Heterospecific Songs Research Articles

An Acoustic Password Enhances Auditory Learning in Juvenile Brood Parasitic Cowbirds.

How does a naive, young animal decide from which adults to learn behavior? Obligate brood parasitic birds, including brown-headed cowbirds (Molothrus ater), face a particular challenge in learning species-specific behaviors; they lay their eggs in the nest of another species, and juveniles are raised without exposure to adult conspecifics. Nevertheless, male cowbirds need to learn a conspecific song to attract appropriate mates, and female cowbirds need to learn to identify conspecific males for mating. Traditionally, it was thought that parasitic bird species rely purely on instinctual species recognition [1-4], but an alternative is that a species-specific trait serves as a "password" [5], a non-learned cue for naive animals that guides decisions regarding from whom to learn. Here, we tested the hypothesis that the adult "chatter call" enhances the learning of specific songs in juvenile cowbirds. We exposed acoustically naive juvenile male and female cowbirds to songs paired with chatter calls and found that the chatter call enhanced song production learning in males and induced a neurogenomic profile of song familiarity in females, even for heterospecific songs. Thus, a combination of experience-independent and -dependent mechanisms converges to explain how young cowbirds emerge from another species' nest yet learn behaviors from conspecifics. Identifying whether such password-based mechanisms relate to perceptual and behavioral learning in non-parasitic taxa will contribute to our general understanding of the development of social recognition systems.

Partners coordinate territorial defense against simulated intruders in a duetting ovenbird.

Duets in breeding pairs may reflect a situation of conflict, whereby an individual answers its partner's song as a form of unilateral acoustic mate guarding or, alternatively, it may reflect cooperation, when individuals share in territory defense or safeguard the partnership. The degree of coordination between the sexes when responding to solo versus paired intruders may elucidate the function of songs in duets. We examined this issue in a study with rufous horneros (Furnarius rufus), a duetting, socially monogamous Neotropical species with low levels of extrapair paternity. We exposed social pairs during the nonbreeding season to playbacks of duets, male solos, female solos, and control heterospecific songs. Partners approached all conspecific stimuli together and responded by singing quickly, at higher rates and by coordinating ~80% of their songs into duets. For both sexes, most response variables (seven of nine) did not vary across conspecific treatments. These results suggest that partners duet and coordinate behaviors to cooperatively defend common territories. However, females spent more time in territorial vigilance, and partners were highly coordinated (correlated responses) in response to duets and female solos in comparison with male solos. This indicates that female intrusions (paired or solo) might be more threatening than male intrusions in the nonbreeding season, especially for territorial females, and that females are less cooperative with their partners in territory defense against male intruders.

Female cognitive performance and mass are correlated with different aspects of mate choice in the zebra finch (Taeniopygia guttata).

A female's cognitive ability may influence her mate preferences through various mechanisms. These mechanisms include the direct effect of cognitive ability on the information-processing skills used during mate choice, and the indirect effect of cognitive ability on quality when females mate assortatively. Here, we examined whether the ability to learn a novel foraging task, a cognitive skill which has been associated with reproductive success in other capacities, was correlated with song preferences in female zebra finches (Taeniopygia guttata). Female preferences were measured in an operant testing chamber where hops on a perch triggered song playback. Females were given the choice of (1) conspecific vs. heterospecific song and (2) high-quality male vs. low-quality male conspecific song. We found that female performance on the novel foraging task was positively correlated with preference for conspecific song, but not with preference for high-quality male song. Instead, female mass was positively correlated with preference for high-quality male song, potentially signifying that female mass is a stronger predictor of female quality in assortative mating than female cognitive performance. Female mass and cognitive performance were unrelated. Our results suggest that the particular traits of a female that affect conspecific preference do not necessarily affect preference for high-quality males.

