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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. peerReviewed

BackgroundTracing 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.ResultsWe 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.ConclusionsOur 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.

Drosophila obscura is a common fruit fly that inhabits the temperate forests of Europe. While it is abundant in the north compared to other Drosophila, its density decreases southwards, where it is gradually replaced by other Drosophila species. This study describes variation in the mitochondrial Cyt b gene of D. obscura from several European populations. We observed a large number of haplotypes, together with the structuring of genetic variation. Genetic variation is higher in the west where O1 and related divergent haplotypes dominate. In the east, the O2 haplotype is most frequent, together with haplotypes that recently arose from it. In the central part of the species range, both O1 and O2 are equally present, along with many others. These data reveal signs of population expansions that probably happened earlier in the west, and more recently in the east. Though our conclusions are based on only one genetic marker, limiting the power of the analysis, the results imply either postglacial expansion from two unique sources or, more likely, eastwards stepping-stone expansion. This study adds important information on genetic variation and phylogeography to the obscure biology of D. obscura, a species that has the potential to become an interesting model in evolutionary biology and conservation genetics. peerReviewed

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. peerReviewed

Simple Summary Most laboratory experiments on insects to date have been conducted using constant temperature settings. Even when the purpose of the study was to investigate effects of temperature, insects have mostly been kept at different but constant temperatures ignoring natural variation in temperature. Here we investigated effects of simple daily temperature fluctuation (22.5/27.5 °C and 20/30 °C) on some development characteristics in five species of fruit flies (Drosophila) originating from areas with different temperature profiles. We demonstrated how species of the same genus can show substantial differences when developing at fluctuating temperatures not always predictable by development at comparable constant temperature (25 °C). Abstract Temperature has profound effects on biochemical processes as suggested by the extensive variation in performance of organisms across temperatures. Nonetheless, the use of fluctuating temperature (FT) regimes in laboratory experiments compared to constant temperature (CT) regimes is still mainly applied in studies of model organisms. We investigated how two amplitudes of developmental temperature fluctuation (22.5/27.5 °C and 20/30 °C, 12/12 h) affected several fitness-related traits in five Drosophila species with markedly different thermal resistance. Egg-to-adult viability did not change much with temperature except in the cold-adapted D. immigrans. Developmental time increased with FT among all species compared to the same mean CT. The impact of FT on wing size was quite diverse among species. Whereas wing size decreased quasi-linearly with CT in all species, there were large qualitative differences with FT. Changes in wing aspect ratio due to FT were large compared to the other traits and presumably a consequence of thermal stress. These results demonstrate that species of the same genus but with different thermal resistance can show substantial differences in responses to fluctuating developmental temperatures not predictable by constant developmental temperatures. Testing multiple traits facilitated the interpretation of responses to FT in a broader context.

Funding: This work was supported by Academy of Finland projects 268214 and 322980 to MK and a NERC (UK) grant NE/P000592/1 to MGR. Organisms can plastically alter resource allocation in response to changing environmental factors. For example, in harsh conditions, organisms are expected to shift investment from reproduction toward survival; however, the factors and mechanisms that govern the magnitude of such shifts are relatively poorly studied. Here we compared the impact of cold on males and females of the highly cold-tolerant species Drosophila montana at the phenotypic and transcriptomic levels. Although both sexes showed similar changes in cold tolerance and gene expression in response to cold treatment, indicating that the majority of changes are concordant between the sexes, we identified a clear reduction in sexually dimorphic gene expression, suggesting that preparing for the colder season involves reducing investment in sex-specific traits. This reduction was larger in males than females, as expected if male sexual traits are more condition-dependent than female traits, as predicted by theory. Gene expression changes were primarily associated with shifts in metabolic profile, which likely play a role in increasing cold tolerance. Finally, we found that the expression of immune genes was reduced following cold treatment, suggesting that reduced investment in costly immune function may be important in helping flies survive colder periods. Publisher PDF

