Sexual selection on bushcricket genitalia operates in a mosaic pattern

Abstract In most species with internal fertilization, male genitalia evolve faster than other morphological structures. This holds true for genital titillators, which are used exclusively during mating in several bushcricket subfamilies. Several theories have been proposed for the sexual selection forces driving the evolution of internal genitalia, especially sperm competition, sexually antagonistic coevolution (SAC), and cryptic female choice (CFC). However, it is unclear whether the evolution of genitalia can be described with a single hypothesis or a combination of them. The study of species‐specific genitalia action could contribute to the controversial debate about the underlying selective evolutionary forces. We studied female mating behaviors in response to experimentally modified titillators in a phylogenetically nested set of four bushcricket species: Roeseliana roeselii, Pholidoptera littoralis littoralis, Tettigonia viridissima (of the subfamily Tettigoniinae), and Letana inflata (Phaneropterinae). Bushcricket titillators have several potential functions; they stimulate females and suppress female resistance, ensure proper ampulla or spermatophore attachment, and facilitate male fixation. In R. roeselii, titillators stimulate females to accept copulations, supporting sexual selection by CFC. Conversely, titillator modification had no observable effect on the female's behavior in T. viridissima. The titillators of Ph. l. littoralis mechanically support the mating position and the spermatophore transfer, pointing to sexual selection by SAC. Mixed support was found in L. inflata, where manipulation resulted in increased female resistance (evidence for CFC) and mating failures by reduced spermatophore transfer success (evidence for SAC). Sexual selection is highly species‐specific with a mosaic support for either cryptic female choice or sexually antagonistic coevolution or a combination of both in the four species.

We still know little about how species-specific genitalia contribute to the controversial debate about the underlying selective evolutionary forces. Given the species-specific morphology and the proposed varying function of genitalia, it is possible that criteria supporting different sexual selection theories might be fulfilled in closely related species or even within a single species. Such a mosaic of sexual selection forces acting between and within species might in part explain the long-standing controversy around genitalia evolution.
Moreover, comparative studies found that males bearing titillators copulated longer than those without (Vahed et al., 2011), and the female's refractory period was shorter in species with more complex titillators . Consequently, the compiled data for the bushcricket R. roeselii show that titillators in this species evolved under cryptic female choice (Eberhard & Lehmann, 2019), but sexually antagonistic coevolution might also act in bushcrickets. In the first case, titillators should be used as copulatory courtship devices to stimulate the females, while in the latter case, they could be used for grasping and position securing, allowing males to control the copulation duration, or even wound the females (Dougherty et al., 2017).
It has been suggested that genital evolution is influenced simultaneously or sequentially by different sexually selective forces (Eberhard, 2011;Hosken & Stockley, 2004) and that these may have unequal effects on reproductive behavior and genital morphology (Eberhard, 2011). In this paper, we examine whether the species-specific morphology of the bushcricket titillators can be explained by a unifying function or if sexual selection has led to a variety of functions.
We address this through experiments that alter the titillator structures in three bushcricket species that have stepwise phylogenetic relationships to our model species Roeseliana roeselii (Wulff et al., 2017(Wulff et al., , 2015(Wulff et al., , 2018Wulff & Lehmann, 2014, 2016 (Figure 1). Two species were selected from the same subfamily Tettigoniinae, which have paired titillators with numerous spines. A third species was chosen from the different subfamily Phaneropterinae, bearing a single titillator.
Mating in bushcrickets can be described along behavioral landmarks (compare Lehmann & Lehmann, 2008;Wulff & Lehmann, 2016); once a male and a female have physical contact with their antennae, the male tries to achieve the mating position. Copula is initiated by grasping the female with a male's cerci, sometimes supported by the subgenital plate holding her ovipositor. Once a firm coupling is established, the female opens her subgenital plate to give the male access to her genital chamber. The male pulls near the female to make close contact and insert his titillators into the female' genital chamber.
The titillator together with the male's phallobasis is then rhythmically moved forwards and backwards. Two types of titillator movements can be distinguished. During the small ones, the titillator is moved inside the female, whereas in the big ones, the titillator is moved in and out, becoming visible during retraction phases. Both types of titillator movements can be observed without manipulation (Video S1).
In the three Tettigoniinae species, the males transfer a large spermatophore at the end of the mating, containing a spermatophylax and the ampulla with the male's sperm Vahed et al., 2011). While the female eats the spermatophylax, the sperm migrates from the ampulla into the female's seminal receptacle (Lehmann, 2012). In the subfamily Phaneropterinae, the majority of the roughly 3,000 species (Cigliano, Braun, Eades, & Otte, 2019) have no titillators. One notable exception is the species Letana inflata. Males have one spiny titillator and transformed genital lobes, which they use as claspers to restrain the female after the transfer of the sperm-containing ampulla (Heller & Liu, 2015). The prolonged mate guarding in this species prevents the females from eating the ampulla and gives the sperm the time it needs to migrate successfully into the female's body (Lehmann, Heller, & Mai, 2016).
To test for selective forces likely to explain the evolution of titillators, we observed the responses of females mated to males of the wild type or with experimentally altered genital titillators. If they are sexually selected, we hypothesize that titillator manipulations affect female behavior during or after copula. Based on the main hypotheses for sexual selection on genitalia, cryptic female choice, and sexually antagonistic coevolution, we developed a matrix for likely copulatory and postcopulatory responses (see Table 5