Symmetrical discrimination despite weak song differentiation in 2 suboscine bird sister species

Abstract Song mediates territorial competition and mate choice in birds and population divergence in this signal can have important evolutionary consequences. For example, divergent songs can act in specific recognition and limit gene flow and, hence, have a fundamental role on the origin and/or integrity of evolutionary lineages. Especially interesting systems to test the role of song in specific recognition are species pairs that present small structural differences in this signal. Here, we perform song play-back experiments on males of a long-diverged sister pair of Neotropical Suboscine species, the squamate antbird (Myrmoderus squamosus) and the white-bibbed antbird (Myrmoderus loricatus), which occur in parapatry in the Atlantic Forest and that overlap extensively in song variation. Previous evidence indicates that genetic introgression between these species is either absent or negligible, suggesting that vocal discrimination or other mechanisms function as effective barriers to gene flow. Our results show that responses to heterospecific songs were symmetrical and intermediary compared with responses to conspecific songs in both species. A stronger response to conspecific territorial songs suggests that conspecific individuals pose greater competitive threat than heterospecifics. An important implication of our study is that even small song differences can play an important role in specific recognition.

A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster

Many animals utilize auditory signals to communicate with conspecific individuals. During courtship, males of the fruit fly Drosophila melanogaster and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In D. melanogaster, a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. Traditionally, the fly’s response to the song is measured under grouped conditions, in which the effect of sensory modalities other than audition cannot be excluded. Here, to quantify the individual ability to discriminate a conspecific song, we systematically analyzed the auditory response of single male flies to sound with various parameters. Moreover, we applied this method, termed SMART (Single Male Auditory Response Test), to two sister species for potential application in a comparative approach. By quantifying the locomotor activity of single D. melanogaster males during sound exposure, we detected increased locomotor activity in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, D. sechellia, exhibited a locomotor response to pulse song, while D. simulans exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among Drosophila species.

Neural activation in response to conspecific songs in zebra finch (Taeniopygia guttata) embryos and nestlings.

Classic studies on the effects of auditory stimulation in embryonic birds have largely been limited to precocial taxa. In altricial taxa, physiological responses of embryos and, subsequently, the behavioral responses of nestlings have begun to receive increasing attention, yet it remains unclear whether and to what specificity neural responses are generated in ovo. Using in-situ hybridization for an immediate early gene, ZENK, we detected significant neural activation in both the embryos and nestlings of an altricial songbird, the zebra finch (Taeniopygia guttata) when exposed to conspecific song playbacks relative to silence. In turn, embryonic ZENK responses to heterospecific songs were intermediate in strength. These results are consistent with physiological evidence for conspecific song selectivity in embryos of other altricial songbird taxa.

Nestling and adult sparrows respond differently to conspecific dialects

Baby birds prefer local dialects. Songbirds have predispositions to learn songs of their own species over others, but what about species that have regional dialects? And how different are responses of baby and adult birds? We show that golden-crowned sparrow chicks, before song learning, treat foreign songs like heterospecific songs. Adults, on the other hand, show graded responses to local, foreign and heterospecific songs. This study tells us how song responses change across life stages.

Elevated temperatures reduce discrimination between conspecific and heterospecific sexual signals

Heat waves are rapidly increasing in frequency and length around the globe. These periods of elevated temperatures are inducing extreme thermal stress in many organisms. One dramatic result of heat waves is mass mortality events, notably in birds. Although much attention is given to lethal events, little is known about how more common sublethal temperatures affect behaviour, especially in endotherms. For example, male song sparrows, Melospiza melodia atlantica, sing at lower rates at higher temperatures. We hypothesized that simulated heat waves may also reduce the ability of females to discriminate between sexual signals. We tested this hypothesis by quantifying whether female zebra finches, Taeniopygia guttata, responded more to conspecific song or heterospecific song while at their typical housing temperature compared to temperatures above their upper critical temperature. We collected data on how often the birds selected conspecific over heterospecific songs and how much effort the birds invested in sampling those songs using a matched-pairs design. During housing temperature trials, females preferred conspecific song compared to heterospecific song. In contrast, females no longer showed a preference for conspecific song at temperatures above their upper critical limit, although the birds invested similar effort in sampling songs in both treatments. Our findings provide the first experimental evidence that high temperatures affect female preference for a mating signal in an endotherm. Our results highlight the need for future work to investigate this overlooked effect of climate change on endotherms. Because thermoregulatory processes are phylogenetically conserved, our results generate useful predictions for future studies of heat wave effects on mating behaviours in endotherms.