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. peerReviewed

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses., M.W. was supported by the UK Natural Environmental Research Council through the E3 doctoral training programme (NE/L002558/1), and S.R. was supported by Wellcome Trust PhD programme (108905/Z/15/Z). A.B. received funding from BBSRC (grant number BB/P00685X/1). T.F. received funding from Swiss National Science Foundation (grant numbers 31003A-182262, PP00P3_165836, and PP00P3_133641/1). C.G. received funding from Agence Nationale de la Recherche (grant number ANR-15-CE32-0011-01). J.G. received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900) and from the Fundación Española para la Ciencia y la Tecnologia-Ministerio de Economía y Competitividad (FCT-15-10187). S.G. received funding from Deutsche Forschungsgemeinschaft (grant number GR 4495/2). M.K. received funding from Academy of Finland projects (268214 and 322980). M.K. received funding from Austrian Science Fund (FWF; grant number P32275). V.L. received funding from Danish Research council for natural Sciences (FNU; grant number 4002-00113B). B.S.O. received funding from the Scientific and Technological Research Council of Turkey (TUBITAK; grant number 214Z238). J.P. received funding from Deutsche Forschungsgemeinschaft (grant number PA 903/8). M.S.-R., M.S.V., and M.J. received funding from the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 451-03-68/2020-14/200178). F.S. received funding from Deutsche Forschungsgemeinschaft (grant number STA1154/4-1; Projektnummer 408908608). M.T., A.P., and K.E. received funding from the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 451-03-68/2020-14/200007). The DrosEU consortium has been funded by a Special Topics Network (STN) grant by the European Society of Evolutionary Biology (ESEB).

Funding: UK Natural Environment Research Council (Grant Number(s): NE/L501852/1, NE/P000592/1); Academy of Finland (GrantNumber(s): 267244, 268214, 322980), Ella ja Georg Ehrnroothin Säätiö. Detecting signatures of ecological adaptation in comparative genomics is challenging, but analysing population samples with characterised geographic distributions, such as clinal variation, can help identify genes showing covariation with important ecological variation. Here, we analysed patterns of geographic variation in the cold-adapted species Drosophila montana across phenotypes, genotypes and environmental conditions and tested for signatures of cold adaptation in population genomic divergence. We first derived the climatic variables associated with the geographic distribution of 24 populations across two continents to trace the scale of environmental variation experienced by the species, and measured variation in the cold tolerance of the flies of six populations from different geographic contexts. We then performed pooled whole genome sequencing of these six populations, and used Bayesian methods to identify SNPs where genetic differentiation is associated with both climatic variables and the population phenotypic measurements, while controlling for effects of demography and population structure. The top candidate SNPs were enriched on the X and fourth chromosomes, and they also lay near genes implicated in other studies of cold tolerance and population divergence in this species and its close relatives. We conclude that ecological adaptation has contributed to the divergence of D. montana populations throughout the genome and in particular on the X and fourth chromosomes, which also showed highest interpopulation FST. This study demonstrates that ecological selection can drive genomic divergence at different scales, from candidate genes to chromosome-wide effects. Publisher PDF

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. peerReviewed

Alternative splicing, in which one gene produce multiple transcripts, may influence how adaptive genes respond to specific environments. A newly produced transcriptome of Drosophila montana shows the Gs1-like (Gs1l) gene to express multiple splice variants and to be down regulated in cold acclimated flies with increased cold tolerance. Gs1l’s effect on cold tolerance was further tested by injecting cold acclimated and non-acclimated flies from two distantly located northern and southern fly populations with double stranded RNA (dsRNA) targeting Gs1l. While both populations had similar cold acclimation responses, dsRNA injections only effected the northern population. The nature of splicing expression was then investigated in the northern population by confirming which Gs1l variants are present, by comparing the expression of different gene regions and by predicting the protein structures of splices using homology modelling. We find different splices of Gs1l not only appear to have independent impacts on cold acclimation but also elicit different effects in populations originating from two very different environments. Also, at the protein level, Gs1l appears homologous to the human HDHD1A protein and some splices might produce functionally different proteins though this needs to be verified in future studies by measuring the particular protein levels. Taken together, Gs1l appears to be an interesting new candidate to test how splicing influences adaptations.

