosome as has been found in other systems. Our results show how ancient and recent introgressions confuse phylogenetic reconstruction, but may play an important role during early radiation of a group.

Plasticity in Photoperiodism: Drosophila montana Females Have a Life-Long Ability to Switch From Reproduction to Diapause.

Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. We studied the duration of SP for diapause induction and the females' ability to enter post-reproductive diapause at short day lengths in Drosophila montana strains from different latitudes in Europe, North America, and Japan. Our study shows that the females of this species have a life-long SP and that they retain an ability to switch between reproduction and diapause as a response to back-and-forth changes in day length for at least 3 months. D. montana strains from different latitudes showed high variation in females' ability to enter post-reproductive diapause; females of the southern strains generally requiring longer time and/or lower temperature to enter this stage than those of the northern strains. Moreover, the proportion of females that switched to post-reproductive diapause in 3 weeks in short day conditions at 16 °C showed positive correlation with the critical day length (CDL) for diapause induction and the latitudinal and continental origin of the strains. Life-long SP increases females' flexibility to respond to short-term changes in environmental conditions and enables reproducing females to switch to post-reproductive diapause when the days get shorter and colder toward the autumn. This ability can play a major role in species phenology and should be taken into account in theoretical and empirical studies on insect adaptation to seasonal variation.

Adaptation and ecological speciation in seasonally varying environments at high latitudes: Drosophila virilis group.

Living in high latitudes and altitudes sets specific requirements on species' ability to forecast seasonal changes and to respond to them in an appropriate way. Adaptation into diverse environmental conditions can also lead to ecological speciation through habitat isolation or by inducing changes in traits that influence assortative mating. In this review, we explain how the unique time-measuring systems of Drosophila virilis group species have enabled the species to occupy high latitudes and how the traits involved in species reproduction and survival exhibit strong linkage with latitudinally varying photoperiodic and climatic conditions. We also describe variation in reproductive barriers between the populations of two species with overlapping distributions and show how local adaptation and the reinforcement of prezygotic barriers have created partial reproductive isolation between conspecific populations. Finally, we consider the role of species-specific chromosomal inversions and the X chromosome in the development of reproductive barriers between diverging lineages.

Multiple paths to cold tolerance: the role of environmental cues, morphological traits and the circadian clock gene vrille.

BACKGROUND: Tracing the association between insect cold tolerance and latitudinally and locally varying environmental conditions, as well as key morphological traits and molecular mechanisms, is essential for understanding the processes involved in adaptation. We explored these issues in two closely-related species, Drosophila montana and Drosophila flavomontana, originating from diverse climatic locations across several latitudes on the coastal and mountainous regions of North America. We also investigated the association between sequence variation in one of the key circadian clock genes, vrille, and cold tolerance in both species. Finally, we studied the impact of vrille on fly cold tolerance and cold acclimation ability by silencing it with RNA interference in D. montana. RESULTS: We performed a principal component analysis (PCA) on variables representing bioclimatic conditions on the study sites and used latitude as a proxy of photoperiod. PC1 separated the mountainous continental sites from the coastal ones based on temperature variability and precipitation, while PC2 arranged the sites based on summer and annual mean temperatures. Cold tolerance tests showed D. montana to be more cold-tolerant than D. flavomontana and chill coma resistance (CTmin) of this species showed an association with PC2. Chill coma recovery time (CCRT) of both species improved towards northern latitudes, and in D. flavomontana this trait was also associated with PC1. D. flavomontana flies were darkest in the coast and in the northern mountainous populations, but coloration showed no linkage with cold tolerance. Body size decreased towards cold environments in both species, but only within D. montana populations largest flies showed fastest recovery from cold. Finally, both the sequence analysis and RNAi study on vrille suggested this gene to play an essential role in D. montana cold resistance and acclimation, but not in recovery time. CONCLUSIONS: Our study demonstrates the complexity of insect cold tolerance and emphasizes the need to trace its association with multiple environmental variables and morphological traits to identify potential agents of natural selection. It also shows that a circadian clock gene vrille is essential both for short- and long-term cold acclimation, potentially elucidating the connection between circadian clock system and cold tolerance.

Nanda-Hamner Curves Show Huge Latitudinal Variation but No Circadian Components in Drosophila Montana Photoperiodism.

