Dispersal influences genetic and acoustic spatial structure for both males and females in a tropical songbird

Animals exhibit diverse dispersal strategies, including sex‐biased dispersal, a phenomenon common in vertebrates. Dispersal influences the genetic structure of populations as well as geographic variation in phenotypic traits. Patterns of spatial genetic structure and geographic variation may vary between the sexes whenever males and females exhibit different dispersal behaviors. Here, we examine dispersal, spatial genetic structure, and spatial acoustic structure in Rufous‐and‐white Wrens, a year‐round resident tropical bird. Both sexes sing in this species, allowing us to compare acoustic variation between males and females and examine the relationship between dispersal and song sharing for both sexes. Using a long‐term dataset collected over an 11‐year period, we used banding data and molecular genetic analyses to quantify natal and breeding dispersal distance in Rufous‐and‐white Wrens. We quantified song sharing and examined whether sharing varied with dispersal distance, for both males and females. Observational data and molecular genetic analyses indicate that dispersal is female‐biased. Females dispersed farther from natal territories than males, and more often between breeding territories than males. Furthermore, females showed no significant spatial genetic structure, consistent with expectations, whereas males showed significant spatial genetic structure. Overall, natal dispersal appears to have more influence than breeding dispersal on spatial genetic structure and spatial acoustic structure, given that the majority of breeding dispersal events resulted in individuals moving only short distances. Song sharing between pairs of same‐sex animals decreases with the distance between their territories for both males and females, although males exhibited significantly greater song sharing than females. Lastly, we measured the relationship between natal dispersal distance and song sharing. We found that sons shared fewer songs with their fathers the farther they dispersed from their natal territories, but that song sharing between daughters and mothers was not significantly correlated with natal dispersal distance. Our results reveal cultural differences between the sexes, suggesting a relationship between culture and sex‐biased dispersal.     

Divergence in Female Duetting Signals in the Enchenopa binotata Species Complex of Treehoppers (Hemiptera: Membracidae)

Sexual communication often involves signal exchanges between the sexes, or duetting, in which mate choice is expressed through response signals. With both sexes acting as signalers and receivers, variation in the signals of males and females may be important for mate choice, reproductive isolation, and divergence. In the Enchenopa binotata species complex – a case study of sympatric speciation in which vibrational duetting may have an important role – male signals are species‐specific, females choose among males on the basis of signal traits that reflect species and individual differences, and female preferences have exerted divergent selection on male signals. Here, we describe variation in female signals in the E. binotata species complex. We report substantial species differences in the spectral and temporal features of female signals, and in their timing relative to male signals. These differences were similar in range to differences in male signals in the E. binotata complex. We consider processes that might contribute to divergence in female signals, and suggest that signal evolution in the E. binotata complex may be influenced by mate choice in both sexes.     

Acoustic Monitoring Reveals Congruent Patterns of Territorial Singing Behaviour in Male and Female Tropical Wrens

Many animals defend territories against conspecific individuals using acoustic signals. In birds, male vocalizations are known to play a critical role in territory defence. Territorial acoustic signals in females have been poorly studied, perhaps because female song is uncommon in north‐temperate ecosystems. In this study, we compare male vs. female territorial singing behaviour in Neotropical rufous‐and‐white wrens Thryothorus rufalbus, a species where both sexes produce solo songs and often coordinate their songs in vocal duets. We recorded free‐living birds in Costa Rica using an eight‐microphone Acoustic Location System capable of passively triangulating the position of animals based on their vocalizations. We recorded 17 pairs of birds for 2–4 consecutive mornings and calculated the territory of each individual as a 95% fixed kernel estimate around their song posts. We compared territories calculated around male vs. female song posts, including separate analyses of solo vs. duet song posts. These spatial analyses of singing behaviour reveal that males and females use similarly sized territories with more than 60% overlap between breeding partners. Territories calculated based on solo vs. duet song posts were of similar size and similar degrees of overlap. Solos and duets were performed at similar distances from the nest for both sexes. Overall, male and female rufous‐and‐white wrens exhibit very similar spatial territorial singing behaviour, demonstrating congruent patterns of male and female territoriality.     

Call divergence is correlated with geographic and genetic distance in greenish warblers (Phylloscopus trochiloides): a strong role for stochasticity in signal evolution?

