Variation in complex courtship traits across a hybrid zone between grasshopper species of the Chorthippus albomarginatus group

Closely related grasshopper species of the Chorthippus albomarginatus group are notable for their extremely complex courtship songs, accompanied by a visual display. Two species of this group, Ch. albomarginatus and Ch. oschei, were previously shown to hybridize in a wide mosaic hybrid zone in Ukraine and Moldova. In this paper, variation in five courtship song characters, one character of visual display and the number of stridulatory pegs were analysed across the hybrid zone to estimate selection against hybrids and strength of assortative mating. Comparison of cline width and position across the hybrid zone showed concordant and coincident clines in four traits, such as three song characters and one morphological character, and discordant and non‐coincident clines in two other song characters and the character of visual display. Concordance of clines in different characters suggests an equal strength of selection acting on underlying loci. Increase of variance and covariance between phenotypic traits at the cline centre could more likely result from assortative mating than from selection against hybrids. Most pairwise cases showed the highest covariance for the oschei‐like, than for the albomarginatus‐like hybrid populations. This indicates that introgression of the oschei genes into the albomarginatus genome is stronger than vice versa, and may be evidence of the movement of the hybrid zone in favour of Ch. albomarginatus. Analysis of associations between phenotype and local vegetation showed that mosaic structure of the hybrid zone is explained to a great extent by habitat–phenotype associations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 275–291.     

Acoustic signals in insects: A reproductive barrier and a taxonomic character

In singing insects, the song is an important component of the specific mate recognition system (SMRS). In communities of sympatric singing species, there is a partitioning of communication channels, the so-called “acoustic niches.” Within one community, the songs of different species always differ in temporal or frequency characters, i.e. occupy different acoustic niches. However, conspecific songs do not always act as an interspecific reproductive barrier, despite always being a SMRS component. The species that do not communicate acoustically due to allopatry, different timing of vocalization, inhabiting different biotopes, or unmatched food specializations can produce similar songs while forming reproductively isolated communities. Individuals of different sexes need not only to recognize a conspecific mate but also to evaluate its “quality.” The close-range signal (courtship song) provides more opportunities for choosing the “best” male than does the distant signal (calling song). In many species of Orthoptera, courtship includes not only acoustic but also vibrational, visual, chemical, and mechanical signals. An analysis of cricket songs showed the courtship songs to be on average more elaborate and variable than the calling songs. At the same time, due to the difference in mating behavior between the two groups, the acoustic component of courtship is used for mate quality evaluation to a greater extent in grasshoppers than in crickets. The courtship songs of grasshoppers are generally more elaborate in temporal structure than cricket songs; moreover, they may be accompanied by visual displays such as movements of various body parts. Thus, song evolution in grasshoppers is more strongly driven by sexual selection than that in crickets. According to the reinforcement hypothesis, the premating barrier between hybridizing species becomes stronger in response to reduced hybrid fitness. However, our behavioral experiments with two groups of hybridizing grasshopper species did not confirm the reinforcement hypothesis. We explain this, firstly, by a low level of genetic incompatibility between the hybridizing species and secondly, by high hybrid fitness when attracting a mate. A high competitive capability of hybrids may be accounted for by attractiveness of new elements in hybrid courtship songs. When we divide similar forms based on their songs, we in fact distinguish biological species using the criterion of their reproductive isolation. Acoustic differences between species are usually greater than morphological ones. Therefore, song analysis allows one to determine the real status of doubtful species-rank taxa, to distinguish species in a medley of sibling forms, and to reveal cryptic species in the cases when morphological studies fail to provide a univocal result. At the same time, songs are subject to intraspecific variation the range of which is different in different groups. Therefore, it is necessary to study which degree of difference corresponds to the species level before interpreting the status of some forms based on song comparisons. Besides, song similarities cannot indicate conspecificity of acoustically isolated forms; on the other hand, song differences between these forms prove that they are full-rank species.     