The work has been supported by the Academyof Finland to A.H. (project 267244) and Natural Environment Research Council (NERC) funding (NE/J020818/1 to M.G.R.; NE/L501852/1 to R.A.W.W.). 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. Publisher PDF

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. peerReviewed

Introduction Organisms use environmental cues to match their phenotype with the future availability of resources and environmental conditions. Changes in the magnitude and frequency of environmental cues such as photoperiod and temperature along latitudes can be used by organisms to predict seasonal changes. While the role of temperature variation on the induction of plastic and seasonal responses is well established, the importance of photoperiod for predicting seasonal changes is less explored. Materials and methods Here we studied changes in life‐history and thermal stress resistance traits in Drosophila subobscura in response to variation in photoperiod (6:18, 12:12 and 18:6 light:dark cycles) mimicking seasonal variations in day length. The populations of D. subobscura were collected from five locations along a latitudinal gradient (from North Africa and Europe). These populations were exposed to different photoperiods for two generations, whereafter egg‐to‐adult viability, productivity, dry body weight, thermal tolerance, and starvation resistance were assessed. Results We found strong effects of photoperiod, origin of populations, and their interactions on life‐history and stress resistance traits. Thermal resistance varied between the populations and the effect of photoperiod depended on the trait and the method applied for the assessment of thermal resistance. Perspectives Our results show a strong effect of the origin of population and photoperiod on a range of fitness‐related traits and provide evidence for local adaptation to environmental cues (photoperiod by population interaction). The findings emphasize an important and often neglected role of photoperiod in studies on thermal resistance and suggest that cues induced by photoperiod may provide some buffer enabling populations to cope with a more variable and unpredictable future climate. peerReviewed

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. peerReviewed

To function properly, organisms must adjust their physiology, behavior and metabolism in response to a suite of varying environmental conditions. One of the central regulators of these changes is organisms' internal circadian clock, and recent evidence has suggested that the clock genes are also important in the regulation of seasonal adjustments. In particular, thermosensitive splicing of the core clock gene timeless in a cosmopolitan fly, Drosophila melanogaster , has implicated this gene to be involved in thermal adaptation. To further investigate this link we examined the splicing of timeless in a northern malt fly species, Drosophila montana , which can withstand much colder climatic conditions than its southern relative. We studied northern and southern populations from two different continents (North America and Europe) to find out whether and how the splicing of this gene varies in response to different temperatures and day lengths. Interestingly, we found that the expression of timeless splice variants was sensitive to differences in light conditions, and while the flies of all study populations showed a change in the usage of splice variants in constant light compared to LD 22:2, the direction of the shift varied between populations. Overall, our findings suggest that the splicing of timeless in northern Drosophila montana flies is photosensitive, rather than thermosensitive and highlights the value of studying multiple species and populations in order to gain perspective on the generality of gene function changes in different kinds of environmental conditions.

This work was supported by the Academy of Finland to AH (projects 132619 and 267244) and to MK (projects 268214 and 272927) and NERC (UK) funding to MGR (grants NE/E015255/1 and NE/J020818/1) and PhD studentship to DJP (NE/I528634/1). 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. 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 look for evidence of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, and 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. We also re-sequenced three populations of D. montana from 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. Publisher PDF