Divergence in signalling systems might play a central role in speciation. To assess the importance of possible causes of signal divergence, we examine two types of vocalizations within a geographically variable species complex, the greenish warblers (Phylloscopus trochiloides Sundevall). Calls, which are used by both sexes throughout the year, and songs, which are sung primarily by breeding males, differ distinctly between two distinct Siberian forms. Through a ring of southern populations that connect the northern forms, signal divergence is correlated with both geographic distance and genetic divergence. Calls and songs differ in their particular patterns of geographic variation, probably because of the larger influence of sexual selection on songs than on calls. These patterns are supportive of neither acoustic adaptation nor morphology being major drivers of divergence in vocalizations. Rather, these results support the importance of stochastic evolution of communication systems in the evolution of new species.     

Melodic males and flashy females: Geographic variation in male and female reproductive behavior in red‐eyed treefrogs (Agalychnis callidryas)

Geographic variation in courtship behavior can affect reproductive success of divergent phenotypes via mate choice. Over time, this can lead to reproductive isolation and ultimately to speciation. The Neotropical red‐eyed treefrog (Agalychnis callidryas) exhibits high levels of phenotypic variation among populations in Costa Rica and Panama, including differences in color pattern, body size, and skin peptides. To test the extent of behavioral premating isolation among differentiated populations, we quantified male advertisement calls from six sites and female responses to male stimuli (acoustic and visual signals) from four sites. Our results show that both male advertisement calls and female behavior vary among populations: Discriminant function analyses can predict the population of origin for 99.3% ± 0.7 of males based on male call (dominant frequency and bandwidth) and 76.1% ± 6.6 of females based on female response behavior (frequency and duration of visual displays). Further, female mate choice trials (n = 69) showed that population divergence in male signals is coupled with female preference for local male stimuli. Combined, these results suggest that evolved differences among populations in male call properties and female response signals could have consequences for reproductive isolation. Finally, population variation in male and female behavior was not well explained by geographic or genetic distance, indicating a role for localized selection and/or drift. The interplay between male courtship and female responses may facilitate the evolution of local variants in courtship style, thus accelerating premating isolation via assortative mating.     

The effects of cultural drift on geographic variation in echolocation calls of the Chinese rufous horseshoe bat (Rhinolophus sinicus)

Drift, selection, or their combined effects commonly drive geographic variation in traits. Clarifying the relative roles of each process is a long‐standing research goal in evolutionary biology. Acoustic signals of bats are a phenotypic characteristic that plays an important role in social organization and species recognition. We extensively sampled the Chinese rufous horseshoe bat (Rhinolophus sinicus) throughout China and Vietnam and reconstructed a species phylogeny to better understand the patterns and causes of the geographic variation of acoustic signals. Our results showed that the resting frequency (RF) of calls varied with latitude, sex, and distance among different colony locations. RF differences were not correlated with genetic distance (based on only one nuclear locus and the mitochondrial locus), climatic factors (mean annual temperature and mean annual precipitable water), or body size, although differences in calls increased with distance among various populations. This suggests that cultural drift may play more important roles than genetic drift and acoustic adaptation in shaping acoustic differences within regions in R. sinicus.     

Behavioural response of a migratory songbird to geographic variation in song and morphology

Introduction

Sexually selected traits contribute substantially to evolutionary diversification, for example by promoting assortative mating. The contributing traits and their relevance for reproductive isolation differ between species. In birds, sexually selected acoustic and visual signals often undergo geographic divergence. Clines in these phenotypes may be used by both sexes in the context of sexual selection and territoriality. The ways conspecifics respond to geographic variation in phenotypes can give insights to possible behavioural barriers, but these may depend on migratory behaviour. We studied a migratory songbird, the Stonechat, and tested its responsiveness to geographic variation in male song and morphology. The traits are acquired differently, with possible implications for population divergence. Song can evolve quickly through cultural transmission, and thus may contribute more to the establishment of geographic variation than inherited morphological traits. We first quantified the diversity of song traits from different populations. We then tested the responses of free-living Stonechats of both sexes to male phenotype with playbacks and decoys, representing local and foreign stimuli derived from a range of distances from the local population.

Results

Both sexes discriminated consistently between stimuli from different populations, responding more strongly to acoustic and morphological traits of local than foreign stimuli. Time to approach increased, and time spent close to the stimuli and number of tail flips decreased consistently with geographic distance of the stimulus from the local population. Discriminatory response behaviour was more consistent for acoustic than for morphological traits. Song traits of the local population differed significantly from those of other populations.