A narrow hybrid zone between the grasshoppers Stenobothrus clavatus and Stenobothrus rubicundus: courtship song analysis

The closely related grasshopper species Stenobothrus rubicundus and Stenobothrus clavatus are known to hybridize in a very narrow contact zone on Mt. Tomaros in northern Greece. These species produce very different and complex courtship songs accompanied with visual display. We analyzed the courtship songs and underlying stridulatory movements of the hind legs in natural hybrids from Mount Tomaros. The two species were also hybridized in the laboratory and their songs were compared with the songs of the natural hybrids. Some hybrid songs were shown to have intermediate features between parental songs, whereas other hybrid songs comprised completely new elements. The clavatus‐like song elements were found to dominate in hybrid songs. These song features may influence the mating success of hybrid males in the contact zone. A comparison of hybrid songs with the song pattern of the north European S. rubicundus populations allowed us to suggest a scenario of S. rubicundus and S. clavatus origin. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Stable and variable parameters in courtship songs of grasshoppers of the subfamily Gomphocerinae (Orthoptera,Acrididae)

The courtship behavior of seven grasshopper species of the subfamily Gomphocerinae from different localities of Russia, Ukraine and Greece was described. Not only the sounds but also the corresponding stridulatory movements of the hind legs and visual display accompanying the courtship song were analyzed. Comparison of the degree of variation in different courtship parameters showed that the most stable traits were the syllable and pulse periods. The potential role of stable and variable traits in the grasshopper courtship songs is discussed.     

A narrow hybrid zone between the grasshoppers Stenobothrus clavatus and Stenobothrus rubicundus (Orthoptera: Gomphocerinae): female preferences for courtship songs

Two grasshopper species, Stenobothrus rubicundus and Stenobothrus clavatus, were previously shown to hybridize in a narrow contact zone on Mount Tomaros in northern Greece. The species are characterized by complex and completely different courtship songs. In the present study, we investigated female preferences for the courtship songs of S. rubicundus, S. clavatus and hybrids in playback experiments. Playback of the courtship songs revealed assortative preferences in females of the parental species: they significantly more often preferred the songs of conspecific males. Hybrid females showed a lower selectivity than parental females, responding somewhat equally eager to playback of the songs of S. clavatus, S. rubicundus, and natural hybrid song, although less actively to the F₁ hybrid song. The results suggest that hybrid males may lose to males of parental species, whereas hybrid females would even have an advantage over parental females. Comparison of responses of females from allopatric populations and Mount Tomaros to different song types shows no evidence for reinforcement. Asymmetry found in female preferences may have implications for the structure of the hybrid zone. © 2013 The Linnean Society of London     

Inheritance of species differences in female receptivity and song requirement between Drosophila virilis and D. montana

Females of two Drosophila virilis group species, D. virilis and D. montana, have different requirements for the courting males. In the present study we have examined species differences in female receptivity and male courtship song requirement using females' acceptance signal instead of copulation for measuring female readiness to mate. Behavior of D. virilis and D. montana females and F1 and backcross hybrid females was observed in a single-pair courtships with D. virilis and D. montana males and normal and wingless (mute) F1 hybrid males. D. virilis females were very receptive and they commonly accepted the courtship of males unable to produce courtship song. D. montana females, on the contrary, had a low receptivity and these females accepted the courting male only after hearing his song. Interspecific F1 and backcross (BCm) females resembled D. virilis more than D. montana in their receptivity. These females, however, resembled D. montana in their song requirement. These findings suggest that female song requirement and female receptivity are determined by different genetic factors.     

A hybrid zone between Chorthippus parallelus parallelus and Chorthippus parallelus erythropus (Orthoptera: Acrididae): behavioural characters

Two subspecies of the grasshopper Chorthippus parallelus have ranges that abut in the Pyrenees. Males produce two types of song—calling song and courtship song. We have examined the use and structure of these songs in males from nine sites near Mont-Louis, Pyrénées-Onentales, France. These sites were previously used to identify the position of the hybrid zone on morphological characters. The subspecies differ in the use and structure of both types of song. Clines in these characters correspond in position with the morphological cline. At least one character may be relevant to mate choice but none of the characters show evidence of reinforcement.     

