**4. Discussion**

60 Zoology

Songs were composed of isolated syllables (Table 4, Fig. 6D and E) emitted alternately by different individuals. The frequency spectrum occupied a broad band, with the peak of maximum amplitude at around 10 kHz (Fig. 6F). The leg movements to produce this song were similar to that performed by *R. pictus*, usually one or two quick asynchronous up and

This song was emitted in different circumstances, such as coinciding with or at the end of a

For disturbance songs, indicated the existence of overall significant differences among

No disturbance songs produced by females have been recorded. One or two mute up and down movements of their hind legs were observed after a male interaction or courtship song or when interacting with other female. These movements have been also observed

Fig. 6. Disturbance song. *Rhammatocerus pictus* (A-C) and *Rhammatocerus brunneri* (D-F). Sequence of syllables (A and D); syllable detail (B and E) and frequency spectra (C and F).

performed by males when close to other individuals or interacting with them.

**3.3.2** *Rhammatocerus brunneri*

calling song of other male or as rivalry song.

species (MANOVA: F8, 15= 53.073; P<0.001).

down movements.

The stridulatory file of *R. pictus* and *R. brunneri* are well developed in both males and females. The peg spread, regular all along the file except at the ends, fits with that of other Gomphocerinae (Clemente et al., 1989; Pitkin, 1976). It can be pointed that the metrical characteristics referred to the stridulatory file are not species specific for males of *R. pictus* and *R. brunneri* but can serve to differentiate the females of both species. Nevertheless, there seem to be some morphological differences between stridulatory pegs of males. In both cases pegs are conic shaped, but *R. pictus* have pegs clearly more elongated, with a more acute apex, and the alveolus have the raised margin more irregular than *R. brunneri*.

The acoustic repertoire of *R. pictus* and *R. brunneri* is similar to that of other neotropical Gomphocerinae species, such as *Parapellopedon instabilis* (Rehn, 1906), *Euplectrotettix ferrugineus* Bruner, 1900 and *Fenestra bohlsi* Giglio-Tos, 1895 (García et al., 2003; Lorier, 1996; Riede, 1987), being made up of:


The function of these different types of signals linked to the reproductive behaviour in Orthoptera and other insects has been analysed and interpreted in the context of the sexual selection theory. Sound can evolve, among other possibilities, through sexual selection. Evolutionary pressures have to act towards a minor energetic cost and a minimum risk of predation in the mating (Bailey, 1991). The significance of the male calling songs of Orthoptera lies in the fact that they are believed to provide the main means of mater recognition and hence of reproductive isolation of sympatric species (Ragge, 1987; Ragge & Reynolds, 1998). Acoustic signals provide enough relevant information on species identification and sexual selection. So, sound is considered essential to solve taxonomical problems at the specific level. Closely related, sympatric species often use strikingly different acoustic criteria to discriminate the same set of conspecific and heterospecific signals (Gehardt & Huber, 2002). A marked difference between songs is a strong indicative of different species (Ragge & Reynolds, 1998). When two morphologically similar populations of Orthoptera have consistently different calling song, it is likely that they belong to different species. Conversely, when two populations show small morphological differences but have exactly the same calling song, they are probably forms of a single species (Ragge, 1987).

The Acoustic Behaviour as a Tool for Biodiversity and Phylogenetic Studies: Case of

the *Rhammatocerus* Species Inhabiting Uruguay (Orthoptera, Acrididae, Gomphocerinae) 63

Table 4. Data summary concerning the physical characteristics of the songs emitted by the males of *Rhammatocerus pictus* and *R. brunneri* on the time and frequency domains (data of which are expressed in Hz)

In most Gomphocerinae some acoustic signals identify the species and can avoid the crossing between sympatric close species. This is the case of *Rhammatocerus* species here studied, which show differences in physical characteristics of all types of song. Both species have overall different songs, as shown in Table 4 and Figures 4, 5 and 6. Differences are more or less evident depending on the type of song.

The calling song of *R. brunneri* is very similar to the courtship song (Fig. 5). Thus, this species uses a similar song, with little differences (Table 4), in two different behavioural situations. In *R. pictus* a similar situation exists between the calling song and one of the courtship songs recorded (pictus 1) (Fig. 4A-F). At the light of the recorded songs, this species may start courting with a song similar to the calling song (pictus 1) changing afterwards to a particular courtship song (pictus 2), accompanied by legs and antennae movements. Although the whole sequence has not been registered, it would be not surprising since the use of a courtship song similar to calling song has been observed in other Gomphocerinae species, such as *Chorthippus binotatus binotatus* (Charpentier, 1924) (García et al., 1995) and *Omocestus antigai* (Bolívar, 1987) (Clemente et al., 1999). In the last case, it could be observed that the courtship started with the calling song and, later, the courtship song was emitted.

This kind of behaviour fits with that observed, for example, in genus *Stenobothrus* Fischer, 1853, the ancestor of which should have had produced simple and likely identical calling and courtship songs. The complex courtship songs observed in many species resulted from the addition of new acoustic traits derived from the common calling song pattern and from the addition of visual signals produced by the movement of legs and antennae (Berger, 2008 as cited in Nattier et al., 2011). In *R. pictus*, during the courtship new traits related to the spectral characteristics of the sound and, on the other hand, visual signals by legs and antennae movements would have been added.