Socially regulated estrogen in an eavesdropping brood parasite

Social regulation of reproductive hormones is a means by which conspecific males and females orchestrate successful reproductive efforts. We investigate whether social cues modify activity within the hypothalamic-pituitary-gonadal (HPG) axis and the specificity of this response in a social parasite that is known to eavesdrop on the communication signals of other species: the brown-headed cowbird (Molothrus ater). Brown-headed cowbirds are obligate brood parasites that do not build nests or care for their own young. Instead, obligate brood parasites leave their eggs in the nest of a host species and therefore must coordinate their breeding attempts with conspecifics as well as potential heterospecific hosts. Here, we explore whether the vocal signals of potential host species can also be used as a social cue that modifies the HPG axis of female brown-headed cowbirds. Results reveal that both conspecific and heterospecific song-exposed females exhibit significantly greater circulating estradiol concentrations as compared to silence-exposed females. While conspecific song induces the greatest elevation in circulating estradiol, there is no significant difference in circulating estradiol levels in females exposed to either conspecific or heterospecific songs. This pattern suggests both song types are effective at evoking a reproductive physiological response. On the other hand, circulating progesterone concentrations did not differ among the song- and silence-exposed groups nor did the size of the female’s ovarian follicles. These results indicate that heterospecific vocal communication signals can effectively be used as a social cue that simultaneously provides necessary information regarding breeding status of hosts and modifies breeding condition of the eavesdropper.

Early singers attend to conspecific but not heterospecific behavioural cues at dawn

During the dawn chorus songbirds initiate singing activities just prior to sunrise. Environmental factors affect the timing of the dawn chorus, but relatively little is known about how behavioural cues influence chorus timing. We assessed whether early playback of conspecific and heterospecific song influenced chorus start times in two early‐singing temperate species, hermit thrush Catharus guttatus and veery C. fuscescens. We used arrays of GPS time‐synchronized recorders to record the dawn chorus at 30 locations. In each location, thirty minutes prior to natural chorus initiation, we broadcast: 1) an early singing species (hermit thrush or veery), 2) a late singing species (black‐throated green warbler Setophaga virens or Nashville warbler Oreothlypis ruficapilla), 3) a noise control, and 4) also recorded without playback (silent control). Playback treatments were separated by two‐three days at each location. Both hermit thrush and veery sang significantly earlier in response to conspecific playback compared to noise or silent controls. Both species also sang earlier in response to conspecific playback compared to the day before or the day after playback. Neither species sang earlier in response to heterospecific playback (regardless of whether the broadcast species was a naturally early or late song initiator) compared to noise or silent controls. Our results support the theory that the dawn chorus is a communication network where individuals attend primarily to conspecific cues; heterospecific song appears to have minimal impacts on the chorus start times of the two early‐singing species we investigated.

Subspecies status and methods explain strength of response to local versus foreign song by oscine birds in meta-analysis

To understand the implications of geographical variation in vocal culture in songbirds, researchers have often compared territorial responses to playback of local songs versus responses to playback of songs from ‘foreign’ conspecifics. This body of work has the potential to help us move towards a general understanding of factors driving divergence in signal recognition. We conducted a systematic review and meta-analysis of 57 playback studies to explain variation in strength of response to local versus foreign songs. Studies with incomplete reporting of results had elevated effects due to selective reporting. Studies that used small numbers of stimuli as exemplars (pseudoreplication) had more variable effects than studies without severe pseudoreplication. Whether or not we controlled for pseudoreplication, we found greater response to playback of local song than to foreign song. In investigating potential biological drivers of the variation in strength of experimental effects, we found that the difference in territorial response to local versus foreign song was stronger if the foreign song was recorded from another subspecies than if the foreign song was recorded from the same subspecies as the focal individuals. Indexes of risk of accidental response to heterospecific song did not coherently explain response to foreign conspecific songs, nor did factors expected to influence individual experience with foreign conspecific songs. Thus, although oscine songbirds clearly react more aggressively to local song than to foreign song and variation in the strength of this effect is influenced by methodological choices and subspecies status, considerable variation in the strength of response to local versus foreign song playback remains to be explained.