Interspecific genetics of speciation phenotypes: song and preference coevolution in Hawaiian crickets

Understanding the genetic architecture of traits involved in premating isolation between recently diverged lineages can provide valuable insight regarding the mode and tempo of speciation. The repeated coevolution of male courtship song and female preference across the species radiation of Laupala crickets presents an unusual opportunity to compare the genetic basis of divergence across independent evolutionary histories. Previous studies of one pair of species revealed a polygenic basis (including a significant X chromosome contribution) to quantitative differences in male song and female acoustic preference. Here, we studied interspecific crosses between two phenotypically less-diverged species that represents a phylogenetically independent occurrence of intersexual signalling evolution. We found patterns consistent with an additive polygenic basis to differentiation in both song and preference (n(E) = 5.3 and 5.1 genetic factors, respectively), and estimate a moderate contribution of the X chromosome (7.6%) of similar magnitude to that observed for Laupala species with nearly twice the phenotypic divergence. Together, these findings suggest a similar genetic architecture underlying the repeated evolution of sexual characters in this genus and provide a counterexample to prevailing theory predicting an association between early lineage divergence and sex-linked 'major genes'.     

Courtship-sounds and behaviour in the four species of the Drosophila bipectinata complex

The D. bipectinata complex contains four species which are sympatric over parts of their range. They are morphologically identical and hybridize in no-choice mating situations. They have similar but distinct courtship patterns and males of all species sing two songs, long song early and short song late in courtship. Each species has a unique song profile due to differences in at least one song parameter. This is circumstantial evidence that sounds function to maintain sexual isolation within the complex.     

Genetics of divergence in male wing pigmentation and courtship behavior between Drosophila elegans and D. gunungcola

Many sex-specific traits involved in mating consist of functionally coordinated morphologies and behaviors. How the components of these complex traits evolve and become coordinated during evolution is unknown. In order to understand how such trait complexes evolve and diversify, we must decipher the genetic underpinnings of their components. In this study, we begin to elucidate the genetic architecture underlying differences in functionally related male pigmentation and behavior between two Asian Drosophila melanogaster group species, D. elegans and D. gunungcola. D. elegans possesses a male-specific wing melanin spot and a stereotypical wing display element in male courtship, whereas D. gunungcola lacks both of these traits. Using reciprocal F1 male hybrids, we demonstrate that the X-chromosome contains a major locus or loci required for wing spot formation and that autosomal loci largely determine the male courtship display. Using phenotypic and genetic analysis of backcross progeny, we further demonstrate that both the wing spot and courtship differences between the two species are polygenic and both depend at least in small part on genetic factors on both the X and the autosomes. Finally, we find that male wing spot size and courtship wing display are highly correlated in backcross progeny, suggesting that linkage or pleiotropy may have been involved in their coordinated evolution.     

Dominance effects strengthen premating hybridization barriers between sympatric species of grasshoppers (Acrididae,Orthoptera)

Sexual selection can lead to the rapid evolution of premating hybridization barriers and allows accelerated diversification and speciation within an evolutionary lineage. Especially during early stages of divergence, hybridization may impede further divergence, which strongly depends on the reproductive success of hybrids. Behavioural sterility of hybrids can limit or even prevent homogenizing gene flow. In this study, we investigated the attractiveness of male courtship songs for females of the grasshopper species Chorthippus biguttulus and C. brunneus and their interspecific F1 and F2 hybrids. Song preferences of females of both species are highly species specific and differ in three parameters: shape of the preference function, preference for syllable pattern and phrase duration. F1 hybrid females of both reciprocal crosses as well as F2 hybrid females resembled closely pure C. biguttulus females in respect of shape of the preference function and preference for syllable pattern, while preference for phrase duration showed an intermediate expression. This resulted in song preferences of hybrid females that closely resembled those of one parental species, that is C. biguttulus females. Such strong dominance effects were rarely reported so far. They represent an effective barrier limiting gene flow between the two species, since hybrid females will backcross to only one parental species and discriminate against hybrid males, which are behaviourally sterile. Such taxon‐specific modes of inheritance may have facilitated the rapid divergence of acoustically communicating grasshoppers of the species group of Chorthippus biguttulus. Our findings have novel implications on the expression of neuronal filters and the evolution of complex courtship signals.     

