Abstract The pattern and the genetic basis of variation in courtship song of D. virilis were studied using three different approaches: a candidate gene, a biometrical and a quantitative trait locus (QTL) method. Nucleotide variation in a candidate song gene, no-on-transientA, was analysed both within the species (D. virilis and D. littoralis) and between the species of the D. virilis group. Nucleotide variation showed no signs of selection and there was no association between the nucleotide or repeat length variation in nonA gene region and the song characters of the D. virilis group species. Molecular markers (microsatellites) were isolated for D. virilis and their cross-species amplification was tested in all members of the D. virilis group. Intraspecific variation in D. virilis was studied at the phenotypic level in male song characters and at the genetic level in microsatellites. Significant geographic variation was detected in both levels, grouping the strains according to the main continents of the species' distribution range: America, Asia, Europe and Japan. The strains with most extreme song phenotypes were chosen for further analysis. The inheritance of two courtship song characters, the number of pulses in a pulse train (PN) and the length of a pulse train (PTL) was studied by analysing the means and variances of these characters between parental and reciprocal F1, F2 and backcross males. This biometrical analysis showed the genetic basis of these song characters to be polygenic with significant dominance, epistatic and Y-chromosomal effects on both characters. A subset of these data (F2 generation males) were used to conduct a QTL study with the aid of a recombination linkage map constructed for the microsatellites. Composite interval mapping (CIM) revealed significant QTLs, which were shared in both characters. Altogether, significant QTLs, located on the X, 2nd, 3rd and 4th chromosome, were found to affect PN, whereas only QTLs on the 3rd chromsome was found to affect PTL. The effect of the same QTL on the 3rd chromosome on both characters accounted for 31.8% and 49.1% of the mean difference between the parental strains in PN and PTL, respectively. These results suggest the genetic basis for these song characters is caused mainly by autosomal QTLs with a relatively large effect.

Abstract Divergence of behavioral traits (e.g. courtship rituals, habitat choice) has had a major impact on species formation and isolation. Species-specific courtship rituals preventing species hybridization may arise as a by-product of natural selection during spatial isolation or through direct action of natural selection to prevent species hybridization after a secondary contact. Coordination leading to the assumption of coevolution of signals and preferences is a prerequisite for effective courtship signaling between a male and a female of the same species. We found a reasonable amount of variation in the mate traits within the Drosophila montana and D. virilis species, but our findings did not reveal evidence of the coevolution between the male courtship signals and female preference for these signals. Variation also did not cause isolation within species. The form of female preference for carrier frequency of male song was found to be stabilizing even in changing environments, when female preference for the carrier frequency and pulse length of the male song was measured at ambient temperature. D. montana females always preferred males with high frequency songs, which probably advertises the male condition. The frequency of male song decreased due to male aging, although males with larger body size were able to maintain frequencies better. Ageing also had a deterioration effect on male reproductive success. The importance of male courtship song was not only highlighted in mate choice, but also in species-recognition. We were able to persuade D. montana females to copulate with alien D. lummei species by playing them simulated song resembling the song of conspecific males. Simulated courtship songs were demonstrated to be a practical tool in studies of preference between sexually isolated Drosophila species. / Tiivistelmä Kosintapiirteiden (esim. kosintarituaalit, habitaatin valinta) vaihtelevuudella on ollut suuri vaikutus lajien muodostumisessa ja eriytymisessä. Lajien välisiä risteytymisiä estäviä lajispesifisiä kosintarituaaleja voi syntyä luonnonvalinnan sivutuotteena spatiaalisen eristäytymisen aikana tai luonnonvalinnan suoralla vaikutuksella estämään lajiristeymät lajien uudelleen kohdatessa. Tehokkaan koiraan ja naaraan välisen kosintaviestinnän edellytyksenä on koordinointi, minkä vuoksi on oletettavaa, että signaalien ja vasteiden välillä on yhteisevoluutiota. Löysimme kohtuullisen määrän vaihtelevuutta kosintapiirteissä Drosophila montana – ja D. virilis – lajeilla, mutta emme löytäneet todisteita koiraan kosintasignaalien ja naaraan näihin signaaleihin kohdistamien vasteiden välisestä yhteisevoluutiosta. Vaihtelu ei myöskään aiheuttanut isolaatiota lajien välille. Naaraan koiraan kosintalaulun kantofrekvenssiin osoittaman vasteen funktion muodon havaittiin olevan tasapainottava jopa vaihtelevissa ympäristöissä, kun naaraan kantotaajuuteen osoittamaa vastetta mitattiin eri lämpötiloissa. D. montana naaraat suosivat koiraita, joilla oli korkeataajuinen kosintalaulu. Laulun korkea taajuus kertoo luultavasti naaraalle koiraan fyysisestä kunnosta. Koiraan laulun frekvenssin havaittiin laskevan koiraan ikääntyessä, mutta isommat koiraat pystyivät paremmin säilyttämään korkean taajuuden laulussaan. Ikääntyminen heikensi myös koiraan lisääntymismenestystä. Koiraan kosintalaulun tärkeys ei korostunut pelkästään parinvalinnassa, vaan myös lajintunnistuksessa. Pystyimme suostuttelemaan D. montana –naaraat parittelemaan vieraan D. lummei –lajin kanssa käyttämällä simuloituja kosintalauluja, jotka osoittautuivat käytännöllisiksi välineiksi tutkittaessa naaraan vasteita seksuaalisesti eriytyneillä Drosophila-lajeilla.

Abstract In sympatric Drosophila species, songs produced by male wing vibration during courtship are an effective mechanism preventing interspecific matings and maintaining sexual isolation between different species. These songs can vary greatly even between closely related species. The aim of this study was to localise X chromosomal and autosomal genes affecting species differences in male courtship song and to study their interaction in the D. virilis group species. Various genes were probed by in situ hybridisation on the X chromosomes of six species of the group, which enabled us to use localised RFLP markers in QTL studies, as well as to compare gene arrangements of different species. Genetic analyses of differences between the songs of D. virilis and D. littoralis showed that species-specific song traits are affected both by X chromosomal and autosomal genes. The X chromosomal gene(s) having a major impact on pause and pulse length in male song were found to be located at the proximal region of the chromosome. Precise localisation of the song genes was, however, not possible due to multiple chromosome rearrangements restricting recombination between RFLP markers located on this area. The same problem was faced when studying hybrids between D. flavomontana and D. montana with less diverged X chromosomal gene arrangements. Interaction between the X chromosomal and autosomal song genes in determining male song traits was studied in four species belonging to the virilis and montana phylads of D. virilis group. The long pauses in courtship song were found to be mainly caused by X chromosomal song genes (or maternal / cytological factors), while pulse length was determined by X chromosomal genes interacting with autosomal genes. This confirms the important role of X chromosomal gene(s) in song evolution in the montana phylad species. The direction of dominance in hybrid songs suggests that the songs of the montana phylad species have been affected by directional selection favouring shorter pulses and longer pauses between sound pulses during their evolution. The levels and patterns of DNA polymorphism in an X-linked fused (fu) gene was studied in different D. montana populations. These studies revealed that D. montana populations are significantly but not completely isolated, and that a selective sweep at fu (or at a gene linked to fu) may be the reason for the reduced levels and patterns of variability of this gene in Finnish D. montana populations. The methods used in this study will be utilized to study variation in 'song genes' in the future.