Conclusions

Evaluating an individual’s perception of geographic variation in sexually selected traits is a crucial first step for understanding reproductive isolation mechanisms. We have demonstrated that in both sexes of Stonechats the responsiveness to acoustic and visual signals decreased with increasing geographic distance of stimulus origin. These findings confirm consistent, fine discrimination for both learned song and inherited morphological traits in these migratory birds. Maintenance or further divergence in phenotypic traits could lead to assortative mating, reproductive isolation, and potentially speciation.

     

Detection of female mating status using chemical signals and cues

Males of many species choose their mate according to the female's reproductive status, and there is now increasing evidence that male fitness can depend on this discrimination. However, females will also aim to regulate their mating activity so as to maximize their own fitness. As such, both sexes may attempt to dictate the frequency and timing of female mating, reflecting the potentially different costs of female signaling to both sexes. Here, I review evidence that chemical cues and signals are used widely by males to discriminate between mated and unmated females, and explore the mechanisms by which female odour changes post‐mating. There is substantial empirical evidence that mated and unmated females differ in their chemical profile, and that this variation provides males with information on a female's mating status. Although there appears to be large variation among species regarding the mechanisms by which female odour is altered post‐mating, the transfer of male substances to females during or subsequent to copulation appear to play a major role. This transfer of substances by males may be part of their strategy to suppress reproduction by competing males, particularly in species where females mate more than once.     

Cross‐sex genetic correlations and the evolution of sex‐specific local adaptation: Insights from classical trait clines in Drosophila melanogaster

Natural selection varies widely among locations of a species’ range, favoring population divergence and adaptation to local environmental conditions. Selection also differs between females and males, favoring the evolution of sexual dimorphism. Both forms of within‐species evolutionary diversification are widely studied, though largely in isolation, and it remains unclear whether environmental variability typically generates similar or distinct patterns of selection on each sex. Studies of sex‐specific local adaptation are also challenging because they must account for genetic correlations between female and male traits, which may lead to correlated patterns of trait divergence between sexes, whether or not local selection patterns are aligned or differ between the sexes. We quantified sex‐specific divergence in five clinally variable traits in Drosophila melanogaster that individually vary in their magnitude of cross‐sex genetic correlation (i.e., from moderate to strongly positive). In all five traits, we observed parallel male and female clines, regardless of the magnitude of their genetic correlation. These patterns imply that parallel spatial divergence of female and male traits is a reflection of sexually concordant directional selection imposed by local environmental conditions. In such contexts, genetic correlations between the sexes promote, rather than constrain, local adaptation to a spatially variable environment.     

BOTH MALE AND FEMALE NOVEL TRAITS PROMOTE THE CORRELATED EVOLUTION OF GENITALIA BETWEEN THE SEXES IN AN ARTHROPOD

The correlated evolution of genitalia between sexes has been demonstrated in many taxa. However, it remains unclear whether female rather than male genitalia can play a key role in the correlated evolution of male and female genitalia. We conducted an extensive cross‐population analysis of the divergence patterns of genital structures, weights of whole genital organs, and the bodies of both sexes, and male genital length in a group of xystodesmid millipedes showing diverse genital morphologies. We demonstrate that the correlated evolution of male and female genitalia toward exaggerated states has occurred in the millipedes, which have evolved novel traits in both males (forceps‐like gonopods) and females (retractable bellows). Enlargement and elongation of forceps‐like gonopods may be advantageous in sperm competition, whereas enlargement and elongation of the bellows may facilitate acceptance/rejection of insemination for ensuring the female's fitness. These male and female genital parts have affected the correlated evolution in the opposite sex, resulting in diversification and exaggeration of genital morphology. Our study suggests that evolutionary novel traits in not only males but also in females could play an important role in the correlated evolution of genitalia between the sexes.     

