Song Traits Research Articles

The songs of male pied flycatchers: exploring the legacy of the fathers.

Singing is a key element of songbirds’ behavioral repertoire, particularly for males, which sing during the breeding season to defend resources against other males and to attract females. Different song traits may convey honest information about males’ qualities or conditions, which may be used by females to select their mates. Traits under strong sexual selection have an important component of additive genetic variation (i.e., the main genetic inheritance from parents), and so relatively high heritability; therefore, it can be expected that song traits also do. Although the act of singing is an innate behavior, and thus, genetically determined, songbirds need to learn their songs and therefore the genetic contribution to song traits may be reduced by the effect of environmental factors. We tested this hypothesis in seven song traits recorded in the long-distance migratory bird, the pied flycatcher (Ficedula hypoleuca). From a 23-year database (1992–2015), we obtained songs for 28 father–son pairs, and for each song trait we applied parent–offspring regressions to estimate heritability. The type of syllables sung are learned from tutors, and here we also determined the cultural contribution of fathers to the song repertoires of their sons, by quantifying the percentage of syllables that sons shared with their fathers, and compared this with what sons shared with other males in the population (e.g., neighbors). The heritabilities of song traits were highly variable (ranging from −0.22 to 0.56), but most of these were around zero and none of them were significant. These results indicate that the seven song traits are most likely determined by environmental factors. Sons shared more syllables with their fathers than with neighbors (21% vs. 3%), suggesting that fathers are important song tutors during the nestling period. We conclude that there is a cultural inheritance from fathers to their sons’ syllable repertoires, but there is no strong evidence for a genetic contribution of fathers to the seven song traits studied.

MHC-mediated sexual selection on birdsong: Generic polymorphism, particular alleles and acoustic signals.

Several hypotheses predict that the major histocompatibility complex (MHC) drives mating preference in females. Olfactory, colour or morphological traits are often found as reliable signals of the MHC profile, but the role of avian song mediating MHC-based female choice remains largely unexplored. We investigated the relationship between several MHC and acoustic features in the collared flycatcher (Ficedula albicollis), a European passerine with complex songs. We screened a fragment of the class IIB second exon of the MHC molecule, of which individuals harbour 4-15 alleles, while considerable sequence diversity is maintained at the population level. To make statistical inferences from a large number of comparisons, we adopted both null-hypothesis testing and effect size framework in combination with randomization procedures. After controlling for potential confounding factors, neither MHC allelic diversity nor the presence of particular alleles was associated remarkably with the investigated qualitative and quantitative song traits. Furthermore, genetic similarity among males based on MHC sequences was not reflected by the similarity in their song based on syllable content. Overall, these results suggest that the relationship between features of song and the allelic composition and diversity of MHC is not strong in the studied species. However, a biologically motivated analysis revealed that individuals that harbour an MHC allele that impairs survival perform songs with broader frequency range. This finding suggests that certain aspects of the song may bear reliable information concerning the MHC profile of the individuals, which can be used by females to optimize mate choice.

Multiple signaling functions of song in a polymorphic species with alternative reproductive strategies.

Vocal traits can be sexually selected to reflect male quality, but may also evolve to serve additional signaling functions. We used a long‐term dataset to examine the signaling potential of song in dimorphic white‐throated sparrows (Zonotrichia albicollis). We investigated whether song conveys multifaceted information about the vocalizing individual, including fitness, species identity, individual identity, and morph. We also evaluated whether song traits correlate differently with fitness in the two morphs, as the more promiscuous strategy of white, relative to tan, morph males might impose stronger sexual selection. Males with high song rates achieved higher lifetime reproductive success, and this pattern was driven by white morph males. In addition, males that sang songs with many notes survived longer, but this pattern was less robust. Thus, song traits reflect differences in fitness and may more strongly affect fitness in the white morph. Song frequency was unrelated to fitness, body size, or morph, but was individual specific and could signal individual identity. Songs of the two morphs displayed similar frequency ratios and bandwidths. However, tan morph males sang songs with longer first notes, fewer notes, and higher variability. Thus, song could be used in morph discrimination. Variation in frequency ratios between notes was low and could function in conspecific recognition, but pitch change dynamics did differ between four different song types observed. Our results support a multiple messages model for white‐throated sparrow song, in which different song traits communicate discrete information about the vocalizing individual.