Early social experience alters transcriptomic responses to species-specific song stimuli in female songbirds

Amongst an array of stimuli from countless species, animals must recognize salient signals, including those of their own species. In songbirds, behavioral tests have demonstrated that preferences for conspecific male songs are determined by both preexisting biases and social experience with a male ‘tutor’ during the sensitive period for learning. Although immediate early gene expression (e.g. ZENK) and electrophysiological experiments generally find greater neural responses for conspecific songs, it remains unclear whether distinct mechanisms, such as sensory gating, are engaged to filter out irrelevant heterospecific songs. Here we compare the transcriptomic profiles, via RNA-seq, of non-singing females of a songbird, the zebra finch (Taeniopygia guttata), by focusing on the auditory forebrain, a region known to be critical in the processing of conspecific vs. heterospecific songs. Gene expression profiles demonstrate that different neural mechanisms are involved in the processing of conspecific versus heterospecific Bengalese finch (Lonchura striata) songs. In particular, one gene known to mediate sensory gating, the alpha 3 subunit member of nicotinic cholinergic receptors (CHRNA3), was significantly downregulated in response to hearing Bengalese finch song, but not when young females were tutored by a Bengalese male during early development. Overall, our results confirm previous behavioral and physiological studies, such that heterospecific-tutored individuals processed both conspecific and tutor songs similarly. Using transcriptomic profiling of peripheral blood samples, we also demonstrate the methodological potential of non-terminal sampling to identify transcriptomic biomarkers for conspecific auditory recognition. These results show how experience and inherited preferences facilitate the neural processing of salient songs by female songbirds.

Song ontogeny in Nuttall’s white-crowned sparrows tutored with individual phrases

Behavioral ontogeny involves the interaction of innate predispositions and experience. In bird song learning, one approach to exploring this interaction is to examine the songs rehearsed by young birds whose exposure to tutor models has been carefully controlled. Here, I analyzed the rehearsed repertoire in Nuttall’s white-crowned sparrows (Zonotrichia leucophrys nuttalli) tutored with individual phrases of conspecific and heterospecific songs. The proportions of phrase types rehearsed indicate that the learning biases evident in crystallized song are manifest early on, suggesting preferential memorization rather than preferential retention during attrition. The proportion of songs beginning with whistles increased during song rehearsal and phrase sequence variability decreased, consistent with the idea that innate syntax specifications guide song rehearsal. Single-phrase tutored birds overproduced phrases to the same extent previously observed in birds tutored with full, normal song but retained fewer phrase types in their crystallized repertoires. This suggests that in this subspecies, acquired syntax information does not affect the number of phrase types memorized and rehearsed but does affect repertoire attrition at the end of the sensorimotor phase. I discuss these results with a focus on the action of innate templates in song development and subspecies differences in this process.

The neural response of female zebra finches (Taeniopygia guttata) to conspecific, heterospecific, and isolate song depends on early-life song exposure

The auditory forebrain regions caudo-medial nidopallium (NCM) and caudo-medial mesopallium (CMM) of songbirds exhibit differential expression of the immediate-early gene ZENK in response to playback of different song stimuli, and dependent on early-life auditory experience. Similarly, song preferences depend both on auditory experience and unlearned biases for particular song features. We explored the contributions of early-life auditory experience and the type of song stimuli on the Zenk response in the auditory forebrain of female zebra finches. Females were raised in three different early tutoring conditions: conspecific tutors that sang isolate song, heterospecific tutors, or conspecific tutors that sang wild-type song. At maturity, these females were exposed to one of five different playback conditions: wild-type song, isolate song, tutor song, heterospecific song, or white noise. Subsequently, the number of cells immunoreactive for ZENK in CMM and NCM was measured. We predicted that birds exposed to conspecific song early in life, and during the song playback in adulthood, would have the highest neural response. Instead, we found that the Zenk response varied across playback conditions with the highest response to conspecific wild-type and conspecific isolate song. In addition, we found a main effect of tutoring, with the lowest overall Zenk response in females tutored by males singing isolate song. Most importantly, there was a significant interaction in that females tutored by wild-type conspecific or heterospecific songs showed a similar increased response to zebra finch songs (wild-type or isolate), but females tutored by isolate song showed no differential response to conspecific song and only showed elevated Zenk response to the particular songs they were tutored with. Combined, our results indicate that unlearned response biases to conspecific song elements depend on previous auditory experience. That is, early experience appears to modulate the expression of innate biases.

Familiar But Unexpected: Effects of Sound Context Statistics on Auditory Responses in the Songbird Forebrain.