| Study species
Four bushcricket species were used three European Tettigoniinae The males of the three Tettigoniinae species bear paired titillators with several spines on the tips (Harz, 1969;Lehmann et al., 2017;Vahed et al., 2011), whereas L. inflata males possess a single titillator with several spines, which is merged with the surrounding tissue of the phallobasis (Heller & Liu, 2015) ( Figure 2).
Individuals of the three tettigoniids were caught as juveniles in the wild and reared to adulthood in the laboratory (Table 1). The individuals of L. inflata originated from a single female captured in Sri Lanka (Heller & Liu, 2015). Animals were reared until adulthood Before reaching sexual maturity, adults were separated and individually accommodated in 0.5-L plastic containers covered with gauze. All individuals were fed their species-specific diet ad libitum (Table 1), and water was sprinkled once to twice a day on the walls of the boxes and plastic jars. Ambient temperature in the laboratory was 22-25°C with a light-dark cycle of 16:8 hr.

| Titillator manipulations
To test for changes in female mating behaviors as a response to manipulations, the male's titillator(s) were shortened or covered with UV-hardening glue before mating experiments ( 2015, see Figure 1). As removal of the titillator was therefore not an option, we covered the spines on the single titillator ( s T glued ) with UV-hardening glue (UV-Star, Marston-Domsel GmbH). The glue was applied precisely on the spines with the tip of a fine long brush-hair under the stereo microscope and hardened for 30 s TA B L E 2 Manipulation scheme for the four bushcricket species. Wild type: sham operation; p T -2 : both titillators ablated; p T -1 : the left titillator ablated; p T -left spines : spines on the tips of the left titillator removed; s T glued : spines of the single titillator covered with UV-hardening glue with a UV-Lamp ("UV-Beamer," Marston-Domsel GmbH). In the wild-type group, the single titillator was touched with the tip of the fine brush-hair, and, to control for possible side effects of the glue on the males without interfering with the copulation, it was applied on the basal part of the male's genital lobe. After application and hardening of the UV-glue, its correct and firm placement was verified.