Short- and long-term repeatability and pseudo-repeatability of bird song: sensitivity of signals to varying environments

To provide reliable information about individual-specific characteristics, sexual signals should be consistently displayed within an individual at least within a particular social context or time window. However, some male traits, like bird song, depict extreme within-individual variations even within a very short time frame. To investigate the extent by which acoustic displays in male collared flycatchers (Ficedula albicollis) reveal consistent individual-specific attributes or more flexible characteristics, we assessed the repeatability of various song traits at different sampling regimes (within-day, between-day, and between-year contexts). Repertoire size showed considerably high and significant repeatability (R > 0.25) in all temporal contexts suggesting that it can potentially indicate an individual attribute that is shaped by genetic background, permanent environmental effects, or long-term experience. Song rate, song length, mean frequency and frequency bandwidth, tempo of syllables, and within-song complexity had small-to-moderate repeatability (R < 0.25) that was significant for the within-day scenario only. Environmental effects can confound repeatability estimates, as males that changed nest boxes between song recordings performed with lower consistency than those that systematically sang on the same territory. Hence, the characteristics of particular songs could reveal aspects that are sensitive to changes in the environment rendering a signal function to the within-individual variance of songs. The consistency of acoustic signals and their sensitivity to the environment determines what kind of information can be conveyed to the receivers. We investigated the repeatability of several song traits in the collared flycatcher to determine signal consistency in different temporal contexts: within days, between days, and between years. We found that repeatability greatly varied across traits, but it generally showed a decreasing tendency as sampling window increased. Repeatability was confounded by effects arising from territory identity, because individuals that changed residency between days sang with less consistency. This result highlights the often-neglected scenario that pseudo-repeatability may arise due to unstandardized environment. In general, our findings imply that the consistency of behaviors and the signal value of song traits may depend on the considered temporal and/or environmental context. The degree of plasticity of acoustic signals determines whether information on a long-term aspect of individual quality or on an immediate state can be conveyed to the receivers.

Evolution and plasticity: Divergence of song discrimination is faster in birds with innate song than in song learners in Neotropical passerine birds.

Plasticity is often thought to accelerate trait evolution and speciation. For example, plasticity in birdsong may partially explain why clades of song learners are more diverse than related clades with innate song. This "song learning" hypothesis predicts that (1) differences in song traits evolve faster in song learners, and (2) behavioral discrimination against allopatric song (a proxy for premating reproductive isolation) evolves faster in song learners. We tested these predictions by analyzing acoustic traits and conducting playback experiments in allopatric Central American sister pairs of song learning oscines (N = 42) and nonlearning suboscines (N = 27). We found that nonlearners evolved mean acoustic differences slightly faster than did leaners, and that the mean evolutionary rate of song discrimination was 4.3 times faster in nonlearners than in learners. These unexpected results may be a consequence of significantly greater variability in song traits in song learners (by 54-79%) that requires song-learning oscines to evolve greater absolute differences in song before achieving the same level of behavioral song discrimination as nonlearning suboscines. This points to "a downside of learning" for the evolution of species discrimination, and represents an important example of plasticity reducing the rate of evolution and diversification by increasing variability.

Condition-dependence and sexual ornamentation: Effects of immune challenges on a highly sexually dimorphic grasshopper.

Sexual ornaments contribute substantially to phenotypic diversity and it is particularly relevant to understand their evolution. Ornaments can assume the function of signals-of-quality that the choosy sex uses to evaluate potential mating partners. Often there are no obvious direct benefits and investment into mate choice is primarily rewarded by beneficial alleles that are inherited to the offspring. Inter-sexual communication via sexual ornaments requires honesty of the sexual signal, yet the question of what maintains honesty remains only partially solved. One solution is that honesty is maintained by trait expression being dependent on individual condition, since condition-dependent trait expression offers an effectively inexhaustible source of genetic variability. Here we test in the highly sexually dimorphic club-legged grasshopper Gomphocerus sibiricus if putative sexual ornaments, in particular the striking front-leg clubs, are more strongly affected by a lipopolysaccharide (LPS) immune challenge than putatively not sexually selected traits. Our results show overall little condition-dependent expression of morphological and song traits, with sexually selected traits exhibiting effects comparable to nonsexually selected traits (with the possible exception of stridulatory file length and syllable-to-pause ratio in advertisement songs). Interestingly, field observations of individuals of lethally parasitized individuals suggest that a very strong environmental challenge can specifically affect the expression of the front-leg clubs. The presence of 1% of males in natural populations with missing or heavily deformed clubs plus 5% with minor club deformations furthermore indicate that there are risks associated with club development during final ecdysis and this might act as a filter against deleterious alleles.