Patterns of Inheritance of Mating Signals in Sailfin and Shortfin Mollies (Poeciliidae: Poecilia: Mollienesia)

Divergence in mating signals is a primary factor leading to reproductive isolation, and thus, speciation. However, the genetic changes underlying such divergence are poorly understood, especially in vertebrates. We used two species of poeciliid fishes, Poecilia velifera and P. mexicana , to explore the link between genes and mating behaviors that has resulted in pre-mating reproductive isolation between these species. Using backcross hybrids created from the F1 male offspring of reciprocal interspecific crosses between a sailfin molly ( P. velifera ) and a shortfin molly ( P. mexicana ), we examined the effects of Y-linkage and autosomal contributions on the expression of two male mating behaviors: courtship displays and gonopodial thrusts. The F1 hybrid males displayed a strong influence of sire on courtship display rates, with F1 males sired by the sailfin species showing courtship display rates that were up to three times higher than the rates of displays performed by F1 males sired by the shortfin species. These results suggest a Y-linked genetic effect on the expression of courtship display behavior. Comparisons between backcross hybrid males with sailfin Y-chromosomes or shortfin Y-chromosomes suggested that the interaction of autosomal genes also influences the inheritance of courtship display rates. Sailfin autosomal genes significantly increased the probability of performing courtship displays for hybrid males, and increased display rate for males from the sailfin Y-chromosome line. Autosomal genes had less of an impact on gonopodial thrusting behavior, however, thrust rates did significantly decrease with an increasing proportion of sailfin autosomes in males from the shortfin Y-chromosome line. These results suggest that the inheritance of species differences in mating signals between shortfin and sailfin mollies involves both genes found on the Y-chromosome and autosomal gene influences on their expression.     

Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa

Sexual isolation has been considered one of the primary causes of speciation and its genetic study has the potential to reveal the genetics of speciation. In Drosophila, the importance of courtship songs in sexual isolation between closely related species has been well investigated, but studies analysing the genetic basis of the difference in the courtship songs associated with sexual isolation are less well documented. Drosophila ananassae and Drosophila pallidosa are useful for studies of sexual isolation, because of their sympatric distribution and absence of postmating isolation. Courtship songs are known to play a crucial role in sexual isolation between these two species, and the female discrimination behaviour against the courting male has been revealed to be controlled by a very narrow region on the second chromosome. In this study we investigated the genetic basis controlling the song differences associated with their sexual isolation, using intact and wingless males with chromosomes substituted between species. The results obtained from F₁ hybrid males between these species indicate the dominance of the song characters favoured by D. pallidosa females. In addition, the results obtained from backcross F₂ males indicate that chromosome 2 had a major effect on the control of the song characters associated with sexual isolation.     

Divergence between the Courtship Songs of the Field Crickets Gryllus texensis and Gryllus rubens (Orthoptera, Gryllidae)

Acoustic mating signals are often important as both interspecific prezygotic isolating mechanisms and as sexually selected traits in intraspecific mate choice. Here, we investigate the potential for cricket courtship song to act as an isolating mechanism by assessing divergence between the courtship songs of Gryllus texensis and Gryllus rubens , two broadly sympatric cryptic sister species of field crickets with strong prezygotic isolation via the calling song and little or no postzygotic isolation. We found significant species-level differences in the courtship song, but the song has not diverged to the same extent as the calling song, and considerable overlap remains between these two species. Only two related courtship song characters are sufficiently distinct to play a possible role in prezygotic species isolation.     

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.     

Genetics of incipient speciation in Drosophila mojavensis. I. Male courtship song, mating success, and genotype x environment interactions

Few studies have examined genotype by environment (GxE) effects on premating reproductive isolation and associated behaviors, even though such effects may be common when speciation is driven by adaptation to different environments. In this study, mating success and courtship song differences among diverging populations of Drosophila mojavensis were investigated in a two-environment quantitative trait locus (QTL) analysis. Baja California and mainland Mexico populations of D. mojavensis feed and breed on different host cacti, so these host plants were used to culture F2 males to examine host-specific QTL effects and GxE interactions influencing mating success and courtship songs. Linear selection gradient analysis showed that mainland females mated with males that produced songs with significantly shorter L(long)-IPIs, burst durations, and interburst intervals. Twenty-one microsatellite loci distributed across all five major chromosomes were used to localize effects of mating success, time to copulation, and courtship song components. Male courtship success was influenced by a single detected QTL, the main effect of cactus, and four GxE interactions, whereas time to copulation was influenced by three different QTLs on the fourth chromosome. Multiple-locus restricted maximum likelihood (REML) analysis of courtship song revealed consistent effects linked with the same fourth chromosome markers that influenced time to copulation, a number of GxE interactions, and few possible cases of epistasis. GxE interactions for mate choice and song can maintain genetic variation in populations, but alter outcomes of sexual selection and isolation, so signal evolution and reproductive isolation may be slowed in diverging populations. Understanding the genetics of incipient speciation in D. mojavensis clearly depends on cactus-specific expression of traits associated with courtship behavior and sexual isolation.     