These similarity of calling and courtship songs in *R. brunneri* and, partly, in *R. pictus* could be explained by the signals flexibility observed in acridids by which the same signal can be used in different contexts, as here occurred. This would imply following the opinion of Otte (1977), a further stage in an evolutionary process not ended still, probably accompanying a sympatric speciation process. Probably, sounds should not be the unique barrier to avoid hybridizing but there could also take part other kind of signals, such as those visual or chemical (Otte, 1977), although behavioural traits are often evolutionary labile and hence communication systems often diverge more rapidly than do morphological and molecular characteristics (Gehardt & Huber, 2002). Nevertheless, the question of which of the two song types calling or courtship song is the phylogenetic older one remains open (Berger & Gottsberger, 2010) but a simplification of courtship repertoires in some species suggests that the evolution of courtship song, and that of mating behaviour as a whole, could also result from a dynamic process (Berger, 2008 as cited in Nattier et. al., 2011).

As regards the disturbance song, in some species phonotaxis is reduced when other males interfere, diminishing the efficiency of calling or courtship song. It could be pointed, for male disturbance songs – either spacing or answering other male courtship –, that the answering male, when alternating its song, uses its interference potential of acoustic channel


Table 4. Data summary concerning the physical characteristics of the songs emitted by the males of *Rhammatocerus pictus* and *R. brunneri* on the time and frequency domains (data of which are expressed in Hz)

62 Zoology

In most Gomphocerinae some acoustic signals identify the species and can avoid the crossing between sympatric close species. This is the case of *Rhammatocerus* species here studied, which show differences in physical characteristics of all types of song. Both species have overall different songs, as shown in Table 4 and Figures 4, 5 and 6. Differences are

The calling song of *R. brunneri* is very similar to the courtship song (Fig. 5). Thus, this species uses a similar song, with little differences (Table 4), in two different behavioural situations. In *R. pictus* a similar situation exists between the calling song and one of the courtship songs recorded (pictus 1) (Fig. 4A-F). At the light of the recorded songs, this species may start courting with a song similar to the calling song (pictus 1) changing afterwards to a particular courtship song (pictus 2), accompanied by legs and antennae movements. Although the whole sequence has not been registered, it would be not surprising since the use of a courtship song similar to calling song has been observed in other Gomphocerinae species, such as *Chorthippus binotatus binotatus* (Charpentier, 1924) (García et al., 1995) and *Omocestus antigai* (Bolívar, 1987) (Clemente et al., 1999). In the last case, it could be observed that the courtship started with the calling song and, later, the

This kind of behaviour fits with that observed, for example, in genus *Stenobothrus* Fischer, 1853, the ancestor of which should have had produced simple and likely identical calling and courtship songs. The complex courtship songs observed in many species resulted from the addition of new acoustic traits derived from the common calling song pattern and from the addition of visual signals produced by the movement of legs and antennae (Berger, 2008 as cited in Nattier et al., 2011). In *R. pictus*, during the courtship new traits related to the spectral characteristics of the sound and, on the other hand, visual signals by legs and

These similarity of calling and courtship songs in *R. brunneri* and, partly, in *R. pictus* could be explained by the signals flexibility observed in acridids by which the same signal can be used in different contexts, as here occurred. This would imply following the opinion of Otte (1977), a further stage in an evolutionary process not ended still, probably accompanying a sympatric speciation process. Probably, sounds should not be the unique barrier to avoid hybridizing but there could also take part other kind of signals, such as those visual or chemical (Otte, 1977), although behavioural traits are often evolutionary labile and hence communication systems often diverge more rapidly than do morphological and molecular characteristics (Gehardt & Huber, 2002). Nevertheless, the question of which of the two song types calling or courtship song is the phylogenetic older one remains open (Berger & Gottsberger, 2010) but a simplification of courtship repertoires in some species suggests that the evolution of courtship song, and that of mating behaviour as a whole, could also result from a dynamic process (Berger, 2008 as

As regards the disturbance song, in some species phonotaxis is reduced when other males interfere, diminishing the efficiency of calling or courtship song. It could be pointed, for male disturbance songs – either spacing or answering other male courtship –, that the answering male, when alternating its song, uses its interference potential of acoustic channel

more or less evident depending on the type of song.

courtship song was emitted.

cited in Nattier et. al., 2011).

antennae movements would have been added.

The Acoustic Behaviour as a Tool for Biodiversity and Phylogenetic Studies: Case of

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Assis-Pujol, C.V. de & Lecoq, M. (2000). Comparative study of spermathecae in eleven

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(Cade, 1985; Greenfield, 1997). Thus, the emitters compete for the receipt's attention, in this case the silent female. Thereby it could be understood, for *R. brunneri*, the courtship interruption provoked by rivalry songs (McGregor & Peake, 2000). Differences between disturbance songs of the two species, *R. pictus* and *R. brunneri*, support the specific communicative value even of this type of song.

Anyway, since the acoustic communication involves a particular signal recognisable by the conspecifics to avoid the interspecific mating, its characteristics have to be specific. In the studied cases, it has been proved that the sound characteristics, both in the time and in the frequency domains, allow clearly discriminating cryptic species, and offer useful elements for phylogenetic analysis. Up to now, the only characters to separate between *R. pictus* and *R. brunneri* was the colour of hind femora and tibiae and the spermatheca shape (Assis– Pujol, 1998), but the real status of both taxa remained obscure. So, our results can have consequences on taxonomy since they bring new elements to the genus revision, justifying the separation of the two species, *R. pictus* and *R. brunneri*, as valid species (Carbonell pers. comm.).