Sex‐biased transcriptome divergence along a latitudinal gradient

Sex‐dependent gene expression is likely an important genomic mechanism that allows sex‐specific adaptation to environmental changes. Among Drosophila species, sex‐biased genes display remarkably consistent evolutionary patterns; male‐biased genes evolve faster than unbiased genes in both coding sequence and expression level, suggesting sex differences in selection through time. However, comparatively little is known of the evolutionary process shaping sex‐biased expression within species. Latitudinal clines offer an opportunity to examine how changes in key ecological parameters also influence sex‐specific selection and the evolution of sex‐biased gene expression. We assayed male and female gene expression in Drosophila serrata along a latitudinal gradient in eastern Australia spanning most of its endemic distribution. Analysis of 11 631 genes across eight populations revealed strong sex differences in the frequency, mode and strength of divergence. Divergence was far stronger in males than females and while latitudinal clines were evident in both sexes, male divergence was often population specific, suggesting responses to localized selection pressures that do not covary predictably with latitude. While divergence was enriched for male‐biased genes, there was no overrepresentation of X‐linked genes in males. By contrast, X‐linked divergence was elevated in females, especially for female‐biased genes. Many genes that diverged in D. serrata have homologs also showing latitudinal divergence in Drosophila simulans and Drosophila melanogaster on other continents, likely indicating parallel adaptation in these distantly related species. Our results suggest that sex differences in selection play an important role in shaping the evolution of gene expression over macro‐ and micro‐ecological spatial scales.     

Effect of a novel repellent,acetylated glyceride,on courtship behaviours and acoustic signals of Bemisia tabaci

The sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), attacks a wide range of vegetables and ornamental plants in the tropics and subtropics. This study involved observations of adult whitefly courtship behaviours to determine the impact of acetylated glyceride treatment, which is currently being developed as a repellent for adults, on their progeny. In addition, the effect of acetylated glyceride treatment on acoustic‐based communication was evaluated, as this type of communication is known to play a pivotal role in the search for mating partners on host‐plant leaves in adult whiteflies. Adults were able to land on grape tomato leaves a few days after treatment, when the residual repellency had dissipated. Almost no male courtship behaviour in terms of searching for sexually mature females was observed throughout the observation period (60 min) under conditions in which adults of both sexes remained on a treated leaf or those in which an adult of either sex remained on a non‐treated leaf. By contrast, these behaviours accounted for 26.5% of the observation time under conditions in which both sexes remained on a non‐treated leaf. In cross‐tests performed for 4 days using one non‐mated female and two non‐mated males, the sex ratio (male/female) of the newly emerged adults on treated leaves (2.4) was 63.6% lower than that of the non‐treated controls (0.9). During courtship, acoustic signals produced by both sexes were exchanged rhythmically on non‐treated leaves, whereas the signals on treated leaves were irregular. The frequency of the vibratory sound produced by males was 66–81% lower on the treated leaves than on the non‐treated leaves. A large reduction in the acoustic signals produced by males on treated leaves caused a decrease in courting pair formation, leading to a significant reduction in the number of female progeny due to arrhenotokous parthenogenesis in virgin B. tabaci females.     

Clinal variation in body size and sexual dimorphism in an Indian fruit bat, Cynopterus sphinx (Chiroptera: Pteropodidae)

Geographic variation in body size and sexual dimorphism of the short‐nosed fruit bat (Cynopterus sphinx) was investigated in peninsular India. Bats were sampled at 12 localities along a 1200 km latitudinal transect that paralleled the eastern flanks of the Western Ghats. The geographic pattern of variation in external morphology of C. sphinx conforms to the predictions of Bergmann's Rule, as indicated by a steep, monotonic cline of increasing body size from south to north. This study represents one of the first conclusively documented examples of Bergmann's Rule in a tropical mammal and confirms that latitudinal clines in body size are not exclusively restricted to temperate zone homeotherms. Body size was indexed by a multivariate axis derived from principal components analysis of linear measurements that summarize body and wing dimensions. Additionally, length of forearm was used as a univariate index of structural size to examine geographic variation in a more inclusive sample of bats across the latitudinal transect. Multivariate and univariate size metrics were strongly and positively correlated with body mass, and exhibited highly concordant patterns of clinal variation. Stepwise multiple regression on climatological variables revealed that increasing size of male and female C. sphinx was associated with decreasing minimum temperature, increasing relative humidity, and increasing seasonality. Although patterns of geographic size variation were highly concordant between the sexes, C. sphinx also exhibited a latitudinal cline in the magnitude and direction of sexual size dimorphism. The size differential reversed direction across the latitudinal gradient, as males averaged larger in the north, and females averaged larger in the south. The degree of female‐biased size dimorphism across the transect was negatively correlated with body size of both sexes. Canonical discriminant analysis revealed that male‐ and female‐biased size dimorphism were based on contrasting sets of external characters. Available data on geographic variation in the degree of polygyny in C. sphinx suggests that sexual selection on male size may play a role in determining the geographic pattern of sexual size dimorphism.     