| Mating experiments
The mating partners were mated in a dome-shaped meshed arena Total time from coupling the male cerci to the female until separation of the pair after spermatophore transfer. c As couples repeatedly separate during copula, all single copula events were summed up. d Defined as the number a pair interrupts the cerci coupling and reengage in copula. e Does not occur in T. viridissima.
f Duration of the last copula attempt, leading to the spermatophore transfer or the termination of mating. g Visible retraction of parts or the total male titillators out of the female and reinsertion (in-and-out movement). h Titillator movements are not external visible in L. inflata . i Visible movement of the male titillators inside the female without retraction. j Occurrence of female walking, jumping, kicking, and eventually biting during copulation. Percentage of females showing this behavior. k Failed mechanical anchoring of the titillator, leading to slipping out of the copula position with a full retraction of the titillators. l Percentage of mating couples successfully ending their mating by transfer of a spermatophore. m The bushcricket spermatophore consists of the sperm-bearing ampulla and a gelatinous nutritious spermatophylax. n However, in L. inflata the spermatophore is deposited inside the female genital chamber and is built only of the ampulla. o Spermatophores were removed after copulation using fine forceps and immediately weighed on a precision balance (Kern EG 300 -3 M, 0.001/300 g). p Transfers an internal ampulla that is not accessible without dissection . q Spermatophore consisting of the sperm-containing ampulla and a surrounding gelatinous spermatophylax. r No precise data taken-the females took several hours to finish ingestion. s Spermatophore build only by the sperm-containing ampulla. t Females were presented every day a virgin male ready-to-mate to test for female willingness to remate. u Number of eggs laid until remating. mating. Individuals of the single titillator possessing L. inflata were used immediately after UV hardening, because some individuals were able to remove the glue from their genitalia over time. In line with their natural activity time, R. roeselii was tested during the daytime, whereas T. viridissima, Ph. littoralis, and L. inflata were mated at night between 10 p.m. and 6 a.m. Prior to the experiments, all males and females were weighed on a precision balance (Kern EG 300 -3 M, 0.001/300 g). Randomization of males and females was successful regarding body mass of three species, only in T. viridissima were males of one out of three groups significantly lighter (Table 3).
To test for the different hypotheses of sexual selection acting on bushcricket titillators, we have developed specific predictions for the six copulatory and four postcopulatory traits (Table 5). Many of the predictions can be deduced from our list supporting cryptic female choice in insect genitalia of tsetse flies and the bushcricket R. roeselii (Eberhard & Lehmann, 2019). Cryptic female choice and sexually antagonistic coevolution make distinct predictions for the outcomes in mating with genitalia-manipulated males. As we have seen different responses between symmetrically and asymmetrically manipulated titillators in R. roeselii (Wulff & Lehmann, 2016;Wulff et al., 2018), we extended the predictions for the number of titillator movements regarding symmetry.

TA B L E 5
Relevance of the mating-related parameters during and after copulation for sexual selection, especially to distinguish between cryptic female choice (CFC) and sexually antagonistic coevolution (SAC)

TA B L E 6 (Continued)
Statistical analysis was performed using Excel and SPSS version 24 (IBM SPSS Statistics 24).

| RE SULTS
Female responses during copulations toward titillator-manipulated males were highly species-specific (Tables 6 and 7). No evidence for sexual selection on titillators was found in T. viridissima, as the removal of one or both titillators had no effect on the mating outcome, nor female or male mating behaviors. However, the altered female behaviors in the other three species showed no consistent pattern as responses were not correlated with the morphology of the titillators, asymmetric or symmetric alterations, nor phylogenetic relationships (see Tables 6 and aggregated summary in   Table 7).

TA B L E 7
Changes in six mating-related traits as a response to male titillator manipulations for the Tettigoniinae Roeseliana roeselii, Pholidoptera l. littoralis, Tettigonia viridissima, and the Phaneropterinae species Letana inflata Note: Directional changes toward longer copula duration and increased female mating resistance (marked blue), trait values reduced, shortened or less successful compared to wild mating (marked brown), unchanged characters (yellow), and those not applicable or not visible faded out. The implications for sexual selection by either cryptic female choice (CFC) or sexually antagonistic coevolution (SAC) are given; CFC = The observed changes support sexual selection on titillators by cryptic female choice, SAC = The observed changes support sexual selection on titillators by sexually antagonistic coevolution, CFC/SAC: The observed changes are compatible with both cryptic female choice and sexually antagonistic coevolution.

