The DAIBAM MITE element is involved in the origin of one fixed and two polymorphic Drosophila virilis phylad inversions

Chromosomal inversions can originate from breakage and repair by non-homologous end-joining. Nevertheless, they can also originate from ectopic recombination between transposable elements located on the same chromosome inserted in opposite orientations. Here, we show that a MITE element (DAIBAM), previously involved in the origin of one Drosophila americana polymorphic inversion, is also involved in the origin of one fixed inversion between D. virilis and D. americana and another D. americana polymorphic inversion. Therefore, DAIBAM is responsible for at least 20% of the chromosomal rearrangements that are observed within and between species of the virilis phylad (D. virilis, D. lummei, D. novamexicana and D. americana), having thus played a significant role in the chromosomal evolution of this group of closely related species.     

Analysis of courtship behavior in closely related species of Drosophila virilis group: a new approach arises new questions

Courtship behavior of males was studied in four closely related Drosophila species: D. virilis, D. lummei, D. a. americana and D. littoralis. Using a video-computing approach, we compared behavior in males courting conspecific and heterospecific females. In males of all species studied, touching and licking were found to be the most prolonged courtship elements. Touching and licking were typically proceeding together; wing vibration was usually produced against a background of touching and licking. We found only minor interspecific variations in courtship rituals. Heterospecific courtships in D. virilis and D. lummei were almost as active as conspecific ones; however, isolation between D. a. americana and D. littoralis appeared to be much stronger than between D. virilis and D. lummei. Analysis of prolonged touching and licking raises a question about chemical and tactile sensory stimuli exchanged between sexes in the developed courtship of D. virilis group.     

Melanic pigmentation and light preference within and between two Drosophila species

Environmental adaptation and species divergence often involve suites of co‐evolving traits. Pigmentation in insects presents a variable, adaptive, and well‐characterized class of phenotypes for which correlations with multiple other traits have been demonstrated. In Drosophila, the pigmentation genes ebony and tan have pleiotropic effects on flies'' response to light, creating the potential for correlated evolution of pigmentation and vision. Here, we investigate differences in light preference within and between two sister species, Drosophila americana and D. novamexicana, which differ in pigmentation in part because of evolution at ebony and tan and occupy environments that differ in many variables including solar radiation. We hypothesized that lighter pigmentation would be correlated with a greater preference for environmental light and tested this hypothesis using a habitat choice experiment. In a first set of experiments, using males of D. novamexicana line N14 and D. americana line A00, the light‐bodied D. novamexicana was found slightly but significantly more often than D. americana in the light habitat. A second experiment, which included additional lines and females as well as males, failed to find any significant difference between D. novamexicana‐N14 and D. americana‐A00. Additionally, the other dark line of D. americana (A04) was found in the light habitat more often than the light‐bodied D. novamexicana‐N14, in contrast to our predictions. However, the lightest line of D. americana, A01, was found substantially and significantly more often in the light habitat than the two darker lines of D. americana, thus providing partial support for our hypothesis. Finally, across all four lines, females were found more often in the light habitat than their more darkly pigmented male counterparts. Additional replication is needed to corroborate these findings and evaluate conflicting results, with the consistent effect of sex within and between species providing an especially intriguing avenue for further research.     

Dominance status of shape of male genitalia in interspecific crosses of some Drosophila virilis group species

The heritability of the shape of the main species-specific morphological trait for the Drosophila virilis group-the male mating organ has been analyzed using the hybrid males D. virilis × D. lummei and D. virilis × D. novamexicana. The results suggest an increase in the share of the characters with a recessive status in the evolutionarily younger species and demonstrate the role of sex chromosomes in the implementation of a dominant or recessive status of the trait. The roles of additive and epistatic components of the total variation in the evolution of the dominance status, shown in several known theoretical models and confirmed by our data, are considered. The published data on sterility of hybrid males in interspecific crosses are discussed from the standpoint of the evolution of dominance.     