How weather instead of urbanity measures affects song trait variability in three European passerine bird species.

Previous studies detected an influence of urban characteristics on song traits in passerine birds, that is, song adjustments to ambient noise in urban areas. Several studies already described the effect of weather conditions on the behavior of birds, but not the effect on song traits. We investigate, if song trait variability changes along a continuous urbanity gradient in Frankfurt am Main, Germany. We examined, for the first time on a larger scale, the influence of weather on song parameters. We made song recordings of three common passerine species: the blue and great tit (Cyanistes caeruleus (Linnaeus, 1758) and Parus major Linnaeus, 1758) and the European blackbird (Turdus merula Linnaeus, 1758). We measured different song traits and performed statistical analyses and modeling on a variety of variables—among them urbanity and weather parameters. Remarkably, we found only few cases of a significant influence of urbanity parameters on song traits. The influence of weather parameters (air pressure, atmospheric humidity, air and soil temperatures) on song traits was highly significant. Birds in Frankfurt face high noise pollution and might show different adaptations to high noise levels. The song trait variability of the investigated species is affected more by weather conditions than by urban characteristics in Frankfurt. However, the three species react differently to specific weather parameters. Smaller species seem to be more affected by weather than larger species.

Multimodal signalling in estrildid finches: song, dance and colour are associated with different ecological and life-history traits.

Sexual traits (e.g. visual ornaments, acoustic signals, courtship behaviour) are often displayed together as multimodal signals. Some hypotheses predict joint evolution of different sexual signals (e.g. to increase the efficiency of communication) or that different signals trade off with each other (e.g. due to limited resources). Alternatively, multiple signals may evolve independently for different functions, or to communicate different information (multiple message hypothesis). We evaluated these hypotheses with a comparative study in the family Estrildidae, one of the largest songbird radiations, and one that includes many model species for research in sexual selection and communication. We found little evidence for either joint evolution or trade-offs between song and colour ornamentation. Some negative correlations between dance repertoire and song traits may suggest a functional compromise, but generally courtship dance also evolved independently from other signals. Instead of correlated evolution, we found that song, dance and colour are each related to different socio-ecological traits. Song complexity evolved together with ecological generalism, song performance with investment in reproduction, dance with commonness and habitat type, whereas colour ornamentation was shown previously to correlate mostly with gregariousness. We conclude that multimodal signals evolve in response to various socio-ecological traits, suggesting the accumulation of distinct signalling functions.

Genes, environments and their interaction: song and mate choice in canaries

Male secondary sexual traits and female mate choice traits must contain heritable variation for sexual selection to operate. However, for female mate choice, especially, this is poorly known. To complicate matters, both male sexual traits and female mate choice typically show condition dependence, implying that environmental effects probably play an important synergistic role. Using a cross-fostering design, we therefore aimed to disentangle genetic, environmental and their potential interacting effects to investigate how they affect the expression of a sexually selected trait (here birdsong) and female mate choice. To assess environmental effects, we focused on the role of the social environment and thus on learned components. Among the different male song traits investigated, we found a high heritability for song bout length and song bout repertoire, as well as an intriguing gene-by-environment interaction for song bout repertoire. Specifically, the tutor appeared to negatively affect the song bout repertoire of the tutee when his genetic father had a large song bout repertoire but had a positive effect on descendants from fathers with small song bout repertoires. In contrast, we did not detect significant heritability in female mate choice. Female mate choice contained a learned component as females significantly disfavoured their foster father in the mate choice tests, indicating a learned inbreeding avoidance. Thus, our study provides important insights into the role of the social environment for both birdsong and female mate choice. However, the absence of a heritable component in female mate choice suggests a need for studies investigating the heritability of female preferences for (heritable) male song traits in order to gain a better understanding of a potential coevolution between male sexual traits and female mate choice. Overall, our results suggest that indirect genetic effects acting during the pre- and postfledging social contexts may play a prominent role in sexual selection.