Insect species with a wide distribution offer a great opportunity to trace latitudinal variation in the photoperiodic regulation of traits important in reproduction and stress tolerances. We measured this variation in the photoperiodic time-measuring system underlying reproductive diapause in Drosophila montana, using a Nanda-Hamner (NH) protocol. None of the study strains showed diel rhythmicity in female diapause proportions under a constant day length (12 h) and varying night lengths in photoperiods ranging from 16 to 84 h at 16°C. In the northernmost strains (above 55°N), nearly all females entered diapause under all photoperiods and about half of them even in continuous darkness, while the females of the southern strains showed high diapause proportions only in the circadian 24 h photoperiod. Significant correlation between the strains' mean diapause proportions in ≥ 24 h photoperiods and critical day length (CDL; half of the females enter diapause) suggests at least partial causal connection between the traits. Interestingly, females of the northern strains entered diapause even in ≤ 24 h photoperiods, where the night length was shorter than their critical night length (24 h - CDL), but where the females experienced a higher number of Light:Dark cycles than in 24 h photoperiods. NH experiments, performed on the control and selection lines in our previous selection experiment, and completed here, gave similar results and confirmed that selection for shorter, southern-type CDL decreases female diapausing rate in non-circadian photoperiods. Overall, our study shows that D. montana females measure night length quantitatively, that the photoperiodic counter may play a prominent but slightly different role in extra short and extra long photoperiods and that northern strains show high stability against perturbations in the photoperiod length and in the presence of LD cycles. These features are best explained by the quantitative versions of the damped external coincidence model.

Diapause affects cuticular hydrocarbon composition and mating behavior of both sexes in Drosophila montana.

Environmental cues, mainly photoperiod and temperature, are known to control female adult reproductive diapause in several insect species. Diapause enhances female survival during adverse conditions and postpones progeny production to the favorable season. Male diapause (a reversible inability to inseminate receptive females) has been studied much less than female diapause. However, if the males maximized their chances to fertilize females while minimizing their energy expenditure, they would be expected to be in diapause at the same time as females. We investigated Drosophila montana male mating behavior under short-day conditions that induce diapause in females and found the males to be reproductively inactive. We also found that males reared under long-day conditions (reproducing individuals) court reproducing postdiapause females, but not diapausing ones. The diapausing flies of both sexes had more long-chain and less short-chain hydrocarbons on their cuticle than the reproducing ones, which presumably increase their survival under stressful conditions, but at the same time decrease their attractiveness. Our study shows that the mating behavior of females and males is well coordinated during and after overwintering and it also gives support to the dual role of insect cuticular hydrocarbons in adaptation and mate choice.

Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence.

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 inter-related 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.

Strength of sexual and postmating prezygotic barriers varies between sympatric populations with different histories and species abundances.

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.

Direct and correlated responses to bi-directional selection on pre-adult development time in Drosophila montana.

Selection experiments offer an efficient way to study the evolvability of traits that play an important role in insects' reproduction and/or survival and to trace correlations and trade-offs between them. We have exercised bi-directional selection on Drosophila montana flies' pre-adult development time under constant light and temperature conditions for 10 generations and traced the indirect effects of this selection on females' diapause induction under different day lengths, as well as on the body weight and cold tolerance of both sexes. Overall, selection was successful towards slow, but not towards fast development. However, all fast selection line replicates showed at the end of selection increased variance in females' photoperiodic diapause response and about one hour increase in the critical day (CDL), where more than 50% of emerging females enter diapause. Indirect effects of selection on flies' body weight and cold-tolerance were less clear, as the flies of the slow selection line were significantly heavier and less cold-tolerant than the control line flies after five generations of selection, but lighter and more cold-tolerant at the end of selection. Changes in females' diapause induction resulting from selection for fast development could be due to common metabolic pathways underlying these traits, collaboration of circadian clock and photoperiodic timer and/or by the interaction between the endocrine and circadian systems.

Inter and Intraspecific Genomic Divergence in Drosophila montana Shows Evidence for Cold Adaptation.

The genomes of species that are ecological specialists will likely contain signatures of genomic adaptation to their niche. However, distinguishing genes related to ecological specialism from other sources of selection and more random changes is a challenge. Here, we describe the genome of Drosophila montana, which is the most extremely cold-adapted Drosophila species known. We use branch tests to identify genes showing accelerated divergence in contrasts between cold- and warm-adapted species and identify about 250 genes that show differences, possibly driven by a lower synonymous substitution rate in cold-adapted species. We also look for evidence of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, but do not find strong evidence for divergent selection on coding sequence variation. Divergent genes are involved in a variety of functions, including cuticular and olfactory processes. Finally, we also resequenced three populations of D. montana from across its ecological and geographic range. Outlier loci were more likely to be found on the X chromosome and there was a greater than expected overlap between population outliers and those genes implicated in cold adaptation between Drosophila species, implying some continuity of selective process at these different evolutionary scales.

Transcriptional Differences between Diapausing and Non-Diapausing D. montana Females Reared under the Same Photoperiod and Temperature.