Interspecies variability of number of bristles on dorsal surface of aedeagus in D. virilis species group and its genetic mapping with interspecies hybrids of D. virilis and D. lummei

The results of morphologic and hybrid analyses of the feature of the male reproductive system of sibling species in the virilis group were presented. Bristles appeared on the surfaces of male genitals (aedeagus). The occurrence of a specific expression of the examined feature in the phyllades of D. virilis group, the correspondence of both the number and distribution pattern of the bristles on surfaces of the aedeagus and developmental temperature in D. virilis and D. lummei, as well as the link between feature and sexual behavior, have been shown. Dominance of D. lummei phenotype in the interspecies D. virilis × D. lummei was found. The interspecies hybrids D. virilis and D. lummei were used for a genetic analysis of the variability of the examined feature. The significant influence of chromosomes 2 and 6 on the number of bristles on the aedeagus in hybrid males was shown. Furthermore, the correspondence between the effects of the autosomes 2 and 6 on the variability of the examined feature and the genetic status of the other chromosomes (the effect of interaction between genetic factors, chromosomes here) was revealed. The adaptive value of the examined feature related to the involvement in the formation of isolating barriers at the copulation stage is under discussion.     

Genetic architecture of a body colour cline in Drosophila americana

Phenotypic variation within a species is often structured geographically in clines. In Drosophila americana, a longitudinal cline for body colour exists within North America that appears to be due to local adaptation. The tan and ebony genes have been hypothesized to contribute to this cline, with alleles of both genes that lighten body colour found in D. americana. These alleles are similar in sequence and function to the allele fixed in D. americana's more lightly pigmented sister species, Drosophila novamexicana. Here, we examine the frequency and geographic distribution of these D. novamexicana‐like alleles in D. americana. Among alleles from over 100 strains of D. americana isolated from 21 geographic locations, we failed to identify additional alleles of tan or ebony with as much sequence similarity to D. novamexicana as the D. novamexicana‐like alleles previously described. However, using genetic analysis of 51 D. americana strains derived from 20 geographic locations, we identified one new allele of ebony and one new allele of tan segregating in D. americana that are functionally equivalent to the D. novamexicana allele. An additional 5 alleles of tan also showed marginal evidence of functional similarity. Given the rarity of these alleles, however, we conclude that they are unlikely to be driving the pigmentation cline. Indeed, phenotypic distributions of the 51 backcross populations analysed indicate a more complex genetic architecture, with diversity in the number and effects of loci altering pigmentation observed both within and among populations of D. americana. This genetic heterogeneity poses a challenge to association studies and genomic scans for clinal variation, but might be common in natural populations.     

Sites of the 5s Ribosomal Genes in Drosophila. I. the Multiple Clusters in the VIRILIS Group

Drosophila melanogaster ¹²⁵I-5S RNA was annealed to salivary gland preparations of 6 species in the virilis group of Drosophila. Two patterns of annealing were found. D. virilis, D. montana and D. borealis showed three 5S gene clusters on chromosome 5; S_d–f and W_c–j were strongly labeled, but X_a–e was weakly labeled. D. montana and D. borealis have a greater percentage of their total 5S cistrons at S _d–f than does D. virilis. D. americana americana, D. americana texana and D. novamexicana showed 2 sites labeled; no label was seen at S_d–f while W_c–j was weakly labeled and X_a–e was strongly labeled, the reciprocal of the previous pattern in the W-X region. Hybrids between D. a. americana and D. virilis showed no difference in chromosome banding at the sites of the 5S clusters despite their pattern differences. D. a texana x D. virilis, on the other hand, did show a difference in staining the X_a–e region. These patterns fall squarely into the biosystematic groupings deduced by many previous workers.     

Variation of wing shape in the Drosophila virilis species group (Diptera: Drosophilidae)

Wing shape variation was analysed with geometric morphometric methods in 17 laboratory strains, representing 11 closely related species (including two subspecies) of the Drosophila virilis group: D. virilis, D. lummei, D. novamexicana, D. americana americana, D. americana texana, D. montana, D. lacicola, D. flavomontana, D. borealis, D. littoralis, D. ezoana and D. kanekoi. Overall shape estimated using Procrustes coordinates of 14 landmarks was highly variable among strains and very similar in females and males. The landmarks in the distal part of the wing showed higher variation across strains than those in the proximal part. Procrustes distances between species were not consistent with phylogenetic distances previously suggested for the virilis group. Moreover, Procrustes distances between strains within species and within two major phylads (virilis and montana) were comparable with those between species and between phylads, respectively. The most different from other members of the group was the endemic D. kanekoi species, currently viewed as separate subphylad within the montana phylad. Allometric effects were found to be partly responsible for shape differences between the strains. Three most significant shape transformations were considered using the relative warp analysis and the strains were ordinated in accordance with transformation values. The pattern of relative warp scores could be easily interpreted only for the third warp explaining about 13% of shape variation. It separated the largest species, D. montana, D. ezoana and D. kanekoi, from other ones and was mainly associated with shape changes in the proximal region of the wing. The results of the present work suggest that wing shape in the virilis species group is not related to the speciation process. The observed proximal‐distal contrasts and allometric effects are in agreement with data of other studies, in which wing shape variation was analysed within Drosophila species.     