Maximum frequency of songs reflects body size among male dusky warblers Phylloscopus fuscatus (Passeriformes: Phylloscopidae)

Song plays a vital role in communication in songbirds, primarily for territorial defense and mate attraction. Larger animals, having larger vocal organs, produce low sound frequencies more efficiently. Accordingly, the frequency of vocalizations is often negatively correlated with body size across species, and also among individuals of many species, including several non-songbirds. However, little is known about whether song frequency reveals information about body size among males. We tested for the predicted positive relationship between body size and song traits in male dusky warblers (Phylloscopus fuscatus). In many animal species, some vocalizations are difficult to produce and can therefore indicate quality of signalers. Male dusky warblers produce songs with trilled sequences that are limited in their timing and frequency structure by physiological constraints on vocal performance. We investigated whether there was a relationship between body size and trilled vocalizations in the dusky warbler. We recorded songs of free-living male dusky warblers at dawn, and captured birds with mist nets for morphometric measurements (tarsus length, as an indicator of body size). We conducted correlation analyses between tarsus length and a composite measure of songs. In dusky warblers, body size was not significantly related to the average frequency of their overall songs, low frequency, bandwidth frequency, average note number of songs, or average song duration. In contrast, the maximum frequency of song was found to correlate positively with tarsus length. For trilled vocalizations, we found most trill traits were not significantly related to body size, and only the maximum frequency of trills was positively related to body size. Additionally, the maximal value of frequency bandwidth decreased with increasing trill rates. Overall, these results suggest that in male dusky warblers, the maximum frequency of the entire song is a reliable indicator of body size.

Positive effect of dietary lutein and cholesterol on the undirected song activity of an opportunistic breeder.

Song is a sexually selected trait that is thought to be an honest signal of the health condition of an individual in many bird species. For species that breed opportunistically, the quantity of food may be a determinant of singing activity. However, it is not yet known whether the quality of food plays an important role in this respect. The aim of the present study was to experimentally investigate the role of two calorie-free nutrients (lutein and cholesterol) in determining the expression of a sexually selected behavior (song rate) and other behaviors (locomotor activity, self-maintenance activity, eating and resting) in male zebra finches (Taeniopygia guttata). We predicted that males supplemented with lutein and cholesterol would sing at higher rates than controls because both lutein and cholesterol have important health-related physiological functions in birds and birdsong mirrors individual condition. To control for testosterone secretion that may upregulate birdsong, birds were exposed to a decreasing photoperiod. Our results showed that control males down-regulated testosterone in response to a decreasing photoperiod, while birds treated with lutein or cholesterol maintained a constant singing activity. Both lutein- and cholesterol-supplemented groups sang more than control groups by the end of the experiment, indicating that the quality of food can affect undirected song irrespective of circulating testosterone concentrations. None of the other measured behaviors were affected by the treatment, suggesting that, when individuals have full availability of food, sexually selected song traits are more sensitive to the effect of food quality than other behavioral traits. Overall the results support our prediction that undirected song produced by male zebra finches signals access to high-quality food.

Sky island bird populations isolated by ancient genetic barriers are characterized by different song traits than those isolated by recent deforestation.

Various mechanisms of isolation can structure populations and result in cultural and genetic differentiation. Similar to genetic markers, for songbirds, culturally transmitted sexual signals such as breeding song can be used as a measure of differentiation as songs can also be impacted by geographic isolation resulting in population‐level differences in song structure. Several studies have found differences in song structure either across ancient geographic barriers or across contemporary habitat barriers owing to deforestation. However, very few studies have examined the effect of both ancient barriers and recent deforestation in the same system. In this study, we examined the geographic variation in song structure across six populations of the White‐bellied Shortwing, a threatened and endemic songbird species complex found on isolated mountaintops or “sky islands” of the Western Ghats. While some sky islands in the system are isolated by ancient valleys, others are separated by deforestation. We examined 14 frequency and temporal spectral traits and two syntax traits from 835 songs of 38 individuals across the six populations. We identified three major song clusters based on a discriminant model of spectral traits, degree of similarity of syntax features, as well as responses of birds to opportunistic playback. However, some traits like complex vocal mechanisms (CVM), relating to the use of syrinxes, clearly differentiated both ancient and recently fragmented populations. We suggest that CVMs may have a cultural basis and can be used to identify culturally isolated populations that cannot be differentiated using genetic markers or commonly used frequency‐based song traits. Our results demonstrate the use of bird songs to reconstruct phylogenetic groups and impacts of habitat fragmentation even in complex scenarios of historic and contemporary isolation.

Communication Value of Mistakes in Dark-Eyed Junco Song.