Rapid discrimination of salient acoustic signals in the noisy natural environment may depend, not only on specific stimulus features, but also on previous experience that generates expectations about upcoming events. We studied the neural correlates of expectation in the songbird forebrain by using natural vocalizations as stimuli and manipulating the category and familiarity of context sounds. In our paradigm, we recorded bilaterally from auditory neurons in awake adult male zebra finches with multiple microelectrodes during repeated playback of a conspecific song, followed by further playback of this test song in different interleaved sequences with other conspecific or heterospecific songs. Significant enhancement in the auditory response to the test song was seen when its acoustic features differed from the statistical distribution of context song features, but not when it shared the same distribution. Enhancement was also seen when the time of occurrence of the test song was uncertain. These results show that auditory forebrain responses in awake animals in the passive hearing state are modulated dynamically by previous auditory experience and imply that the auditory system can identify the category of a sound based on the global features of the acoustic context. Furthermore, this probability-dependent enhancement in responses to surprising stimuli is independent of stimulus-specific adaptation, which tracks familiarity, suggesting that the two processes could coexist in auditory processing. These findings establish the songbird as a model system for studying these phenomena and contribute to our understanding of statistical learning and the origin of human ERP phenomena to unexpected stimuli.SIGNIFICANCE STATEMENT Traditional auditory neurophysiology has mapped acoustic features of sounds to the response properties of neurons; however, growing evidence suggests that neurons can also encode the probability of sounds. We recorded responses of songbird auditory neurons in a novel paradigm that presented a familiar test stimulus in a sequence with similar or dissimilar sounds. The responses encode, not only stimulus familiarity, but also the expectation for a class of sounds based on the recent statistics of varying sounds in the acoustic context. Our approach thus provides a model system that uses a controlled stimulus paradigm to understand the mechanisms by which top-down processes (expectation and memory) and bottom-up processes (based on stimulus features) interact in sensory coding.

Introductory whistle is sufficient for early song recognition by golden-crowned sparrow nestlings

Many songbird species have a predisposition to learn conspecific songs, suggesting song learning may be guided by an innate auditory template. Evidence for such a template includes preferential response to conspecific song in early life, even before song learning begins. A prime example of an innate cue for selective song learning is the introductory whistle of white-crowned sparrows, Zonotrichia leucophrys. The songs of its sister species, the golden-crowned sparrow, Zonotrichia atricapilla, also contain an introductory whistle, which differs in structure from that of white-crowned sparrows. Here we tested the ability of nestling golden-crowned sparrows in a sympatric population to discriminate between conspecific and heterospecific songs based on introductory whistles alone, prior to the onset of song learning. Golden-crowned sparrow nestlings responded with more chirps to playbacks of conspecific whistles than to heterospecific (white-crowned sparrow) whistles, and they responded similarly to full conspecific songs and conspecific whistles alone. We suggest that the introductory whistle alone is sufficient for song recognition in the golden-crowned sparrow. We discuss similarities and differences in the role of the introductory whistle between these sister taxa, and how this divergent song phrase may share a role in species recognition in both sister species. Identifying the cues underlying song recognition prior to song learning could be key to understanding the evolution of behavioural isolation between closely related songbird species.

Estradiol modulates neural response to conspecific and heterospecific song in female house sparrows: An in vivo positron emission tomography study.

Although there is growing evidence that estradiol modulates female perception of male sexual signals, relatively little research has focused on female auditory processing. We used in vivo 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging to examine the neuronal effects of estradiol and conspecific song in female house sparrows (Passer domesticus). We assessed brain glucose metabolism, a measure of neuronal activity, in females with empty implants, estradiol implants, and empty implants ~1 month after estradiol implant removal. Females were exposed to conspecific or heterospecific songs immediately prior to imaging. The activity of brain regions involved in auditory perception did not differ between females with empty implants exposed to conspecific vs. heterospecific song, but neuronal activity was significantly reduced in females with estradiol implants exposed to heterospecific song. Furthermore, our within-individual design revealed that changes in brain activity due to high estradiol were actually greater several weeks after peak hormone exposure. Overall, this study demonstrates that PET imaging is a powerful tool for assessing large-scale changes in brain activity in living songbirds, and suggests that after breeding is done, specific environmental and physiological cues are necessary for estradiol-stimulated females to lose the selectivity they display in neural response to conspecific song.