Courtship song analysis in two hybrid zones between sibling species of the <Emphasis Type="Italic">Chorthippus albomarginatus</Emphasis> group (Orthoptera,Gomphocerinae)

Two new hybrid zones between sibling species of the Chorthippus albomarginatus group were described on the basis of the courtship song analysis. Not only the sounds emitted but also the accompanying stridulatory movements of the hind legs were analyzed, which allowed the temporal parameters to be classified in a more reliable way. One hybrid zone between Ch. albomarginatus and Ch. karelini was found in Ulyanovsk and Samara Provinces of Russia. The other hybrid zone, presumably between Ch. karelini and Ch. oschei, was found in the protected Askania-Nova steppe area in Kherson Province of Ukraine. Based on comparison of the natural and laboratory hybrids, a hypothesis on the structure and dynamics of the hybrid zones is proposed.     

Two distinct genomic regions, harbouring the period and fruitless genes, affect male courtship song in Drosophila montana

Acoustic signals often have a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated quantitative trait locus for male courtship song carrier frequency (FRE) in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for 10 candidate genes known to affect song production in Drosophila melanogaster. We also extended the analyses to additional song characters (pulse train length (PTL), pulse number (PN), interpulse interval, pulse length (PL) and cycle number (CN)). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (PTL and PN) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (PL, CN and carrier FRE) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.     

Inheritance of song and stridulatory peg number divergence between Chorthippus brunneus and C. jacobsi, two naturally hybridizing grasshopper species (Orthoptera: Acrididae)

Knowledge of the genetic basis of divergence in mating signal characters that contribute to reproductive isolation is critical to understanding speciation. Here, we describe a semi-automated system for characterizing grasshopper acoustic signals. We used this system to study the genetic basis of divergence in three male calling song components [echeme (EL), syllable (SL) and phrase (PL) lengths] between Chorthippus brunneus and C. jacobsi, two species of grasshoppers that hybridize in northern Spain. We also studied the number of pegs in the stridulatory file. For all characters, additive effects accounted for most of the genetic differentiation between species. However, the three song components also showed small but significant epistatic effects. No sex linkage was detected. Wright-Castle-Lande estimates of the minimum numbers of genetic factors underlying song and peg number divergence were low: peg number (n(e)=5.87+/-5.84), SL (n(e)=2.37+/-4.79) and PL (n(e)=0.87+/-0.86). On the other hand, EL appeared to be controlled by many genes. These results suggest that divergence in SL and PL might be driven by sexual selection whereas EL might not be under selection. This is consistent with experimental results on female song preference in related species. However, the fact that few factors appear to underlie the differences in peg number is surprising. Peg number is not closely related to song characteristics. It often varies between closely related grasshopper species and it has been assumed to be a neutral character. The biometrical approaches used here tend to underestimate the number of factors influencing a trait but provide valuable background for subsequent quantitative trait loci analyses.     

Variation in complex mating signals in an “island” hybrid zone between Stenobothrus grasshopper species

Two grasshopper species Stenobothrus rubicundus and S. clavatus were previously shown to meet in a narrow hybrid zone on Mount Tomaros in northern Greece. The species are remarkable for their complex courtship songs accompanied by conspicuous movements of antennae and wings. We analyzed variations in forewing morphology, antenna shape, and courtship song across the hybrid zone using a geographic information system, and we documented three contact zones on Mount Tomaros. All male traits and female wings show abrupt transitions across the contact zones, suggesting that these traits are driven by selection rather than by drift. Male clines in antennae are displaced toward S. clavatus, whereas all clines in wings are displaced toward S. rubicundus. We explain cline discordance as depending on sexual selection via female choice. The high covariance between wings and antennae found in the centers of all contact zones results from high levels of linkage disequilibria among the underlying loci, which in turn more likely results from assortative mating than from selection against hybrids. The covariance is found to be higher in clavatus‐like than rubicundus‐like populations, which implies asymmetric assortative mating in parental‐like sites of the hybrid zone and a movement of the hybrid zone in favor of S. clavatus.