The ontogeny of color: developmental origins of divergent pigmentation in Drosophila americana and D. novamexicana

Pigmentation is a model trait for evolutionary and developmental analysis that is particularly amenable to molecular investigation in the genus Drosophila. To better understand how this phenotype evolves, we examined divergent pigmentation and gene expression over developmental time in the dark‐bodied D. americana and its light‐bodied sister species D. novamexicana. Prior genetic analysis implicated two enzyme‐encoding genes, tan and ebony, in pigmentation divergence between these species, but questions remain about the underlying molecular mechanisms. Here, we describe stages of pupal development in both species and use this staging to determine when pigmentation develops and diverges between D. americana and D. novamexicana. For the developmental stages encompassing pigment divergence, we compare mRNA expression of tan and ebony over time and between species. Finally, we use allele‐specific expression assays to determine whether interspecific differences in mRNA abundance have a cis‐regulatory basis and find evidence of cis‐regulatory divergence for both tan and ebony. cis‐regulatory divergence affecting tan had a small effect on mRNA abundance and was limited to a few developmental stages, yet previous data suggests that this divergence is likely to be biologically meaningful. Our study suggests that small and developmentally transient expression changes may contribute to phenotypic diversification more often than commonly appreciated. Recognizing the potential phenotypic impact of such changes is important for a scientific community increasingly focused on dissecting quantitative variation, but detecting these types of changes will be a major challenge to elucidating the molecular basis of complex traits.     

Analysis of DNAs from two species of the virilis group of Drosophila and implications for satellite DNA evolution

The DNAs from two virilis group species of Drosophila, D. lummei and D. kanekoi, have been analyzed. D. lummei DNA has a major satellite which, on the basis of CsCl equilibrium centrifugation, thermal denaturation, renaturation and in situ hybridization is identical to D. virilis satellite I. D. kanekoi DNA has a major satellite at the same buoyant density in neutral CsCl gradients as satellite III of D. virilis. However, on the basis of alkaline CsCl gradients, the satellite contains a major and a minor component, neither one of which is identical to D. virilis satellite III. By in situ hybridization experiments, sequences complementary to the major component of the D. kanekoi satellite are detected in only some species and in a way not consistent with the phylogeny of the group. However, by filter hybridization experiments using nick-translated D. kanekoi satellite as well as D. lummei satellite I and D. virilis satellite III DNAs as probes, homologous sequences are detected in the DNAs of all virilis group species. Surprisingly, sequences homologous to these satellite DNAs are detected in DNAs from non-virilis group Drosophila species as well as from yeast, sea urchin, Xenopus and mouse.     

Inheritance of cold shock tolerance in hybrids of Drosophila virilis and Drosophila lummei

The genetic basis of the difference in cold shock tolerance between the southern temperate Drosophila virilis and its boreal relative D. lummei is studied. After adult eclosion, the parental stocks, reciprocal F₁ and backcross hybrids were pretreated for eight days at 18°C or at 6°C. The cold shock used consisted of fast cooling to-10°C and exposure to this temperature for varying lengths of time. D. lummei tolerated such exposure for 40–50% longer than did D. virilis (100–135% after acclimation). Reciprocal F₁ females, differing only in their maternal cytoplasm deviated significantly from each other, and the reciprocal F₁ males even more so, the contribution of the X chromosome being three to four times that of the cytoplasm. The cold resistance scores of the hybrid males were more extreme than those of the parental stocks. Autosomally heterozygous males with the X chromosome and cytoplasm of virilis were the weakest flies studied. The reciprocal males (X chromosome and cytoplasm of lummei) survived better than the parental lummei stock. The reciprocal differences decreased after cold temperature acclimation. The roles of the four major autosomes were analyzed by backcrossing the reciprocal F₁ males with females of the virilis marker stock. The third chromosome of lummei as heterozygous contributed most to cold tolerance, while the other autosomes had a rather weak effect in the opposite direction (virilis homozygotes survived better), which disappeared after acclimation at 6°C. Some of the cold susceptibility of F₁ hybrids disappeared in chromosomally identical backcross flies, indicating complex cytoplasmchromosomal interactions.     