Sisäsiitoksella, eli pariutumisella lähisukulaisten kesken, on monia negatiivisia vaikutuksia sekä populaation että yksilön kelpoisuudelle. Sisäsiitos lisää homotsygotiaa ja mahdollistaa siten väistyvien haitallisten mutaatioiden ilmentymisen fenotyypissä sekä estää heterotsygotiasta johtuvaa paremmuutta lokuksissa, joissa on ylidominanssia. On osoitettu, että sisäsiittoisuus voi vaikuttaa koiraisiin ja naaraisiin eri tavalla siten, että toinen sukupuolista kärsii sisäsiitoksesta toista enemmän. Jotkut tutkimukset ovat näyttäneet, että sisäsiittoisuudella on voimakkaampi negatiivinen vaikutus koiraiden kuin naaraiden lisääntymiskykyyn, kun taas toiset osoittavat että naaraat kärsivät enemmän sisäsiitoksen seurauksena. Tutkimukseni tarkoituksena on selvittää löytyykö sukupuolten väliltä eroja sisäsiitoksen vaikutuksessa lisääntymismenestykseen. Tutkin sisäsiitoksen vaikutuksia pariutumismenestykseen ja jälkeläistuottoon Drosophila littoralis – mahlakärpäspopulaatiossa kahdessa erillisessä kokeessa. Sisäsiitos alensi merkittävästi jälkeläistuottoa naarailla, kun taas sisäsiitoksen vaikutukset koiraiden jälkeläistuottoon olivat lievempiä, joskin negatiivisia. Tämä viittaa siihen, että naaraiden lisääntymismenestys kärsii enemmän sisäsiitoksen vaikutuksesta kuin koiraiden. Nämä tulokset ovat yhteneväisiä sen kanssa, että naaraat panostavat enemmän lisääntymiseen ja sisäsiitoksella voi siten olla enemmän negatiivisia vaikutuksia naaraiden kuin koiraiden lisääntymiseen. Myös sisäsiittoisten naaraiden pariutumismenestys oli heikompi kuin ei-sisäsiittoisten naaraiden, mikä vahvistaa päätelmää että naaraiden lisääntymiskyky vaarantuu sisäsiitoksen negatiivisten vaikutusten takia. Sisäsiittoisten naaraiden alhaisempi lisääntymismenestys tuo esille mielenkiintoisen näkökulman sukupuolten rooleista pariutumisessa. Vaikka naaraita pidetään usein koiraita valikoivampana sukupuolena pariutumistilanteessa, myös koiraat voivat valita parinsa naaraiden ominaisuuksien mukaan. Koiraat voivat yrittää maksimoida oman lisääntymismenestyksensä olemalla valikoivampia parinvalinnan suhteen kuin mitä aiemmin on ajateltu. Koiraat voivat myös hyötyä valikoivasta käyttäytymisestä oletettua enemmän. Sisäsiittoisuus ei heikentänyt merkittävästi koiraiden pariutumismenestystä. Nämä tulokset herättävät monia mielenkiintoisia kysymyksiä sukupuolten rooleista lisääntymisessä ja sisäsiitoksen sukupuoleen perustuvista eroista.

Kylmyys on yksi tärkeimmistä lajien levinneisyyteen ja runsauteen vaikuttavista stressitekijöistä. Karaistuminen ja lisääntymisdiapaussi ovat esimerkkejä hyönteisten fysiologisesta ja biokemiallisesta sopeutumista mataliin lämpötiloihin. Karaistumisella tarkoitetaan lyhyen ajan sisällä tapahtuvaa kylmänkestävyyden kasvamista, ja sillä voi olla huomattava vaikutus kylmistä olosuhteista selviytymiseen. Lisääntymisdiapaussin laukaisee ympäristöstä tulevat ärsykkeet, kuten muutokset päivän pituudessa ja lämpötilassa. Se parantaa naaraiden mahdollisuuksia selvitä epäedullisista vuodenajoista ja siirtää jälkeläisten tuoton ajankohtaan jolloin munien ja jälkeläisten selviytyminen on parhaimmillaan. Tämän työn tavoitteena oli etsiä sopivat lämpötilat ja käsittelyajat Drosophila montana -mahlakärpästen kylmänkestävyyden mittaamiseen ja selvittää vaikuttavatko lyhytaikainen karaistuminen ja lisääntymisdiapaussi naaraiden ja koiraiden kylmänkestävyyteen. Tutkimuslajina käytettiin D. montana -lajia, joka on sopeutunut talvehtimaan pohjoisissa luonnonoloissa. Kärpästen kylmänkestävyyttä tutkittiin mittaamalla niiden kylmäkoomasta selviytymiseen kuluva aika. Tutkittavia yksilöitä pidettiin aluksi kaksi viikkoa kasvatuskaapeissa (+16 C), joista toisessa oli jatkuva valaistus ja toisessa lisääntymisdiapaussin laukaiseva valojakso 16:8 (16 tuntia valoa ja 8 tuntia pimeää). Ennen kylmänkestävyyskoetta puolet yksilöistä sai karaistumiskäsittelyn (1 tunti 0 C:ssa). Varsinaiset kylmänkestävyyskokeet kestivät 16 tuntia ja ne tehtiin kolmessa eri lämpötilassa (−6 C, −7 C ja −8 C). Tämän tutkimuksen perusteella voitiin päätellä, että Drosophila montanan kylmäkoomasta selviämiseen kuluva aika voidaan parhaiten mitata −6 ˚C:ssa altistusajan ollessa 16 tuntia. Tämä lämpötila oli riittävä käsittelyjen välisten erojen selvittämiseksi, mutta ei liian rankka kärpästen selviytymisen kannalta. Tutkimuksen perusteella lisääntymisdiapaussi parantaa naaraiden ja samoissa olosuhteissa kasvatettujen koiraiden kylmänkestävyyttä, karaistumisen vaikutuksen ollessa selkein naarailla, jotka eivät ole diapaussissa. Diapaussin ja karaistumisen aikaansaamat muutokset kärpäsen fysiologiassa voivat olla samankaltaisia, ja näin karaistumisen antama lisähyöty on lähes olematonta tai sattumanvaraista lisääntymisdiapaussissa oleville yksilöille.