BACKGROUND: A wide range of insects living at higher latitudes enter diapause at the end of the warm season, which increases their chances of survival through harsh winter conditions. In this study we used RNA sequencing to identify genes involved in adult reproductive diapause in a northern fly species, Drosophila montana. Both diapausing and non-diapausing flies were reared under a critical day length and temperature, where about half of the emerging females enter diapause enabling us to eliminate the effects of varying environmental conditions on gene expression patterns of the two types of female flies. RESULTS: RNA sequencing revealed large differences between gene expression patterns of diapausing and non-diapausing females, especially in genes involved with metabolism, fatty acid biosynthesis, and metal and nucleotide binding. Differently expressed genes included several gene groups, including myosin, actin and cytochromeP450 genes, which have been previously associated with diapause. This study also identified new candidate genes, including some involved in cuticular hydrocarbon synthesis or regulation (desat1 and desat2), and acyl-CoA Δ11-desaturase activity (CG9747), and few odorant-binding protein genes (e.g. Obp44A). Also, several transposable elements (TEs) showed differential expression between the two female groups motivating future research on their roles in diapause. CONCLUSIONS: Our results demonstrate that the adult reproductive diapause in D. montana involves changes in the expression level of a variety of genes involved in key processes (e.g. metabolism and fatty acid biosynthesis) which help diapausing females to cope with overwintering. This is consistent with the view that diapause is a complex adaptive phenotype where not only sexual maturation is arrested, but also changes in adult physiology are required in order to survive over the winter.

Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes.

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.

Seasonal gene expression kinetics between diapause phases in Drosophila virilis group species and overwintering differences between diapausing and non-diapausing females.

Most northern insect species experience a period of developmental arrest, diapause, which enables them to survive over the winter and postpone reproduction until favorable conditions. We studied the timing of reproductive diapause and its long-term effects on the cold tolerance of Drosophila montana, D. littoralis and D. ezoana females in seasonally varying environmental conditions. At the same time we traced expression levels of 219 genes in D. montana using a custom-made microarray. We show that the seasonal switch to reproductive diapause occurs over a short time period, and that overwintering in reproductive diapause has long-lasting effects on cold tolerance. Some genes, such as Hsc70, Jon25Bi and period, were upregulated throughout the diapause, while others, including regucalcin, couch potato and Thor, were upregulated only at its specific phases. Some of the expression patterns induced during the sensitive stage, when the females either enter diapause or not, remained induced regardless of the later conditions. qPCR analyses confirmed the findings of the microarray analysis in D. montana and revealed similar gene expression changes in D. littoralis and D. ezoana. The present study helps to achieve a better understanding of the genetic regulation of diapause and of the plasticity of seasonal responses in general.

Localization of quantitative trait loci for diapause and other photoperiodically regulated life history traits important in adaptation to seasonally varying environments.

Seasonally changing environments at high latitudes present great challenges for the reproduction and survival of insects, and photoperiodic cues play an important role in helping them to synchronize their life cycle with prevalent and forthcoming conditions. We have mapped quantitative trait loci (QTL) responsible for the photoperiodic regulation of four life history traits, female reproductive diapause, cold tolerance, egg-to-eclosion development time and juvenile body weight in Drosophila montana strains from different latitudes in Canada and Finland. The F2 progeny of the cross was reared under a single photoperiod (LD cycle 16:8), which the flies from the Canadian population interpret as early summer and the flies from the Finnish population as late summer. The analysis revealed a unique QTL for diapause induction on the X chromosome and several QTL for this and the other measured traits on the 4th chromosome. Flies' cold tolerance, egg-to-eclosion development time and juvenile body weight had several QTL also on the 2nd, 3rd and 5th chromosome, some of the peaks overlapping with each other. These results suggest that while the downstream output of females' photoperiodic diapause response is partly under a different genetic control from that of the other traits in the given day length, all traits also share some QTL, possibly involving genes with pleiotropic effects and/or multiple tightly linked genes. Nonoverlapping QTL detected for some of the traits also suggest that the traits are potentially capable of independent evolution, even though this may be restricted by epistatic interactions and/or correlations and trade-offs between the traits.

Reproductive isolation among allopatric Drosophila montana populations.

An outstanding goal in speciation research is to trace the mode and tempo of the evolution of barriers to gene flow. Such research benefits from studying incipient speciation, in which speciation between populations has not yet occurred, but where multiple potential mechanisms of reproductive isolation (RI: i.e., premating, postmating-prezygotic (PMPZ), and postzygotic barriers) may act. We used such a system to investigate these barriers among allopatric populations of Drosophila montana. In all heteropopulation crosses we found premating (sexual) isolation, which was either symmetric or asymmetric depending on the population pair compared. Postmating isolation was particularly strong in crosses involving males from one of the study populations, and while sperm were successfully transferred, stored, and motile, we experimentally demonstrated that the majority of eggs produced were unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of pre- and postmating barriers showed that populations differed in the mode and magnitude of RI barriers. Our results indicate that incipient RI among populations can be driven by different contributions of both premating and PMPZ barriers occurring between different population pairs and without the evolution of postzygotic barriers.