Evolution and arrangement of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in two closely related species of the <Emphasis Type="Italic">virilis</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

To investigate the genetic basis of differing thermotolerance in the closely related species Drosophila virilis and Drosophila lummei, which replace one another along a latitudinal cline, we characterized the hsp70 gene cluster in multiple strains of both species. In both species, all hsp70 copies cluster in a single chromosomal locus, 29C1, and each cluster includes two hsp70 genes arranged as an inverted pair, the ancestral condition. The total number of hsp70 copies is maximally seven in the more thermotolerant D. virilis and five in the less tolerant D. lummei, with some strains of each species exhibiting lower copy numbers. Thus, maximum hsp70 copy number corresponds to hsp70 mRNA and Hsp70 protein levels reported previously and the size of heat-induced puffs at 29C1. The nucleotide sequence and spacing of the hsp70 copies are consistent with tandem duplication of the hsp70 genes in a common ancestor of D. virilis and D. lummei followed by loss of hsp70 genes in D. lummei. These and other data for hsp70 in Drosophila suggest that evolutionary adaptation has repeatedly modified hsp70 copy number by several different genetic mechanisms.     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

Study on the somatic pairing of polytene chromosomes

Plasmids containing Drosophila virilis DNA (pDv118, pDv719, pDv714 and pDv117), characterized and localized on D. virilis chromosomes in Riede et al. (1983) were localized by in situ hybridization with polytene chromosomes of the hybrids D. virilis × D. lummei, D. virilis × D. novamexicana, and D. virilis × D. lacicola. The degree of somatic pairing was determined by comparing the four plasmids in the three hybrids. We found that somatic pairing in the polytene chromosomes decreased with decreasing DNA homology of the bands. — Additional cytological studies indicated that (1) each band can pair independently of its neighboring bands, (2) visible structural differences between bands have no influence on pairing of the surrounding chromosome region, (3) heteromorphic bands can pair by themselves, and (4) inversions of chromosome regions disturb the somatic pairing process but are not the primary cause for nonpairing in hybrids.     

The Importance of Acoustic Signals in Multimodal Courtship Behavior in Drosophila virilis,D. lummei and D. littoralis

The courtship rituals of Drosophila include an exchange of several signals with different modalities, chemical, visual, acoustic and tactile stimuli, between sexes. Using a video recording method, we studied the role of acoustic communication in courtship behavior in three species of the Drosophila virilis group, D. virilis, D. lummei and two populations of D. littoralis. Five series of experiments were performed: tests with intact flies (control), tests with mute flies (wingless males or females), and tests with deaf flies (aristaless males or females). We distinguished the two situations: either a female did not hear a male or vice versa, males did not hear females. When females did not hear males, the reduction in the copulation number was found in D. virilis and both populations of D. littoralis, but not in D. lummei. When males did not hear females, the reduction in the copulation number was only found in D. littoralis. The ablation of the male aristae in D. virilis and D. lummei even increased the mating success as compared to the control, which may be explained by the ‘sensory overload’ hypothesis. The changes in courtship temporal structure usually included the delayed onset of the main courtship elements (tapping, licking, and singing), and the variation in their duration and the total time of courtship. These effects were, however, more substantial in D. virilis and both populations of D. littoralis than in D. lummei. Thus, the effect of blocking the acoustic channel was different in the three species regardless of their phylogenetic relationship, and the role of acoustic communication in courtship behavior seemed to increase in the order D. lummei – D. virilis – D. littoralis.

     