Sex‐specific responses to territorial intrusions in a communication network: Evidence from radio‐tagged great tits

Signals play a key role in the ecology and evolution of animal populations, influencing processes such as sexual selection and conflict resolution. In many species, sexually selected signals have a dual function: attracting mates and repelling rivals. Yet, to what extent males and females under natural conditions differentially respond to such signals remains poorly understood, due to a lack of field studies that simultaneously track both sexes. Using a novel spatial tracking system, we tested whether or not the spatial behavior of male and female great tits (Parus major) changes in relation to the vocal response of a territorial male neighbor to an intruder. We tracked the spatial behavior of male and female great tits (N = 44), 1 hr before and 1 hr after simulating territory intrusions, employing automatized Encounternet radio‐tracking technology. We recorded the spatial and vocal response of the challenged males and quantified attraction and repulsion of neighboring males and females to the intrusion site. We additionally quantified the direct proximity network of the challenged male. The strength of a male's vocal response to an intruder induced sex‐dependent movements in the neighborhood, via female attraction and male repulsion. Stronger vocal responders were older and in better body condition. The proximity networks of the male vocal responders, including the number of sex‐dependent connections and average time spent with connections, however, did not change directly following the intrusion. The effects on neighbor movements suggest that the strength of a male's vocal response can provide relevant social information to both the males and the females in the neighborhood, resulting in both sexes adjusting their spatial behavior in contrasting ways, while the social proximity network remained stable. This study underlines the importance of “silent” eavesdroppers within communication networks for studying the dual functioning and evolution of sexually selected signals.     

Sexual dimorphism modifies habitat‐associated divergence: Evidence from beach and creek breeding sockeye salmon

Studies of parallel or convergent evolution (the repeated, independent evolution of similar traits in similar habitats) rarely explicitly quantify the extent of parallelism (i.e. variation in the direction and/or magnitude of divergence) between the sexes; instead, they often investigate both sexes together or exclude one sex. However, differences in male and female patterns of divergence could contribute to overall variation in the extent of parallelism among ecotype pairs, especially in sexually dimorphic traits. Failing to properly attribute such variation could lead to underestimates of the importance of environmental variation in shaping phenotypes. We investigate the extent of parallelism in the body shape of male and female beach and creek spawning sockeye salmon (Oncorhynchus nerka) from two lake systems in western Alaska that were colonized independently after the last ice age. Although both sexes showed some degree of parallelism, patterns of beach‐creek body shape divergence vary between the sexes and between lake systems. Phenotypic change vector analyses revealed highly parallel aspects of divergence between males from different lake systems (males from beaches had deeper bodies than males from creeks) but weaker parallelism in females and high parallelism between the sexes in one lake system but not the other. Body shape also had population‐specific components, which were mostly, but not entirely, explained by environmental variation in the form of creek depth. Our results highlight the importance of explicitly considering the extent of parallelism between the sexes and environmental variation among sites within habitat types.     

Evolutionary significance of the variation in acoustic communication of a cryptic nocturnal primate radiation (Microcebus spp.)

Acoustic phenotypic variation is of major importance for speciation and the evolution of species diversity. Whereas selective and stochastic forces shaping the acoustic divergence of signaling systems are well studied in insects, frogs, and birds, knowledge on the processes driving acoustic phenotypic evolution in mammals is limited. We quantified the acoustic variation of a call type exchanged during agonistic encounters across eight distinct species of the smallest‐bodied nocturnal primate radiation, the Malagasy mouse lemurs. The species live in two different habitats (dry forest vs. humid forest), differ in geographic distance to each other, and belong to four distinct phylogenetic clades within the genus. Genetically defined species were discriminated reliably on the phenotypic level based on their acoustic distinctiveness in a discriminant function analysis. Acoustic variation was explained by genetic distance, whereas differences in morphology, forest type, or geographic distance had no effect. The strong impact of genetics was supported by a correlation between acoustic and genetic distance and the high agreement in branching pattern between the acoustic and molecular phylogenetic trees. In sum, stochastic factors such as genetic drift best explained acoustic diversification in a social communication call of mouse lemurs.     

