Preferentially mating with one's own species (behavioral isolation) is an important barrier to gene flow separating many animal species 1. The complex behaviors associated with mating allow organisms to choose the best mate and/or to distinguish between species. The evolution of these behaviors is hypothesized to be driven by female choice because of the larger reproductive investment into eggs relative to sperm. A female with a limited number of eggs will maximize her fitness by producing viable, fertile progeny, and should therefore strongly prefer to mate with a conspecific male. In contrast, males often invest little in reproduction and are thus predicted to be less discriminate with respect to their mating partners; their fitness will be maximized by mating with as many females as possible, and the costs of mating with heterospecifics may be much less. However, these oversimplifications ignore male investment of time and effort into courtship rituals that either result in no mating or in sterile progeny. If the courtship or reproductive investment is substantial [e.g. nuptial gifts in insects 2], or if time for mating is limited because of a short reproductive window, then selection would also increase male mating discrimination.

Still, the reigning paradigm in studies of reproductive behavior is that males are often indiscriminate in mating 3. This paradigm has been most recently challenged by innovative experiments testing for the ability of males to learn to avoid heterospecific courtship after repeated exposure to heterospecific females 45. Trinidadian guppies (Poecilia reticulata) living in allopatry show little male mating discrimination, but upon exposure to their sister species, P. picta, quickly learn to avoid interspecific crosses 5. This learned discrimination appears to have become assimilated in sympatric populations 6. Similarly, Drosophila melanogaster males were originally described as indiscriminate [e.g. 7]. However, Dukas 4 found that D. melanogaster males exposed to D. simulans females display significantly fewer mating attempts with heterospecifics than males not exposed to heterospecific females. Thus putatively indiscriminate males can learn to avoid heterospecific courtship, and they imply that there is a cost to indiscriminate male courtship or mating. These findings suggest that male species discrimination may be very common but have been missed because of unnatural laboratory mating designs (i.e., exclusive use of naïve males never exposed to females before).

We test the generality of learned male mating discrimination by examining a pair of North American fruit flies, D. pseudoobscura and D. persimilis. Males of these species have been shown previously to court D. pseudoobscura and D. persimilis females at equal frequencies in several types of both choice and no-choice experiments 89, but all of these experiments employed naïve males. Matings between these two species result in fertile females and sterile males; thus there may be some cost of even successful interspecies mating for males. Nonetheless, F₁ hybrids have been collected from nature 10, and a molecular signature of interspecies introgression has been documented [e.g. 11].

If the findings of Dukas 4 in D. melanogaster are general to the genus, we predict that D. pseudoobscura males will be more reluctant to court heterospecific females after being exposed to and rejected by a heterospecific female. Such discrimination may be more apparent in strains derived from sympatric versus allopatric populations, as has been described for females of these species 12.

If D. pseudoobscura males learn to avoid courting heterospecifics after earlier exposure, we predict that the "experienced" males should exhibit 1) a longer courtship latency, 2) fewer courtship wing vibrations, and 3) fewer attempted copulations than the "naïve" males. We also note that, in all of our experiments, male courtship was both intense and consistent, hence providing no obvious indication that males were at all reluctant to court heterospecific females.

Across most of our mating experiments, we did not observe differences in Drosophila pseudoobscura courtship intensity of heterospecific females by males that had previously courted heterospecific females vs. males that had been maintained in isolation. Hence, we failed to find evidence that D. pseudoobscura males learn to avoid futile courtship of heterospecific females after being previously rejected by them. These findings contrast similar studies in D. melanogaster 4 and guppies 5 and suggest that the process of male learning to avoid courting heterospecifics is not universal. These findings also further evince that D. pseudoobscura males are indiscriminate of species identity in their courtship of females [see also 813].

In one set of experiments, the naïve males of the Mather, California, strain displayed significantly higher intensity of courtship, as measured by frequencies of wing vibration and attempted copulation, and mating success than the experienced males in the first mating assays. However, we interpret this to be an artifact of the removal of a large fraction of males that are most vigorous and most attractive to D. persimilis females from the experienced group (see Results), as this difference was not apparent (and in fact, opposite in direction) in assays of the same strain when fewer males were excluded. The same phenomenon may have also operated in 8-day posteclosion males from the Flagstaff, Arizona, strain (experiment 1), but the smaller sample size of those males tested likely rendered the result statistically nonsignificant (see Table 1). The high mating propensity of older flies [e.g. 1415] contributed to this artifact. Although our three mating assays cannot be statistically compared because they were not temporally controlled, our results seem to confirm the published findings of lower receptivity of young females (see Table 1). Hence, we interpret our results from the second and third mating assays to be generally less biased for evaluating the hypothesis of learned mating discrimination, since a much smaller fraction of (likely more vigorous) males were removed. The only statistically significant difference observed in these latter mating assays was opposite to that predicted by the hypothesis that males learn to avoid fruitless courtship of heterospecific females (i.e. the experienced males of the Mather, California, strain elicited a higher frequency of wing vibrations than the corresponding naïve males in the second mating assays).