| Copula durations and titillator movements
Copula durations (Figure 3) and the number of titillator movements ( Figure 4) varied greatly between the four bushcricket species, but less so between wild-type and manipulated matings (Tables 6 and 7).
Roeseliana roeselii wild-type males exhibited a broad span of copula durations, ranging from 25.93 to 73.50 min (mean ± SD: 41.16 ± 12.87, n = 20) (Figure 3). During copulation, they moved their titillators 9.93 ± 2.00 times per minute (mean ± SD, n = 20) in-and-out of the female genital chamber and performed small movements within the female genital chamber at the double rate (18.92 ± 4.65 per minute, mean ± SD, n = 17) (Figure 4, Video S1). Copula duration was unaltered by titillator manipulations, whereas the number of titillator movements was reduced in symmetric males ( p T -2 ) by around 10 percent, but not in asymmetric males (Table 7, see statistics Tables 6).
Males of Ph. l. littoralis showed the shortest copulation duration of the three Tettigoniinae species, and wild-type matings lasted 17.41 ± 8.17 min (mean ± SD, n = 13), which was less than half of the duration compared to the other Tettigoniinae species (Figure 3).
Despite the short time, Ph. l. littoralis males inserted and retracted their titillators more often from the female genital chamber than males of the other species (Figure 4), with a frequency of 22.32 ± 3.92 movements per minute (mean ± SD, n = 12). This high rate in large titillator movements seems to be compensated by the total lack of small titillator movements within the female's genital chamber (Figure 4). Copula duration was increased by 20 percent for asymmetrically manipulated Ph. l. littoralis ( p T -1 ) males (see Table 6 for statistics), whereas titillator movements did not change (Tables 6 and 7).
Tettigonia viridissima had a similar copula duration as R. roeselii: The males needed between 23.35 and 64.73 min (mean ± SD: 40.39 ± 12.56, n = 20). During mating, males showed the lowest rate of in-and-out titillator movements of all our species (mean ± SD: 4.97 ± 1.60 times per minute, n = 20). In contrast to Ph. l. littoralis, the low number of larger (in-and-out) titillator movements was compensated for by the highest rate of small movements within the female genital chamber (37.05 ± 6.12 per minute; mean ± SD, n = 21). The three Tettigoniinae species therefore demonstrate a negative correlation between the number of big titillator movements in-and-out of the female genital chamber and the number of small rhythmic titillator movements inside the chamber (Figure 4).
Unfortunately, movements of the single titillator were not observable as male and female genitalia were tightly coupled while males used their modified cerci and subgenital plate to securely hold the females. Big titillator movements (in-and-out) mean ± SD/min. behavior by walking, jumping, kicking, or biting prior to spermatophore transfer (Table 6: Fisher's exact test for the proportion of female resistance behaviors in manipulated vs. wild-type matings: p = .0086, n = 41). These seven out of 21 females showed these resistance behaviors in different combinations ( Figure 5), with the majority (57.1%) exhibiting all four behaviors (walking + jumping + kicking + biting the male). The remaining females showed two combinations of three (walking + jumping + kicking, 14.3%, jumping + kicking + biting the male 14.3%), or just the two behaviors of walking and jumping (14.3%). Such female resistance behavior resulted in separation of the couples in four cases. Three of the four couples reengaged in mating afterward. One female did not accept the males' attempts to reengage in copulation, and two females separated for a second time and did not attempt to mate further with the male. Three couples finished the mating attempt without spermatophore transfer, but this number was not significantly lower than in the wild-type group (Fisher's exact test of pairs successfully finishing spermatophore transfer in females mated to manipulated ( p T -left spines ) vs. wild-type males: p = .23, n = 41). However, spermatophore transfer success gradually decreased with the amount of titillator manipulation, slightly, nonsignificantly reduced in asymmetric ( p T -1 , p T -left spines ) but significantly reduced in symmetrically manipulated ( p T -2 ) males in previous experiments (Tables 6 and 7).
The inability to hold the mating position resulted in a failure of spermatophore transfer. In the manipulated groups, the success of the spermatophore transfer was reduced (Fisher's exact test: F I G U R E 5 Resistance behavior of females of the Tettigoniinae species Roeseliana roeselii. One third of females mated with manipulated males (T -left spines ) showed resistance behavior by walking, jumping, kicking, or biting. Among the females that showed resistance, most females showed all four behavioral types, followed by three types of walking + jumping+kicking or jumping + kicking + biting or just two behaviors of walking + jumping F I G U R E 6 Female resistance behavior by walking (light blue) and ampulla transfer success (dark blue) in Letana inflata during copulation with wild-type (n = 16) and with titillator-glued males (n = 18). (Pearson chi-square test: **p < .01)

