**2.1 Microscopy techniques and characters considered in stridulatory file study**

To study the structure of the stridulatory apparatus, 10 specimens of each sex and species (Table 1) were observed under a binocular microscope (Olympus SZH provided with 10X ocular lenses, 0.66-4X zoom objective, 2X lens and graduated eye piece) as well as with a Jeol 6100 scanning microscope, equipped with SEI (secondary electron images), working at an acceleration voltage of 15 kV and at 21 mm (working distance). Images were captured with the LINK ISIS program. Because the samples are hard and have not risk of being dehydrated, they only had to be cleaned as proposed by Clemente et al. (1989) and, then, coated with pure gold.

Measurements were taken by means of a sliding stage mounted on the stereoscopic binocular microscope, the displacement of which was measured by an attached dial calliper in combination with a graduated eye piece. The accuracy of the dial calliper was 0.05 mm.

For this study, the shape of the file and pegs have been taken into account, as well as the number of pegs in the file, femur length (HFL), file length (FL), peg density all along the file (PD) and in its middle area (PDM) and file length / femur length ratio (FLx100/HFL). Measurements are expressed in mm (Table 2).

*R. brunneri* 

*R. pictus* 

Males n=10

Females n=10

> Males n=10

Females n=10

was P≤ 0.05.

species together.

16.96 ± 0.76 (15.80-17.90)

23.86± 0.86 (22.70-25.25)

17.92 ± 0.94 (16.50-19.40)

22.78 ± 1.32 (21.10-24.65)

**2.2 Study of behaviour and sound production** 

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

**HFL (mm) FL (mm) Pegs PD (mm-1) PDM** 

5.09 ± 0.76 (4.5-5.70)

11.61 ± 0.73 (10.70- 12.90)

5.21 ± 0.62 (4.40-6.00)

10.78 ± 1.05 (8.95-12.05)

the *Rhammatocerus* Species Inhabiting Uruguay (Orthoptera, Acrididae, Gomphocerinae) 51

74.4 ± 12.4 (58-99)

> 84 ± 7.1 (72-95)

73 ± 10.70 (58-90)

74.2 ± 3.29 (69-79)

Table 2. Summarized data related to stridulatory apparatus of *Rhammatocerus brunneri* and *R. pictus*. HFL: hind femur length; FL: whole stridulatory file length; Pegs: total number of pegs in the whole file; PD: density of pegs in the whole file; PDM: density of pegs in the

Two-ways multivariate analysis of variance (MANOVA) was used to assess whether there were overall differences between study species regarding the stridulatory file, considering as dependent variables the abovementioned morphological traits. In this case separated analyses were run for each sex. For these and all other statistical analyses, software SPSS (v. 15.0) was used. All variables were log-transformed. The signification value, in all the cases,

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

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.

middle of the file. Values are expressed as mean ± SD and range in parenthesis.

14.60 ± 1.98 (11.60- 18.68)

7.26 ± 0.70 (5.81-8.12)

14.18 ± 2.61 (11.05- 18.88)

6.94 ± 0.69 (6.09-8.16) **(mm-1)** 

19.5 ± 3.2 (13-24)

12.6 ± 1.9 (10-16)

18.6 ± 2.8 (14-22)

9.8 ± 1.8 (8-12)

**FL x 100/HFL** 

29.96 ± 0.97 (28.61- 31.84)

> 49.02 ± 3 (44.95- 53.69)

29.04 ± 2.78 (24.71- 33.33)

47.57 ± 6.10 (36.30- 54.50)


Table 1. Summary of the information concerning origin, collection date and number of specimens used to study the sound producing organs of *Rhammatocerus.* 

Sierra de la Aurora.

Cuchilla de Cuñapirú. Rivera. Uruguay

Ronda Alta. Río

40 km. N de

Rivera. Uruguay

Uruguay

Sª de la Aurora. Rivera. Uruguay

Las Piedras. Canelones. Uruguay

Buena Vista Agraciada. Soriano. Uruguay

Lagoa Vérmelha. Rio

Ayo. Tres Cruces. Artigas. Uruguay

Argentina

Table 1. Summary of the information concerning origin, collection date and number of

Grande do Sul. Brasil 18-II-1964

4 males (nº 61, 62, 63, 65) Cerro Chato Dorado.

1 males (nº 70) Bom Jesús.Río Grande

2 males (nº 56, 59) Cerro Chato Dorado.

2 males (nº 60, 66) Lunarejo. Rivera.

1 male (nº 74) Pto. Pepeají.

1 female (nº 81) Tartagal. Salta.

2 females (nº 82, 84) San Lorenzo. Salta.

1 female (nº 85) Nonoai. Río Grande

specimens used to study the sound producing organs of *Rhammatocerus.* 

1 male (nº 57) V.Lunarejo Rivera.

1 male (nº 64) 2 females (nº 93, 94)

2 males (nº 67, 68) 1 female (nº 91)

1 males (nº 69) 3 female (nº 86, 90, 95)

1 male (nº 71) 4 females (nº 87, 88, 89, 92)

> 1 male (nº 72) 1 female (nº 79)

> 1 male (nº 73) 1 female (nº 76) 1 female (nº 77)

1 male (nº 58)

1 male (nº 75) 1 female (nº 83)

1 female (nº78) 1 female (nº 80)

*Rhammatocerus brunneri* 

*Rhammatocerus pictus* 

**Specimens Locality Date Collector** 

Rivera. Uruguay 14-III-1961

Caaguazú. Paraguay 13-III-1965

Rivera. Uruguay 12-II-2000 Clemente,

Grande do Sul. Brasil 24-II-1964 A. Mesa, M.A.

do Sul. Brasil 26-II-1964 A. Mesa, M.A.

17-18-III-

Uruguay 13-II-2000 Clemente,

14-III-1961 11-III -1961

5-II-1966 5-XI-1966 20-III-1964

12-XI-1955 14-II-1955

> 29,31-I-1965

Argentina 3-II-1965 A. Mesa, R.

do Sul. Brasil 20-II-1964 A. Mesa, M.A.

Paysandú. Uruguay IV-1954 C.S.C.

García, Lorier

C.S.C, A. Mesa, P. San Martín

Monné

Monné

C.S.C, A. Mesa, M.A. Monné

García, Lorier

C.S.C, A. Mesa, R. San Martín

A. Cármenes

Presa

C.S.C, A. Mesa, M.A. Monné

Fac. Humanidades y Ciencias

A. Mesa, R. Sandulski

Sandulski

Monné

21-I-1956 C.S.C.

<sup>2001</sup>E. Lorier

8-II-2001 García, Lorier,

1-IV-1999 E. Lorier


Table 2. Summarized data related to stridulatory apparatus of *Rhammatocerus brunneri* and *R. pictus*. HFL: hind femur length; FL: whole stridulatory file length; Pegs: total number of pegs in the whole file; PD: density of pegs in the whole file; PDM: density of pegs in the middle of the file. Values are expressed as mean ± SD and range in parenthesis.

Two-ways multivariate analysis of variance (MANOVA) was used to assess whether there were overall differences between study species regarding the stridulatory file, considering as dependent variables the abovementioned morphological traits. In this case separated analyses were run for each sex. For these and all other statistical analyses, software SPSS (v. 15.0) was used. All variables were log-transformed. The signification value, in all the cases, was P≤ 0.05.