Sexual signals contain information on individual quality or motivation, and most explanations for their reliability are based on signal costs. A recent suggestion is that signaling mistakes, defined as deviations from typical signal design, provide cues on individual quality, contributing to reliable communication even when signal design is not costly. We describe several atypical song traits in dark-eyed juncos (Junco hyemalis) that may be mistakes during song production or development and occur in up to 6% of songs. These putative mistakes were more frequent in an urban versus a wildland population, and individuals differed in their frequency of mistakes. Some atypical signals were more frequent in younger males or were negatively related to paternity success, supporting the hypothesis that fewer mistakes indicate individual quality. We also discuss unexpected results, such as some atypical signals being more frequent in more ornamented males and in songs with lower performance demands. Song consistency (similarity across syllable renditions) was positively related to male age and paternity success; nonetheless, relations with paternity were stronger when looking at the most deviant syllable renditions, suggesting that the perceptual salience of large mistakes may mediate receiver responses to song consistency. Results indicate that signaling mistakes reveal relevant information to play a role in communication.

Song and Male Quality in Prairie Warblers

AbstractTo determine if the songs of male prairie warblers could potentially reveal to female listeners information about the quality of singers, we compared various aspects of prairie warbler song structure and performance to attributes that might reflect a male singer's potential to enhance the fitness of his mate. We found that all the tested male attributes—arrival date, age, body size, annual survival, and fledging success—were associated with singing, most with multiple aspects of singing. Several of the song traits that were associated with potential indicators of male quality had also been found previously to be good predictors of female social mate choice. In particular, longer songs with longer elements, performed at lower frequency and with greater consistency, were associated with both female mate choice and potential indicators of quality. Thus, female prairie warblers may assess potential mates with the help of a set of song characteristics that collectively reveal an array of attributes that together indicate overall male quality.

Divergence in male cricket song and female preference functions in three allopatric sister species.

Multivariate female preference functions for male sexual signals have rarely been investigated, especially in a comparative context among sister species. Here we examined male signal and female preference co-variation in three closely related, but allopatric species of Gryllus crickets and quantified male song traits as well as female preferences. We show that males differ conspicuously in either one of two relatively static song traits, carrier frequency or pulse rate; female preference functions for these traits also differed, and would in combination enhance species discrimination. In contrast, the relatively dynamic song traits, chirp rate and chirp duty cycle, show minimal divergence among species and relatively greater conservation of female preference functions. Notably, among species we demonstrate similar mechanistic rules for the integration of pulse and chirp time scales, despite divergence in pulse rate preferences. As these are allopatric taxa, selection for species recognition per se is unlikely. More likely sexual selection combined with conserved properties of preference filters enabled divergent coevolution of male song and female preferences.

Development of a Genomic Resource and Quantitative Trait Loci Mapping of Male Calling Traits in the Lesser Wax Moth, Achroia grisella.

In the study of sexual selection among insects, the Lesser Waxmoth, Achroia grisella (Lepidoptera: Pyralidae), has been one of the more intensively studied species over the past 20 years. Studies have focused on how the male calling song functions in pair formation and on the quantitative genetics of male song characters and female preference for the song. Recent QTL studies have attempted to elucidate the genetic architecture of male song and female preference traits using AFLP markers. We continued these QTL studies using SNP markers derived from an EST library that allowed us to measure both DNA sequence variation and map loci with respect to the lepidopteran genome. We report that the level of sequence variation within A. grisella is typical among other Lepidoptera that have been examined, and that comparison with the Bombyx mori genome shows that macrosynteny is conserved. Our QTL map shows that a QTL for a male song trait, pulse-pair rate, is situated on the Z chromosome, a prediction for sexually selected traits in Lepidoptera. Our findings will be useful for future studies of genetic architecture of this model species and may help identify the genetics associated with the evolution of its novel acoustic communication.

Reproduction and immunity trade-offs constrain mating signals and nuptial gift size in a bushcricket

Reproduction and immunity often require significant energetic investment and as a result, trade-offs may occur between them. Reproduction is particularly costly for males of the bushcricket Ephippiger diurnus: not only do they produce loud advertisement calls for long periods of time to attract mates but also they offer females a large nuptial gift, a spermatophore, that can be up to 40% of their body weight. Song traits and spermatophore size vary among males, implying that males may differ in their overall reproductive effort or in how their effort is allocated. Males are long-lived, suggesting that survival traits such as immunity may be particularly critical in fitness. Here, we tested the hypothesis that trade-offs exist between reproductive effort and immune response in E. diurnus and that such trade-offs may constrain male mating calls and nuptial gift size. We investigated the relationship between call syllable number, spermatophore size, and immune response in field-collected individuals. We found an inverse relationship in all pairwise comparisons of call syllable number, spermatophore size, and immune response, as well as between overall reproductive effort and immune response. Our results suggest that males exhibit different energy allocation strategies and that their trade-offs ultimately constrain signal evolution.

