Bioacoustics of Miogryllus piracicabensis

The karyotype of M. itaquiensis n. sp. and M. piracicabensis are similar, both showing the same secondary constriction and small satellite in the p arm. However, there is a small difference in the position of the centromere of the pair 2, probably due to a pericentric inversion, making these bivalents submetacentric in M. itaquiensis n. sp. and metacentric in M. piracicabensis . Although phallic sclerites are traditionally used as specific characters in Gryllinae , their morphology is very similar for South American species of M. itaquiensis n. sp. , M. piracicabensis and M. muranyi ( Mello & Morselli, 2011 ) . On the other hand, there are small morphological differences that allow identifying these species, such as head size and spots pattern, and the tegmina morphology. However, the calling song showed very different parameters among M. itaquiensis n. sp. and M. piracicabensis . Furthermore, the difference in the morphology of the chromosomes of the pair 2 is also a good character to distinguish both species. Thus, the combination of body morphology, calling song parameters and karyotype allowed accurate determination of the two closed related species.

Phallic sclerites ( Figs 29–31 ) . Idem M. itaquiensis n. sp. including same variations. Right tegmen ( Fig. 32 , n=41). Similar to M. itaquiensis n. sp. , however apical region is reduced. Measurements: harp area, 5.4 ± 0.72mm 2 (3.7–6.8); mirror area, 2.1 ± 0.38mm 2 (1.3–3); dorsal field length, 6.7 ± 0.7mm (4.9–7.9); dorsal field width, 2.9 ± 0.4mm (2.3–4,6); lateral field length, 6.5 ± 0.6mm (5.2–7.9); lateral field width, 2.6 ± 0.36mm (1.8–3.7); file length, 1.8 ± 0.17mm (1.3–2.1); teeth number, 97.5 ± 5.2 teeth (87–106); six accessory veins in lateral field. Calling song ( Figs 19–20 , Tab. 1 ) . Calling song emitted only in chirps; 4 to 6 pulses per chirp (percentage in the Table 1 ); chirp rate, 4.8 ± 0.65s (3.9–6, n = 30); chirp period, 0.20 ± 0.026s (0.17–0.25, n = 30); inter-chirp interval, 0.13 ± 0.03s (0.09–0.17, n = 30); dominant frequency 5910 ± 188Hz (5618–6134, n = 30); temperature from 17 to 25°C. Female ( Figs 22 , 33–34 ). Body shape and color pattern similar to males, slightly bigger than male; short tegmina, covering two first abdominal tergites; subgenital plate trapezoid; ovipositor slightly longer than femur III, apex lanceolated. Measurements (mm). Male (n=38): body length, 15.2 ± 1.44 (11.9–18); pronotum width, 4.3 ± 0.27 (3.6–5); pronotum length, 2.3 ± 0.18 (1.8–2.7); head width, 4.2 ± 0.3 (3.4–4.9); interocular distance, 3.3 ± 0.33 (2.5–3.9); femur III length, 8.8 ± 0.61 (7.6–10.3); tibia III length, 5.9 ± 0.41 (5.2–6.9). Female (n = 9): body length, 16.5 ± 2.64 (13.6–20.1); pronotum width, 4.8 ± 0.36 (4.2–5.2); pronotum length, 2.7 ± 0.26 (2.4–3.1); head width, 44.6 ± 0.24 (4.2–4.8); interocular distance, 3.4 ± 0.3 (3.0–3.8); femur III length, 9.7 ± 0.5 (9.1–10.6); tibia III length, 6.4 ± 0.38 (5.6–7.0); dorsal field length, 3.3 ± 0.6 (2.1–4.2); dorsal field width, 2.0 ± 0.3 (1.4–2.6); ovipositor length, 11.5 ± 1.6 (8.8–13.6). Karyotype ( Fig. 36 ) . Diploid chromosome number of 2n = 24 + X ♂ = 25, and 2n = 24 + XX ♀ = 26; three pairs of metacentric chromosomes (pair 1, ci = 43.1, with secondary constriction in p arm, which left a small satellite at the chromosome tip; pair 2, ci = 44.7; pair 4, ci = 44.4), five pairs of submetacentric (pair 3, ci = 34.2; pair 5, ci = 15.1; pair 6, ci = 29; pair 7, ci = 14.8; pair 10, ci = 17.1) and four pairs of acrocentric (pair 8, ci = 10.3; pair 9, ci = 11.5; pair 11, ci = 11.1; pair 12, ci = 10). The X chromosome is metacentric (ci = 43.4), and the largest of the complement. 