Some differences seem apparent in various parameters between our six experiments (two lines × three treatments), such as possible differences between the Flagstaff and Mather lines in courtship latency in our second experiment. However, we stress that Drosophila courtship is labile to subtle environmental factors, and comparisons between experiments that were not temporally controlled are suspect. For the conclusions we present here, we focus only on temporally controlled studies where exactly equal numbers of flies were tested at the same times on the same days on the same batch of media, etc.

In our experiment, we did not exactly replicate the conditions used by Dukas 4 in his study of D. melanogaster, but our changes in design were done to better simulate the natural history differences between D. pseudoobscura and D. melanogaster. Unlike D. melanogaster, courtship in D. pseudoobscura is very rapid, and females appear to evaluate males individually based on a sequential encounter model 16. As a result, we shortened the exposure treatments in our study and also used single males and females. While some of these design differences may affect the comparability of our results to those of Dukas 4, we argue that our approach more accurately reflects (to the best of our knowledge) the behavior of D. pseudoobscura in nature.

The evolutionary reason for a potential difference in male learning ability among Drosophila species is uncertain. D. pseudoobscura males should encounter some cost through futile courtship of heterospecific females, and even if mating would be successful, half the F₁ progeny would be sterile. This mismating cost has putatively driven the evolution of greater female discrimination in this species 12. One could speculate, however, that courtship is much shorter or less energy-intensive in D. pseudoobscura than in D. melanogaster, which would be consistent with the very short mating latencies observed in the laboratory 8 and the field 16 in the former species. Alternatively, a high level of 7-tricosene, a known anti-aphrodisiac for D. melanogaster males 17, in the cuticular hydrocarbon profile of D. simulans virgin females 18 can serve as a strong conditioning agent of male behavior in D. melanogaster 19. 7-tricosene is the major component of the cuticular hydrocarbon profile of D. melanogaster males 20. It is remarkable that the concentration of 7-tricosene increases in D. melanogaster mated females, and hence renders them unattractive to males 1720. Both virgin females of D. pseudoobscura and D. persimilis have a slightly higher proportion of 2-methyl hexacosane (2-MH) in their cuticular hydrocarbon profiles 21 than males, and hence D. persimilis may not provide a strong conditioning agent that can render male behavior in D. pseudoobscura. Finally, there may simply be less detectable phenotypic difference between D. pseudoobscura and D. persimilis than between D. melanogaster and D. simulans, which is consistent with human observations of these species pairs.

Certainly, our assays did not mimic the natural setting perfectly, and available data on reproductive ecology of D. pseudoobscura and D. persimilis in natural populations are limited. Hence, we cannot completely exclude the possibility that male learning to avoid heterospecific courtship does occur in nature and that we could not detect it because of an unknown environmental variable. However, the confinement periods we used were still longer than courtships observed in nature 16. We also used adult males at two different ages, 3-day and 8-day posteclosion, and the courtship intensity of D. pseudoobscura males toward heterospecific females did not change in the second and third mating assays.

Selection should be strongest in species for which males invest large amounts of time into finding mates, courtship, and/or rearing offspring. Thus, the breeding ecology of a species is likely a very influential factor in determining if male discrimination evolves. Studies of P. reticulata show that fish living in sympatry with their sister species discriminate against heterospecifics, while those living in allopatry are indiscriminate but quickly learn to avoid matings with heterospecifics 5. This male discrimination against heretospecific females can become innate in the places where P. reticulata co-occur with P. picta, and lead to reproductive character displacement between the two species 6. While we lack basic natural history information in D. pseudoobscura, our study suggests the possibility that differences in breeding ecology between D. pseudoobscura and D. melanogaster may have led to courtship being less costly in D. pseudoobscura such that there is no selective pressure for males to discriminate.

We failed to find evidence that Drosophila pseudoobscura males moderate their subsequent courtship of heterospecific females after encountering them and being rejected by them. Although some differences in experimental design were employed to more precisely reflect the natural history of our focal species, these findings nonetheless contrast published findings from studies of D. melanogaster 4. As a result, we conclude that learning to avoid heterospecific courtship does not appear to be universal, even within the genus Drosophila, and may possibly be dependent on the natural history of the species.

MAFN conceived the study and coordinated its execution. The first mating assays were performed by KMW. The second and third mating assays were performed NPK and EVK. NPK performed the statistical analyses. All authors contributed to preparation of the manuscript.