**2.2 Study of behaviour and sound production**

This study was conducted with 9 males (Table 3), 6 males of *R. pictus* and 3 males of *R. brunneri*, and 312 different songs (126 from *R. pictus* and 186 from *R. brunneri*), registered in 5 different tapes (Table 3). The specimens were held in cages in the laboratory and fed mainly with grasses that were changed daily. Humidity was provided by daily watering and by cotton imbibed in water. Two types of cages were used: a glass cage, with net top, 20 x 11 x 14 cm, and a wooden cage with metal mesh top and glass front, 35 x 35 x 55 cm, both exposed to natural light or artificial light provided by a 40W bulb 12 hours per day (Table 3).

The sounds produced were recorded under different conditions, such as isolated specimens, a male together a female of the same species, a species together other different species, both species together.

Sound recordings were made in the laboratory (Table 3) using a Uher 4000 and a Uher 6000 analogical tape recorders (Uher Werke München, Barmseestrasse 11, 8000 München 71, Germany), at a tape speed of 9.5 cm/s, with a frequency response (in Hz) of 20-25000 and signal-to-noise ratio better than 66 dB A remote-control Uher M655 and a Uher M518 dynamic microphones, were located 10-20 cm from the specimens.

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

resolution 0.336 Hz.

(http://osf2.orthoptera.org) (Eades 2001).

against some specialized tegminal veins.

several times, when close to other individuals, males or females.

**3. Results** 

the *Rhammatocerus* Species Inhabiting Uruguay (Orthoptera, Acrididae, Gomphocerinae) 53

characteristics of the sound, the analogical signal was digitized with a Sound Blaster® AWE64 Gold at 8 bits and at a 44 kHz sampling frequency, and then studied using the Avisoft® SAS Lab Pro 3.8. PC software for MS-Windows. Oscillograms were obtained using the option One-dimensional functions, selecting the function Time-signal. The spectral characteristics were obtained using the same option than before, selecting, in the function Amplitude spectrum (linear), the Hamming evaluation window, bandwidth 0.526 Hz and

Males of both species produce different types of sound in different behavioural situations fitting with the categories from the literature (García et al., 2003; García et al., 2005; Ragge & Reynolds, 1998). The terminology used to describe songs follows that of Ragge & Reynolds (1998). The variables used for the study of the sounds are: echeme length, number of syllables, syllables length, rate of emission of syllables, peak of maximum amplitude, low quartile, middle quartile, upper quartile, minimum frequency, maximum frequency and band width. The following types of sounds were identified: (1) the calling song, produced spontaneously by a male; (2) the courtship song, produced by males when close to a female and (3) the disturbance song, produced by males when interacting with other individuals.

To explore the relationship among different types of sounds produced by the studied species, a principal component analysis (PCA) was performed considering all the variables used to study the sounds (see before). Since for disturbance songs not all response variables could be recorded, they were not included in the PCA. Two-ways MANOVA was used to formally assess the differences among type of sound (calling song and courtship song) and species (*R. pictus* and *R. brunneri*), considering the variables used for the study of sounds as dependent variables. When cases corresponding to different groups overlapped after running the PCA, a separate MANOVA was performed for these groups considering the same dependent variables and factors as for the overall MANOVA. Variables were logtransformed for both PCA and MANOVA and a significance level of 5% was selected.

Specimens and recordings are kept in the "Colección de Entomología de la Facultad de Ciencias de la Universidad de la República", Uruguay, and in the "Colección del Área de Zoología de la Universidad de Murcia", Spain. Recordings can be reviewed using the recording number (Table 3). Sample songs are also available at OSF

The sounds recorded, emitted by males in different behavioral situations, were all produced by femoro-tegminal mechanism, rubbing the pegs of the stridulatory file of hind femora

No differences between songs have been observed in relation to the recording conditions. No sound produced by females has been registered in any case, although they produce movements with their hind legs. These movements are of moderate amplitude, starting with the leg folded at about 45º respect the vertical and displacing about 45º from the starting position. This movement can be isolated, from resting position to the vertical, or be repeated


Table 3. Summary of the information concerning specimens used to study the acoustic behaviour of *Rhammatocerus* and studied recordings.

Observations of communicative and interactive behaviour and of the general activity of individuals were made in the laboratory and recorded with a JVC GR-AXM23 video-camera for subsequent analysis. Specimens were observed throughout the recordings, including mute periods, and the behaviour of the specimens in each situation was noted.

Sound recordings were analysed using a Mingograph 420 System attached to a digital oscilloscope (Tektronix 2211) and to a Krohn-Hite 3550 filter. To study the physical characteristics of the sound, the analogical signal was digitized with a Sound Blaster® AWE64 Gold at 8 bits and at a 44 kHz sampling frequency, and then studied using the Avisoft® SAS Lab Pro 3.8. PC software for MS-Windows. Oscillograms were obtained using the option One-dimensional functions, selecting the function Time-signal. The spectral characteristics were obtained using the same option than before, selecting, in the function Amplitude spectrum (linear), the Hamming evaluation window, bandwidth 0.526 Hz and resolution 0.336 Hz.

Males of both species produce different types of sound in different behavioural situations fitting with the categories from the literature (García et al., 2003; García et al., 2005; Ragge & Reynolds, 1998). The terminology used to describe songs follows that of Ragge & Reynolds (1998). The variables used for the study of the sounds are: echeme length, number of syllables, syllables length, rate of emission of syllables, peak of maximum amplitude, low quartile, middle quartile, upper quartile, minimum frequency, maximum frequency and band width. The following types of sounds were identified: (1) the calling song, produced spontaneously by a male; (2) the courtship song, produced by males when close to a female and (3) the disturbance song, produced by males when interacting with other individuals.

To explore the relationship among different types of sounds produced by the studied species, a principal component analysis (PCA) was performed considering all the variables used to study the sounds (see before). Since for disturbance songs not all response variables could be recorded, they were not included in the PCA. Two-ways MANOVA was used to formally assess the differences among type of sound (calling song and courtship song) and species (*R. pictus* and *R. brunneri*), considering the variables used for the study of sounds as dependent variables. When cases corresponding to different groups overlapped after running the PCA, a separate MANOVA was performed for these groups considering the same dependent variables and factors as for the overall MANOVA. Variables were logtransformed for both PCA and MANOVA and a significance level of 5% was selected.

Specimens and recordings are kept in the "Colección de Entomología de la Facultad de Ciencias de la Universidad de la República", Uruguay, and in the "Colección del Área de Zoología de la Universidad de Murcia", Spain. Recordings can be reviewed using the recording number (Table 3). Sample songs are also available at OSF (http://osf2.orthoptera.org) (Eades 2001).