1 2 3 4 5 6 (n = 30) 7 (n = 30) 8 9 02DA 0.05 ± 0.001 (0.04–0.05) n = 34, 74% 0.06 ± 0.002 (0.06–0.07) n = 12, 26% – – 4.6 0.2 ± 0.016 (0.18–0.25) 0.16 ± 0.016 (0.13–0.2) 5860 18°C 07DA – 0.06 ± 0.002 (0.05–0.07) n = 32, 60.4% 0.07 ± 0.001 (0.07–0.08) n = 21, 39.6% – 5.3 0.1 9 ± 0.013 (0.16–0.22) 0.13 ± 0.016 (0.10–0.17) 5793 25°C 17DA – – 0.07 ± 0.001 (0.07–0.07) n = 34, 56.7% 0.09 ± 0.001 (0.08–0.09) n = 26, 43.3% 6.0 0.17 ± 0.008 (0.16–0.19) 0.09 ± 0.009 (0.07–0.11) 6082 21°C 18DA – 0.06 ± 0.001 (0.057–0.059) n = 3, 6.4% 0.07 ± 0.001 (0.07–0.07) n = 43, 93.6% – 4.6 0.2 2 ± 0.047 (0.17–0.45) 0.14 ± .0 15 (0.12–0.19) 6065 19°C 21DA – – 0.08 ± 0.07 (0.08–0.002) n = 51, 100% – 5.1 0.1 9 ± 0.008 (0.17–0.21) 0.11 ± 0.008 (0.09–0.123) 6134 21°C 29DA – 0.06 ± 0.001 (0.061–0.063). n = 4, 10% 0.08 ± 0.002 (0.077–0.084) n = 36, 90% – 4.0 0.2 5 ± 0.054 (0.19–0.43) 0.17 ± 0.054 (0.11–0.36) 5770 18°C MMC0 – 0 6 0.06 ±.0 0 0 3 (0.056–0.057) n = 3, 5.9% 0.07 ± 0.0008 (0.07–0.074) n = 48, 94.1% – 4.8 0.1 9 ± 0.02 (0.16–0.24) 0.13 ± 0.02 (0.1–0.17) 5611 23°C Discussion Considering the eight valid species of Miogryllus that occur in South America, six of them, M. verticalis , M. tucumanensis Giglio-Tos, 1894 , M. piracicabensis , M. incertus (Giglio-Tos, 1894) , M. convolutus (Johannson, 1763) and M. bohlsii (Giglio-Tos, 1895) , bear a type locality relatively close to the municipality of Itaqui ( Cigliano et al. 2017 ). However, except for M. piracicabensis , none of the others species had their male genitalia, calling songs or chromosomal information published. Because of their overall similarity, identification was very difficult and ultimately inaccurate. We compared the morphological characteristics of these species with M. itaquiensis n. sp. and verified that the new species bears four yellow occipital stripes, while M. convolutus has only two. The male tegmina of M. itaquiensis n. sp. cover about two thirds of the abdomen, while in M. verticalis and M. bohlsii it covers the entire abdomen. The female tegmina of M. itaquiensis n. sp. is longer than 8mm , while in M. incertus it measures 2mm . The lateral lobe of pronotum of M. itaquiensis n. sp. bears a whitish lateral lobe, while M. tucumanensis has the pronotum with uniform coloration, as wells as both sexes with reduced tegmina, while M. itaquiensis n. sp. has long tegmina in both males and females. Finally, M. itaquiensis n. sp. differs from M. piracicabensis in the characteristics presented in this work, highlighting the differences that occur in the calling song. Otte (2006) and Otte & Perez–Gelabert (2009) described eleven new species of Miogryllus from the Caribbean region, all distinguished by genitalic characters. On the other hand, M. itaquiensis n. sp. , M. piracicabensis ( Mesa et al. 2004 ) and M. muranyi ( Mello & Morselli, 2011 ) , occurring in South America, have the morphology of the phallic sclerites that is very similar. The phallic sclerites of M. itaquiensis n. sp. and M. piracicabensis bear small variations in the morphology of the main lobes of pseudoepiphallus, with the extremities strongly or subtly curved inwards, and the ectophallic fold may or may not be truncated at the base, with the end tapering gradually or abruptly. Probably these small variations will be found in M. muranyi when larger samples are analyzed.