Cuticular hydrocarbons of Drosophila montana: geographic variation, sexual dimorphism and potential roles as pheromones.

Sexual selection within populations can play an important role in speciation when divergence in mating signals and their corresponding preferences occur along different coevolutionary trajectories in different populations. In insects, one potential target of sexual selection is the blend of cuticular hydrocarbons (CHCs), which often show intra- and interspecific variation, sexual dimorphism and may act as pheromones. In Drosophila montana, a cold-adapted, circumboreal member of the Drosophila virilis species group, flies from different populations have been found to show significant premating isolation as well as variation in male mating signal (song) and female preference. While the role of male courtship song in mate choice has been studied extensively, CHCs in this species have received little attention. In this study, we identified most of the CHCs found on the cuticle of D. montana and characterized population divergence and sexual dimorphism of CHC profiles among flies established from three natural populations - one European and two North American. We also studied their potential role as pheromones by analyzing CHCs of flies used in female-choice mating experiments. We report significant population×sex effects on CHC profiles, as well as significant relationships between some CHC principal components and particular mating behaviours, such as female attractiveness and male mating success, providing evidence that CHCs may play a role in mate choice in this species. The study also provides evidence for variation in the degree to which CHCs play a role in chemical communication among these populations, which may have an influence on the speciation process itself, and could be due to variation in interactions with other closely-related species that occur sympatrically with D. montana in some, but not other, parts of its distribution.

Involvement of circadian oscillation(s) in the photoperiodic time measurement and the induction of reproductive diapause in a northern Drosophila species.

An ability to predict forthcoming changes in environmental conditions and get prepared for them in advance is crucial for the survival and reproduction of organisms living in a seasonally changing environment. We have studied the possible involvement of circadian oscillator(s) in the photoperiodic timer controlling seasonal responses by tracing Drosophila montana females' diapause induction in constant darkness and in a classical Nanda-Hamner experiment. Nearly all females developed ovaries in continuous darkness, which shows the direct development to be their default developmental pathway in the absence of photoperiods. In Nanda-Hamner experiment the females' diapause incidence was close to zero in light:dark cycle 12:4 (photoperiod 16 h) and increased to nearly 100% in 12:8 and 12:12 (photoperiods 20 and 24 h). In longer photoperiods (28-72 h) the females' diapause percentages decreased gradually along with an increase in the length of the dark period, showing no peaks of high diapause incidence in the multiples of 24h. These findings suggest that the photoperiodic timer of D. montana is based on heavily damping circadian oscillator(s) or that it lacks strong oscillators. Damping of the photoperiodic timer under prolonged nights and constant darkness fits well with our earlier finding that these flies lose their locomotor activity rhythm in constant darkness, and suggests that the mechanisms underlying females' photoperiodic diapause response and their free-running locomotor activity rhythm may be partly based on same oscillators.

Northern Drosophila montana flies show variation both within and between cline populations in the critical day length evoking reproductive diapause.

Reproductive diapause, and its correct timing, plays an important role in the life cycle of many insect species living in a seasonally varying environment at high latitudes. In the present paper we have documented variation in the critical day length (CDL) for adult reproductive diapause and the steepness of photoperiodic response curves (PPRCs) in seven clinal populations of Drosophila montana in Finland between the latitudes 61 and 67°N, paying special attention to variation in these traits within and between cline populations. The isofemale lines representing these populations showed a sharp transition from 0% to 100% in females' diapause incidence in the shortening day lengths, indicated by steep PPRCs. The mean CDL showed a clear latitudinal cline decreasing by 1.6h from North to South regardless of the age of the lines, variation within the populations (i.e. among lines) in this trait being up to 3h. The steepness of the PPRCs correlated with the age of the line and this trait showed no clear latitudinal cline. Further studies on a large number of lines from one D. montana population confirmed that while maintaining the flies in diapause preventing conditions in the laboratory has no effect on CDL, older lines had steeper PPRCs. High variation in CDL within and between D. montana cline populations is likely to be heritable and provide a good potential for the evolution of photoperiodic responses. Information on genetic variation in life-history traits, such as diapause, is of utmost importance for predicting the ability of insects to survive in seasonally changing environmental conditions and to respond to long term changes in the length of the growing period e.g. by postponing the timing of diapause towards shorter day length and later calendar date.