Sexual differentiation and seasonal variation in response to conspecific and heterospecific acoustic signals

AbstractInterspecific territoriality is frequently reported between closely related species; however, few studies have demonstrated interspecific territoriality between distantly related species living in sympatry. We conducted playback experiments to investigate territorial behaviour in male and female White‐bellied Wrens (Uropsila leucogastra) in response to simulated conspecific and heterospecific intruders during the breeding and non‐breeding seasons. We explored whether heterospecific songs of the Happy Wren (Pheugopedius felix), a distantly related species and ecological competitor, elicited antagonistic responses from focal White‐bellied Wrens, and whether such responses differed between the sexes. We also examined whether male and female responses to conspecific and heterospecific rivals varied with season. We found that male White‐bellied Wrens always responded to conspecific song, and responded significantly more to heterospecific song compared to a control stimulus (Tropical Parula,Setophaga pitiayumi). In contrast, although female White‐bellied Wrens responded strongly to conspecific song, their response to heterospecific song did not differ significantly from the control stimulus. The proportion of males that responded to heterospecific songs and the proportion of females that responded to conspecific songs varied seasonally, showing significantly lower responses during the breeding season. The intense responses of male White‐bellied Wrens to playback of heterospecific songs suggest that they recognise ecological competitors based on their vocal signals. Furthermore, the decrease in agonistic interactions during the breeding season is in line with the hypothesis that aggressive behaviour may be detrimental to reproductive and parental activity, and the hypothesis that heterospecific animals pose less of a threat during the breeding season.

Reproductive character displacement of female, but not male song discrimination in an avian hybrid zone.

Divergence of male sexual signals and female preferences for those signals often maintains reproductive boundaries between closely related, co-occurring species. However, contrasting sources of selection, such as interspecific competition, can lead to weak divergence or even convergence of sexual signals in sympatry. When signals converge, assortative mating can be maintained if the mating preferences of females diverge in sympatry (reproductive character displacement; RCD), but there are few explicit examples. Pied flycatchers (Ficedula hypoleuca) are sympatric with collared flycatchers (F. albicollis) on the Baltic island of Öland, where males from both species compete over nestboxes, their songs converge, and the two species occasionally hybridize. We compare song discrimination of male and female pied flycatchers on Öland and in an allopatric population on the Swedish mainland. Using field choice trials, we show that male pied flycatchers respond similarly to the songs of both species in sympatry and allopatry, while female pied flycatchers express stronger discrimination against heterospecific songs in sympatry than in allopatry. These results are consistent with RCD of song discrimination of female pied flycatchers where they co-occur with collared flycatchers, which should maintain species assortative mating despite convergence of male sexual signals.

The role of courtship song in female mate choice in South American Cactophilic Drosophila.

Courtship songs have undergone a spectacular diversification in the Drosophila buzzatii cluster. Accordingly, it has been suggested that sexual selection has played a significant role in promoting rapid diversification, reproductive isolation and speciation. However, there is no direct evidence (i.e., song playback experiments with wingless males) supporting this tenet. Moreover, several studies have showed that the courtship song in the genus Drosophila is not always used in female mate choice decisions, nor plays the same role when it is taken into account. In this vein, we use an approach that combines manipulative and playback experiments to explore the importance and the role of courtship song in female mate choice in four species of the D. buzzatii cluster and one species of the closely related D. martensis cluster for outgroup comparison. We also investigate the importance of courtship song in sexual isolation in sympatry between the only semi-cosmopolitan species, D. buzzatii, and the other species of the D. buzzatii cluster. Our study revealed that the courtship song is used by females of the D. buzzatii cluster as a criterion for male acceptance or influences the speed with which males are chosen. In contrast, we showed that this characteristic is not shared by D. venezolana, the representative species of the D. martensis cluster. We also found that the studied species of the D. buzzatii cluster differ in the role that conspecific and heterospecific songs have in female mate choice and in sexual isolation. Our findings support the hypothesis that divergence in female preferences for courtship songs has played a significant role in promoting rapid diversification and reproductive isolation in the D. buzzatii cluster. However, evidence from D. venezolana suggests that the use of the courtship song in female mate choice is not a conserved feature in the D. buzzatii complex.