Female resistance (%) Ampulla transfer success (%)
Letana inflata ** χ 2 2,42 = 18.33, p < .001), especially for males with both titillators shortened (post hoc test: wild type vs. p T -2 : χ 2 1,36 = 16.28, p < .001) (Figure 7b). This seems to be the consequence of a malfunction of manipulated titillators. Insertion of the male's titillators of Pholidoptera l. littoralis wild-type males resulted in contact between male and female genitalia, while the retraction of the titillators was followed by a slow slipping out. As the next titillator insertion followed quickly (the wild-type males inserted their titillators in mean 22 times per minute; Table 6, Figure 4), the couples in the wild-type group separated seldom.

| Postcopulatory behavior and outcomes
All observed changes due to titillator manipulations across the four species were restricted to the copulation phase. Postcopulatory female behaviors, such as the ingestion duration of the spermatophore or the ampulla, the female refractory period until the next mating or the number of eggs laid during this refractory period, remained unchanged (Tables 6 and 7).
Our cross-species comparison of four bushcricket species supports such a broadened view on evolutionary forces shaping insect genitalia, as mating-related responses to titillator manipulations are species-specific. In R. roeselii, the titillators apparently function as stimulators (Wulff et al., 2015(Wulff et al., , 2017, which are sensed by female receptors inside the female genital chamber (Wulff et al., 2018) and promote female acceptance of copulation and sperm transfer (Wulff et al. 2016;Wulff et al., 2018). Females' resistance behavior against males with asymmetrical spines in our new experiment is nearly identical to previous mating outcomes when males have one titillator removed (Wulff & Lehmann, 2016;Wulff et al., 2018). The symmetrical stimulation with the spines of both titillators seems to be crucial for determining whether females remain motionless with their genital folds open or disturb the copulation and try to prevent spermatophore transfer (Wulff & Lehmann, 2016;Wulff et al., 2018).
The lack of symmetrical stimulation may therefore cause female rejection behavior. These results support our previous supposition that titillators in R. roeselii function as copulatory courtship devices (Wulff et al., 2015(Wulff et al., , 2018. The best explanation for these cumulative results seems to be female cryptic choice during copulation based on adequate stimulation (Eberhard, 1996;Eberhard & Lehmann, 2019). Furthermore, intact titillators seem to have an additional mechanical function, namely to support the spermatophore transfer, as spermatophore transfer success was lower for males who had both of their titillators altered (Wulff & Lehmann, 2016) do not appear to play the same role. This might suggest that in this species titillators either do not act as stimulators or alternatively that they have effects that do not impact on mating success. So titillator movements seem to be species-specifically sensed by the females and trigger different processes. In T. viridissima, neither symmetrical nor asymmetrical titillator alterations substantially affected female behavioral responses as none of our measured parameters during and after the mating are altered. Consequently, the importance of titillators for mating in this species is unclear. However, as the titillators and the surrounding phallobasis are moved in concert with a fast rhythm, the movements of the phallobasis alone might be sufficient to stimulate the females. Therefore, the possibility of cryptic female choice cannot be excluded. It is clear from our results that we need deeper insights into the mating system of this species to understand the titillator function. The challenge is that finding an effect is easy to interpret, but the lack of a female response does not exclude the possibility that copulatory or postcopulatory selection exists (Eberhard, 2011).
The third Tettigoniinae, Ph. l. littoralis, uses titillators as mechanical anchors. Each titillator insertion induces an approach of the genitalia, while the retraction results in a slow slipping out of the genital chamber. This slow separation movement is counteracted by rapid titillator reinsertion, resulting in a high titillator movement frequency. In the wild-type mating experiments, titillator movement only occasionally leads to a separation of the copulating pair. As the females allow them to remount, all wild-type males transfer their spermatophore. In contrast, experimental shortening of the titillators results in males slipping out regularly, regardless of whether one or both titillators are altered. Males could keep the mating position only for short periods, and several mating partners separate without being able to transfer the spermatophore. As a result, spermatophore transfer is reduced.