Song divergence between subspecies of reed bunting is more pronounced in singing styles under sexual selection

Song divergence between populations of a species can lead to reproductive isolation and speciation. However, birds may have different singing styles used in distinct social contexts, and songs of each style may change at different rates over time and space. Here, we tested whether song divergence between subspecies of reed bunting, Emberiza schoeniclus, differs with singing style, by comparing song traits of its three singing styles among three subspecies breeding in northern and western Europe. We show that the two singing styles under sexual selection (dawn and fast songs, related to obtaining extrapair and social mates, respectively) diverged significantly more than the slow songs (used as an all-clear signal to nest- attending social females). Multiple song traits differed significantly between the subspecies in all singing styles, with E. s. lusitanica generally being intermediate between E. s. schoeniclus and E. s. witherbyi, and the pattern of song complexity opposing the expected latitudinal gradient (of increasing complexity with increasing latitude). Cluster analyses of populations indicate that sexually selected singing styles are better for discriminating subspecies, describing a scenario of a major split in song features between the migratory, northern E. s. schoeniclus and the two resident, southern subspecies, rather than a clinal variation. The greater song divergence in fast and dawn singing styles suggests that sexual selection may be playing an important role in the incipient speciation of reed buntings. (Less)

Phenotypic variation and covariation indicate high evolvability of acoustic communication in crickets.

Studying the genetic architecture of sexual traits provides insight into the rate and direction at which traits can respond to selection. Traits associated with few loci and limited genetic and phenotypic constraints tend to evolve at high rates typically observed for secondary sexual characters. Here, we examined the genetic architecture of song traits and female song preferences in the field crickets Gryllus rubens and Gryllus texensis. Song and preference data were collected from both species and interspecific F1 and F2 hybrids. We first analysed phenotypic variation to examine interspecific differentiation and trait distributions in parental and hybrid generations. Then, the relative contribution of additive and additive-dominance variation was estimated. Finally, phenotypic variance-covariance (P) matrices were estimated to evaluate the multivariate phenotype available for selection. Song traits and preferences had unimodal trait distributions, and hybrid offspring were intermediate with respect to the parents. We uncovered additive and dominance variation in song traits and preferences. For two song traits, we found evidence for X-linked inheritance. On the one hand, the observed genetic architecture does not suggest rapid divergence, although sex linkage may have allowed for somewhat higher evolutionary rates. On the other hand, P matrices revealed that multivariate variation in song traits aligned with major dimensions in song preferences, suggesting a strong selection response. We also found strong covariance between the main traits that are sexually selected and traits that are not directly selected by females, providing an explanation for the striking multivariate divergence in male calling songs despite limited divergence in female preferences.

Phenotypic differentiation in love song traits among sibling species of the Lutzomyia longipalpis complex in Brazil.

BackgroundBrazilian populations of Lutzomyia longipalpis may constitute a complex of cryptic species, and this report investigates the distribution and number of potential sibling species. One of the main differences observed among Brazilian populations is the type of acoustic signal produced by males during copulation. These copulation song differences seem to be evolving faster than neutral molecular markers and have been suggested to contribute to insemination failure observed in crosses between these sibling species. In previous studies, two main types of copulation songs were found, burst-type and pulse-type. The latter type can, in turn, be further subdivided into five different patterns.MethodsWe recorded male song from 13 new populations of the L. longipalpis complex from Brazil and compared the songs with 12 already available.ResultsOut of these 25 populations, 16 produce burst-type and 9 produce pulse-type songs. We performed a principal component analysis in these two main groups separately and an additional discriminant analysis in the pulse-type group. The pulse-type populations showed a clear separation between the five known patterns with a high correspondence of individuals to their correct group, confirming the differentiation between them. The distinctiveness of the burst-type subgroups was much lower than that observed among the pulse-type groups and no clear population structure was observed. This suggests that the burst-type populations represent a single species.ConclusionOverall, our results are consistent with the existence in Brazil of at least six species of the L. longipalpis complex, one with a wide distribution comprising all the populations with burst-type songs, and five more closely related allopatric siblings with different pulse-type song patterns and more restricted distribution ranges.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0900-8) contains supplementary material, which is available to authorized users.