Female preference functions drive interpopulation divergence in male signalling: call diversity in the bushcricket Ephippiger diurnus

Female preferences play a major role in the elaboration and diversification of male traits: as a selective pressure on males, variation in female preferences can generate population divergence and ultimately, speciation. We studied how interpopulation differences in the shape of female mate preference functions may have shaped male advertisement signals in the bushcricket Ephippiger diurnus. This species is distributed as geographically isolated populations with striking interpopulation variation in male acoustic signals, most notably in the number of syllables per call. Here, we asked whether differences in the shape of preference functions exist among populations and whether those differences may have driven male signal evolution resulting in the observed differences in syllable numbers. Our results reveal fundamental differences in female preferences among populations, with differences in the overall preference function shape corresponding to differences in male signals. These differences in female preferences best explain the major differences in male signals among populations. The interpopulation variation in signals and preferences potentially reflects the evolutionary history of the species and may contribute to further divergence among populations and subsequent speciation.     

褐稻虱求偶鸣声和交尾行为

褐稻虱Nilaparvata lugens(stl)成虫交尾前均发出由固体介质(寄主植株)传播的声信号,用于个体间联系。信号产生于伴随腹部振动的摩擦发声过程,具有种的专一性,能联络、识别同种异性个体,刺激性兴奋和为雄虫寻觅雌虫定向,对求偶和完成交尾具有重要意义。为害水稻的与为害李氏禾属杂草Leersia hexandra(Swartz)的生物型之间,鸣声声学特征存在明显差异,构成型间杂交的某些障碍;而为害水稻的四种生物型之间无显著差异。     

Geographic variation in sexual dimorphism in the barn owl Tyto alba: a role for direct selection or genetic correlation?

Geographic variation in sexually selected traits is commonly attributed to geographic variation in the net benefit accrued from bearing such traits. Although natural and sexual selection are potentially important in shaping geographic variation, genetic constraints may also play a role. Although a genetic correlation between two traits may itself be the outcome of natural or sexual selection, it may indirectly reinforce the establishment and maintenance of cline variation with respect to one particular trait when across the cline different values of other traits are selected. Using the barn owl Tyto alba, a species in which the plumage of females is more reddish‐brown and more marked with black spots than that of males, I report results that are consistent with the hypothesis that both direct selection and genetic constraints may help establish and maintain cline variation in sexual dichromatism. In this species, inter‐individual variation in plumage coloration and spottiness has a genetic basis, and these traits are not sensitive to the environment. Data, based on the measurement of skin specimens, is consistent with the hypothesis that the stronger European cline variation in male spottiness than in female spottiness depends on the combined effects of (1) the similar cline variation in male and female plumage coloration and (2) the more intense phenotypic correlation between plumage coloration and spottiness in males (darker birds are more heavily spotted in the two sexes, but especially males) which is a general feature among the globally distributed barn owls. In northern Europe, male and female T. a. guttata are reddish‐brown and heavily spotted, and in southern Europe male and female T. a. alba are white, but only females display many spots. Here, I discuss the relative importance of direct selection, genetic correlation and the post‐ice age invasion of Europe by T. alba, in generating sex‐specific cline variation in plumage spottiness and non‐sex‐specific cline variation in plumage coloration.     

Sexual selection and population divergence III: Interspecific and intraspecific variation in mating signals

A major challenge for studying the role of sexual selection in divergence and speciation is understanding the relative influence of different sexually selected signals on those processes in both intra‐ and interspecific contexts. Different signals may be more or less susceptible to co‐option for species identification depending on the balance of sexual and ecological selection acting upon them. To examine this, we tested three predictions to explain geographic variation in long‐ versus short‐range sexual signals across a 3,500 + km transect of two related Australian field cricket species (Teleogryllus spp.): (a) selection for species recognition, (b) environmental adaptation and (c) stochastic divergence. We measured male calling song and male and female cuticular hydrocarbons (CHCs) in offspring derived from wild populations, reared under common garden conditions. Song clearly differentiated the species, and no hybrids were observed suggesting that hybridization is rare or absent. Spatial variation in song was not predicted by geography, genetics or climatic factors in either species. In contrast, CHC divergence was strongly associated with an environmental gradient supporting the idea that the climatic environment selects more directly upon these chemical signals. In light of recently advocated models of diversification via ecological selection on secondary sexual traits, the different environmental associations we found for song and CHCs suggest that the impact of ecological selection on population divergence, and how that influences speciation, might be different for acoustic versus chemical signals.