The effect is only significant when both titillators are shortened.
We therefore conclude that the titillators in Ph. l. littoralis have a function as anchors, mechanically facilitating male attachment, while also assisting spermatophore transfer. Such genitalia anchoring is reported for several insect species (Simmons, 2014) and might be selected for by sexually antagonistic coevolution (SAC).
Interestingly, titillator anchoring is found only in one of the four bushcricket species tested by us. However, an anchoring function might not explain the repeated retraction and reinsertion of the titillators. The quick in-and-out movement of the titillators therefore hints to some stimulating function as well, even if we have not identified the triggered female copulatory or postcopulatory responses yet.
In our out-group species from the subfamily Phaneropterinae, L. inflata, nonconsensual mating is possible, where males grasp the female on the ventral part of the abdomen and then slowly move downwards until reaching mating position (Heller & Liu, 2015).
Females who move while the male is grasping her abdomen can be injured by the spines on the male's cerci (we observed two out of seven females who struggled during the grasping stage were bleeding afterward). Female resistance at this point therefore can be risky. In contrast to most bushcrickets species, the mating partners do not separate immediately after ampulla transfer but stay in a lengthy copula until the sperm have entered the female spermatheca . In this respect, L. inflata is similar to several other bushcricket species who have replaced the sperm-protecting function of the costly spermatophylax (Lehmann, 2012;Lehmann et al., 2018) with prolonged postcopulatory mate guarding (Vahed, Gilbert, Weissman, & Barrientos-Lozano, 2014). However, in our experiments a significant number of females resisted manipulated males, resulting in reduced copula duration. Therefore, L. inflata also demonstrates cryptic female choice, as properly stimulated females refrain resistance and accept a proper attachment of the sperm-containing ampulla.
Comparing the four species demonstrates that titillator function and the reactions toward titillator-manipulated males show no unifying pattern. Manipulation of the male's titillators had diverse effects. These include affecting female stimulation, the suppression of female resistance to allow stable male fixation, and mechanical support of spermatophore attachment. It is useful to study genital behavior across species in a robust phylogenetic framework, but in contrast to the general expectation of shared outcomes in more closely related species (Eberhard, 2011), our results are independent of the phylogenetic relationships (Hawlitschek et al., 2017;Mugleston et al., 2018). As no clear relationship between the titillator morphology and the responses toward their alterations was found, closer study of both sexes genitalia function for each species is warranted. This is a challenge, as most research focuses on genitalia morphology in males (reviewed in Simmons, 2014) and females as well (Sloan & Simmons, 2019), despite the consideration by Eberhard (2011) that sexual selection on genitalia might act on different female responses. Studying the function of genitalia therefore needs a better understanding of their action. Advanced imaging technologies exist that allow to study the hidden nature of genitalia action inside the female. For example, we have applied the snap-frozen technique in combination with static µCT (Wulff et al., 2015) and synchrotron-assisted live scans of the internal mechanisms in our model species R. roeselii (2017). Applying these advanced imaging techniques successfully revealed the internal mechanisms and made the otherwise hidden genital movements of titillators visible. As understanding the function is crucial to develop testable behavioral paradigms, we strongly encourage researchers of genitalia to move beyond describing static morphologies, which unfortunately still prevails as the major information published for most insect and arthropod species.