THE GENETIC BASIS OF EVOLUTION OF THE MALE COURTSHIP SOUNDS IN THE DROSOPHILA VIRILIS GROUP

When courting, males of the Drosophila virilis group vibrate their wings and emit species-specific courtship sounds consisting of trains of polycyclic sound pulses. To analyze the genetic basis of evolutionary changes in the sounds we made an F₁ diallel set of reciprocal crosses between the members of the virilis phylad of the group (two stocks of D. virilis and one of D. americana americana, D. a. texana, D. novamexicana, and D. lummei). We also crossed the D. virilis stocks with the members of the montana phylad of the same group (D. kanekoi, D. littoralis, D. borealis, D. flavomontana, D. lacicola, and D. montana) and made a backcross (D. virilis x D. littoralis) x D. virilis using a D. virilis marker stock (b; sv t tb gp; cd; pe). The sounds of the hybrids were analyzed using the following parameters: the length of a pulse train (PTL), the number of pulses in a train (PN), the interpulse interval (IPI), the length of a pulse (PL), the number of cycles in a pulse (CN), and the length of a cycle (CL). In the virilis phylad, the differences between species appeared to be determined mainly by autosomal genes in each sound trait. The heritabilities (narrow-/broad-sense) obtained from the diallel tables were the following: PTL 0.662/0.817, PN 0.651/0.841, IPI 0.193/0.546, PL 0.408/0.552, CN 0.425/0.719, and CL 0.361/0.764. The direction of dominance is for longer PTL, higher PN and CN, and shorter IPI and CL. PL shows ambidirectional dominance. In the sounds of the virilis phylad species, PTL and PL seem to be phenotypically the most important parameters, since their components (PN and IPI for PTL, CN and CL for PL) are negatively correlated. In crosses between D. virilis and D. littoralis or D. flavomontana reciprocal hybrids differed from each other in PTL, IPI, PL, and CN indicating X-chromosomal or cytoplasmic inheritance. In the backcrosses between D. virilis and D. littoralis the role of the X chromosome was ascertained to be decisive. We conclude that an X-chromosomal major change allowing variation in IPI has occurred during the separation of the two D. virilis group phylads, the long IPI allowing variation also in PL (and CN). The evolution of the sounds in the virilis phylad has probably gone towards longer and denser pulse trains, while in the montana phylad the sounds have evolved in different directions.     

Local adaptation for body color in Drosophila americana

Pigmentation is one of the most variable traits within and between Drosophila species. Much of this diversity appears to be adaptive, with environmental factors often invoked as selective forces. Here, we describe the geographic structure of pigmentation in Drosophila americana and evaluate the hypothesis that it is a locally adapted trait. Body pigmentation was quantified using digital images and spectrometry in up to 10 flies from each of 93 isofemale lines collected from 17 locations across the United States and found to correlate most strongly with longitude. Sequence variation at putatively neutral loci showed no evidence of population structure and was inconsistent with an isolation-by-distance model, suggesting that the pigmentation cline exists despite extensive gene flow throughout the species range, and is most likely the product of natural selection. In all other Drosophila species examined to date, dark pigmentation is associated with arid habitats; however, in D. americana, the darkest flies were collected from the most humid regions. To investigate this relationship further, we examined desiccation resistance attributable to an allele that darkens pigmentation in D. americana. We found no significant effect of pigmentation on desiccation resistance in this experiment, suggesting that pigmentation and desiccation resistance are not unequivocally linked in all Drosophila species.     

Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.     

Aggression and Mating Success in Three Species of Drosophila1

Three closely related species of Drosophila: D. virilis, D. americana, and D. novamexicana, are known to differ in levels of male-male aggression. Through direct observation in the laboratory, we attempted to determine and characterize the relationships between intrasexual aggression and mating success in males of each of these species. Our results indicated that the most important determinant of male mating success was not the amount of aggression performed by a male, but rather the amount of aggression directed towards him.     

The Genetics of Postmating,Prezygotic Reproductive Isolation Between Drosophila virilis and D. americana