Tapahtuuko lajien toisistaan erillään olevien populaatioiden ilmiasuun liittyvien piirteiden eriytyminen populaatioiden geneettisen eriytymisen sivutuotteena vai nopeutuuko tällaisten piirteiden eriytyminen luonnonvalinnan tai seksuaalivalinnan vaikutuksesta? Tätä kysymystä Jarkko Routtu pohti väitöskirjassaan. Routtu tutki maapallon eri puolilta peräisin olevien mahlakärpäskantojen eriytymistä, D. virilis -lajilla koiraan kosintalaulun ja D. montana -lajilla koiraan kosintalaulun sekä siipien ja genitaalien koon ja muodon perusteella.- Erityisesti Drosophila montanalla seksuaalivalinnan vaikutus populaatioiden eriytymiseen on kiinnostavaa, koska tämän lajin naaraat ovat mieltyneet koiraan tietyntyyppiseen kosiolauluun, Routtu kertoo.D. viriliksellä kärpäskantojen geneettinen erilaistuminen ja laulujen eriytyminen eivät olleet edenneet yhdenmukaisesti, mikä viittaa siihen, että laulut eivät ole erilaistuneet pelkästään neutraalin geneettisen erilaistumisen sivutuotteena. D. montana –lajin populaatiot poikkesivat toisistaan sekä lauluun liittyvien ominaisuuksien että siipien ja genitaalien koon ja muodon suhteen. Eri ominaisuuksissa havaittu populaatioiden välinen näkyvissä ominaisuuksissa tapahtunut eriytyminen ei ollut kulkenut samaan tahtiin populaatioiden genotyyppisen eriytymisen kanssa, vaan niiden evoluutio oli tapahtunut selkeästi erilaisten valintapaineiden, kuten seksuaalisen valinnan tai luonnonvalinnan, alla.D. virilis esiintyy pohjoisella pallonpuoliskolla ihmisen seuralaislajina 30 leveyspiirin alapuolella esimerkiksi panimoissa, leipomoissa tai lautatarhoissa. D. montana esiintyy 40 leveyspiirin pohjoispuolella sekä Euraasiassa että Pohjois-Amerikassa boreaalisella metsävyöhykkeellä järvien, jokien ja purojen rannoilla. Tutkimuksessa käytetyt kannat on kerätty luonnosta ja osaa niistä on säilytetty laboratoriossa useita sukupolvia. Arvokkaita luonnonpopulaatiota kerättiin D. viriliksellä Kiinasta ja Japanista ja D. montanalla Suomesta, Kanadasta ja Yhdysvalloista. Mahlakärpäsillä voi tutkia ilmiöitä mittakaavassa, joka ei ole mahdollinen monille muille tutkimuseliöille.Drosophila virilis ja D. montana -lajien mahlakärpästen geneettinen eriytyminen on tapahtunut eri tahtiin johtuen osittain kärpästen erilaisista elinpiireistä. Ihmisen seuralaislajin, D. viriliksen, eri maantieteellisiltä alueilta peräisin olevat kärpäskannat eivät erotu toisistaan mitokondriaalisen DNAn osalta. Tämän lajin leviäminen ja populaatioiden koon kasvu on todennäköisesti tapahtunut ihmispopulaatioiden kasvun ja uudelle mantereelle levittäytymisen myötä. Lajien myöhemmästä evoluutiosta kertova mikrosatelliittianalyysi ryhmittelee D. virilis -lajin kannat maantieteellisen alkuperän mukaan neljään ryhmään, mikä viittaa populaatioiden viime aikoina tapahtuneeseen erilaistumiseen. Luonnossa vesistöjen äärellä elävän D. montana –lajin Euroopassa ja Pohjois-Amerikassa elävät populaatiot