After studying genital functions, the next necessary step is to test behaviors of the mating partners. The notion that mating behaviors cannot be deduced from morphology alone, but have diversified independently from morphology (Eberhard, 2011), is well supported by our data; despite morphological similarity between the three Tettigoniinae species belonging to the same titillator morphotype Vahed et al., 2011), the behavioral alterations associated with titillator manipulations vary largely. Such plasticity in behavioral responses despite morphological similarities can be attributed to the filter function of the nervous system, showing that behavior connects evolutionary selection pressures with individuals' performance (Orr & Garland, 2017). Again, it is less surprising that genital behavioral parameters and responses vary between species. Similar results have been observed for five Glossina fly species (Briceño & Eberhard, 2009a, 2009bBriceño & Eberhard, 2017;Briceño, Eberhard, Chinea-Cano, Wegrzynek, & Santos Rolo, 2016). The copula duration of our bushcrickets is highly species-specific, varying from moderately short in Ph. l. littoralis to very long in L. inflata. In matings involving males with altered titillators, the copula duration is shortened in the long copulations of L. inflata, but prolonged in the short copulations of Ph. l. littoralis. Whether this response is a general pattern reflecting female cryptic choice selecting against males bearing unfavorable titillators might be analyzed across a greater number of species. A second behavioral response is found in L. inflata for the ampulla transfer duration, which, in accordance with the shorter copula duration, is also reduced in matings with titillator-manipulated males. Despite any sexual selection implications, the combined number of small and large titillator movements seems to be constrained; this is reflected in a negative correlation between the number of large versus the number of small titillator movements across species. It can be assumed that the physical capability for movements limits the combined number of small and large titillator movements.
It is possible that titillator movements are a character representing male fitness, which would make the titillator capacity an honest male signal detectable by females. In this case, the female responses of both L. inflata and R. roeselii can be attributed to cryptic female choice, as females resist males with altered titillators, reducing the sperm transfer success. The exhibited range of female rejection behaviors is plastic and includes female moving during copulation, biting, and a range of other behaviors.
In conclusion, it might help to widen our theoretical approaches and analyze the interplay between males and females during mating within a communication framework, as mating includes the production, hence exchange, and detection by the nervous system, hence reception, of copulatory signals (Briceño & Eberhard, 2017;Rodriguez, 2015). The bushcricket titillators might be a good example for such an approach, as the evidence for the four tested species suggests the evolution of genitalia under a sexual selection mosaic of mainly cryptic female choice, some evidence as well as for sexually antagonistic coevolution, or even a mosaic of both acting within the same species.

ACK N OWLED G M ENTS
We thank William Eberhard and Arne Lehmann for intense discussion on genitalia-related topics and Robert Hickson for discussion and proofreading. We are also acknowledging the great help of the reviewers and the associate editor to improve the presentation of our results. We are grateful to René Mai for his picture of the Letana inflata mating. NW received financial support by the Friedrich-Ebert-Stiftung (Friedrich-Ebert academic foundation).
We acknowledge support by the German Research Foundation (DFG) and the Open Access Publication Fund of Humboldt-Universität zu Berlin.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
NW and GL jointly designed the study. NW collected and reared the specimens, performed the experiments, and initially analyzed the data. GL supervised the study. Both NW and GL interpreted the results, wrote the manuscript, and approved the final version of the manuscript before submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
The behavioral and body mass data from the manuscript are archived with Dryad (https ://doi.org/10.5061/dryad.crjdf n31f). Sampling locations are included in Table 1 of the Section 2.