Many studies have demonstrated the rapid diversification of reproductive genes that function after mating but before fertilization. This process might lead to the evolution of postmating, prezygotic barriers between species. Here, I investigate the phenotypic and genetic basis of postmating, prezygotic isolation between two closely related species of Drosophila, Drosophila virilis and D. americana. I show that a strong barrier to interspecific fertilization results in a 99% reduction in progeny production. A genetic interaction among maternal and paternal alleles at only a few loci prevents the fertilization of D. virilis females by D. americana males. These loci are autosomal and isolation acts recessively; the fertilization incompatibility is caused by at least two loci in the maternal D. virilis parent in combination with at least three loci in the paternal D. americana parent. These findings, together with results from classical experiments, suggest that male–female coevolution within D. americana may have driven postmating, prezygotic isolation between species.AN understanding of speciation requires insight into the origins and mechanisms of reproductive isolation. Divergent selection on traits that facilitate mating or fertilization might eventually lead to incompatibilities between males and females of incipient species. In animals, it has long been recognized that sexual selection can promote the evolution of specialized courtship rituals or elaborate phenotypic displays to attract mates (Darwin 1871). Similarly, sexual selection can be a powerful evolutionary force during or after mating by affecting the many biochemical, physiological, and morphological mechanisms involved in fertilization (Eberhard 1996). Postmating reproductive traits might also be subject to sexually antagonistic coevolution, whereby a difference in the reproductive interests of males and females leads to an evolutionary arms race between the sexes (Rice 1996). Just as divergent sexual selection on mate signals and preferences might give rise to premating (sexual) isolation (reviewed in Ritchie 2007), postcopulatory sexual selection and sexual conflict might promote the evolution of postmating barriers to fertilization or hybrid incompatibilities (Howard 1999; Wu and Davis 1993). Indeed, these evolutionary forces have apparently led to competitive gametic isolation (Price 1997; Price et al. 2000; Fishman et al. 2008) and sperm–egg incompatibilities (Galindo et al. 2003). Moreover, because sexual selection and antagonistic coevolution can act rapidly (Fisher 1930; Rice 1996), they might be particularly important in the early stages of speciation.In diverse animal taxa, sexual selection and/or sexual conflict are thought to drive rapid evolution of a variety of postmating reproductive traits, including male genital morphology (Eberhard 1996), length of sperm and female sperm-storage organs (Pitnick et al. 1997; Miller and Pitnick 2002), ejaculate composition (e.g., Swanson et al. 2001a; Dorus et al. 2004), female reproductive tract proteins (e.g., Lawniczak and Begun 2007; Kelleher et al. 2007), and gamete recognition molecules (e.g., Wyckoff et al. 2000; Swanson et al. 2001b). In recent years, many studies have also documented strong signatures of positive selection in the rapid evolution of reproductive genes (e.g., Haerty et al. 2007; Turner et al. 2008; reviewed in Swanson and Vacquier 2002; Clark et al. 2006). For internally fertilizing species, coevolution between the female reproductive tract and the male ejaculate is particularly dynamic (Pitnick et al. 2007). For example, in Drosophila, hundreds of nonsperm seminal fluid proteins are transferred during mating, including many fast-evolving accessory gland proteins (ACPs) (Swanson et al. 2001a; Wagstaff and Begun 2005). As expected, there is evidence for coordinated evolution of female reproductive tract genes, which also show elevated rates of evolution in Drosophila (Panhuis and Swanson 2006; Prokupek et al. 2008). But what are the consequences of such rapid rates of diversification? How many of these fast-evolving reproductive genes contribute to isolating barriers? Major progress toward addressing these questions would require identifying and characterizing individual loci that cause postmating, prezygotic isolation.A large body of classical work suggests that the Drosophila virilis species group might represent an ideal model for studying the genetics of reproductive isolation (Patterson and Stone 1952); and importantly, the D. virilis genome sequence is now available. There is also evidence that postmating, prezygotic isolation may be significant among D. virilis and the closely related North American species, D. americana and D. novamexicana. Patterson et al. (1942) describe reproductive isolation due to “gamete mortality” in reciprocal crosses between D. virilis and D. americana. In later studies, these authors discovered that very few eggs from interspecific crosses become fertilized or hatch and speculate that sperm become “immobilized in the reproductive tract of the alien female” (Patterson and Stone 1952). Moreover, a recent study has found a similar problem with fertilization in crosses between D. americana and D. novamexicana (Y. Ahmed and B. McAllister, personal communication). Consistent with the evolution of these interspecific barriers, male and female reproductive tract proteins have been shown to evolve rapidly in the D. virilis species group (Civetta and Singh 1995; Haerty et al. 2007). In addition, females of both D. virilis and D. americana produce a large opaque vaginal mass in response to mating (the “insemination reaction”; Wheeler 1947), which almost certainly reflects an evolutionary history of interaction between the female reproductive tract and male ejaculate (Knowles and Markow 2001).Despite the potential importance of postmating, prezygotic isolation in D. virilis group divergence, almost nothing is known about its genetic architecture. On the basis of the results from their crosses between D. virilis and D. americana, Patterson et al. (1942) infer that postmating isolation involves recessive autosomal genes. However, their experiments often cannot distinguish between the effects of the apparent fertilization incompatibility and premating isolation, the latter also being strong between D. americana females and D. virilis males (Stalker 1942). Their genetic mapping studies were also crude.In this study, I have two main objectives. First, I characterize the phenotypic basis of postmating isolation between D. virilis and D. americana. To do so, I perform a series of crosses within and between species. I find that low F₁ hybrid production between D. virilis and D. americana is due primarily to a reduction in interspecific fertilization; females presented with heterospecific males almost always become inseminated, but very few eggs are fertilized. Second, I perform a detailed genetic analysis of the fertilization incompatibility between D. virilis females and D. americana males. Using the D. virilis genome assembly, I developed molecular markers targeted to genomic regions of interest for high-resolution genetic mapping of both the maternal and paternal components of isolation. This study is a first step toward understanding the genetic and evolutionary mechanisms of postmating, prezygotic reproductive isolation in Drosophila.