ovat eriytyneet toisistaan sekä mitokondriaalisen DNAn että mikrosatelliittialleelien osalta, eurooppalaisen populaation edustaessa alkuperäisen populaation tyyppiä. Viimeisessä osatutkimuksessa Routtu kehitti pohjoiseurooppalaisille D. virilis -ryhmän lajeille lajintunnistusmenetelmän helpottamaan luonnosta kerättyjen yksilöiden tunnistusta ja mahdollisesti väärin tunnistettujen laboratoriokantojen identifioimista. Menetelmä perustuu kaikissa D. virilis -ryhmän lajeissa monistuvien polymorfisten mikrosatelliittien käyttöön. Tasaisesti koko genomiin jakautuneet merkkigeenit selkeyttävät D. virilis -ryhmän lajien keskinäisiä sukulaisuussuhteita. Näitä molekyylimerkkigeenejä voi myös käyttää mm. isyystutkimuksiin ja geenien paikallistamiseen samoin kuin luonnonpopulaatioiden laajamittaiseen tutkimiseen.Useammanlaisten merkkigeenien (esim. mitokondriaalisen DNAn ja mikrosatelliitit) yhtäaikainen käyttö antaa hyvän kuvan lajien eri alueille levinneiden populaatioiden historiasta ja sukulaisuussuhteista. Tällainen tutkimus mahdollistaa esim. morfologisten ja käyttäytymiseen liittyvien piirteiden evoluutioon liittyvien valintapaineiden jäljittämisen. Genetic and phenotypic differentiation of Drosophila virilis and D. montana populations has proceeded along evolutionarily different pathways. D. virilis, “a human commensal”, does not show clear diversity or population structure in mtDNA haplotypes, which refers to population growth. Microsatellites group the laboratory strains of this species into four clusters according to their geographical origin, indicating recent population differentiation. In contrast, D. montana, a “wild” species, has populations in Europe and North-America that are genetically diverged. In this species the mtDNA haplotypes are mixed among the two North-American populations, while microsatellites separate all study populations from each other. The courtship songs of the D. virilis laboratory strains showed significant inter-strain and geographic variation in several song traits. The genetic distances and the song divergence of the strains did not show significant association, which suggests that the songs have not diverged solely as a side-effect of genetic divergence. In D. montana the songs of the laboratory strains from different continents showed the highest divergence in song frequency, while the songs of the wild populations varied most prominently in the remaining pulse characters. D. montana populations also demonstrated divergence in male wing and genital morphology. The phenotypic divergence among populations did not coincide with the extent of their genetic divergence, suggesting that the first-mentioned traits are not evolving neutrally. Finally, we constructed a species recognition method for North European D. virilis group species to ease the identification of wild collected flies and to detect possible misclassifications of laboratory strains.