Phallic sclerites ( Figs 29–31 ) . Idem M. itaquiensis n. sp. including same variations. Right tegmen ( Fig. 32 , n=41). Similar to M. itaquiensis n. sp. , however apical region is reduced. Measurements: harp area, 5.4 ± 0.72mm 2 (3.7–6.8); mirror area, 2.1 ± 0.38mm 2 (1.3–3); dorsal field length, 6.7 ± 0.7mm (4.9–7.9); dorsal field width, 2.9 ± 0.4mm (2.3–4,6); lateral field length, 6.5 ± 0.6mm (5.2–7.9); lateral field width, 2.6 ± 0.36mm (1.8–3.7); file length, 1.8 ± 0.17mm (1.3–2.1); teeth number, 97.5 ± 5.2 teeth (87–106); six accessory veins in lateral field. Calling song ( Figs 19–20 , Tab. 1 ) . Calling song emitted only in chirps; 4 to 6 pulses per chirp (percentage in the Table 1 ); chirp rate, 4.8 ± 0.65s (3.9–6, n = 30); chirp period, 0.20 ± 0.026s (0.17–0.25, n = 30); inter-chirp interval, 0.13 ± 0.03s (0.09–0.17, n = 30); dominant frequency 5910 ± 188Hz (5618–6134, n = 30); temperature from 17 to 25°C. 1 2 3 4 5 6 (n = 30) 7 (n = 30) 8 9 02DA 0.05 ± 0.001 (0.04–0.05) n = 34, 74% 0.06 ± 0.002 (0.06–0.07) n = 12, 26% – – 4.6 0.2 ± 0.016 (0.18–0.25) 0.16 ± 0.016 (0.13–0.2) 5860 18°C 07DA – 0.06 ± 0.002 (0.05–0.07) n = 32, 60.4% 0.07 ± 0.001 (0.07–0.08) n = 21, 39.6% – 5.3 0.1 9 ± 0.013 (0.16–0.22) 0.13 ± 0.016 (0.10–0.17) 5793 25°C 17DA – – 0.07 ± 0.001 (0.07–0.07) n = 34, 56.7% 0.09 ± 0.001 (0.08–0.09) n = 26, 43.3% 6.0 0.17 ± 0.008 (0.16–0.19) 0.09 ± 0.009 (0.07–0.11) 6082 21°C 18DA – 0.06 ± 0.001 (0.057–0.059) n = 3, 6.4% 0.07 ± 0.001 (0.07–0.07) n = 43, 93.6% – 4.6 0.2 2 ± 0.047 (0.17–0.45) 0.14 ± 0.015 (0.12–0.19) 6065 19°C 21DA – – 0.08 ± 0.07 (0.08–0.002) n = 51, 100% – 5.1 0.1 9 ± 0.008 (0.17–0.21) 0.11 ± 0.008 (0.09–0.123) 6134 21°C 29DA – 0.06 ± 0.001 (0.061–0.063). n = 4, 10% 0.08 ± 0.002 (0.077–0.084) n = 36, 90% – 4.0 0.2 5 ± 0.054 (0.19–0.43) 0.17 ± 0.054 (0.11–0.36) 5770 18°C MMC0 0 6 – 0.06 ± 0.003 (0.056–0.057) n = 3, 5.9% 0.07 ± 0.0008 (0.07–0.074) n = 48, 94.1% – 4.8 0.1 9 ± 0.02 (0.16–0.24) 0.13 ± 0.02 (0.1–0.17) 5611 23°C Female ( Figs 22 , 33–34 ). Body shape and color pattern similar to males, slightly bigger than male; short tegmina, covering two first abdominal tergites; subgenital plate trapezoid; ovipositor slightly longer than femur III, apex lanceolated. Measurements (mm). Male (n=38): body length, 15.2 ± 1.44 (11.9–18); pronotum width, 4.3 ± 0.27 (3.6–5); pronotum length, 2.3 ± 0.18 (1.8–2.7); head width, 4.2 ± 0.3 (3.4–4.9); interocular distance, 3.3 ± 0.33 (2.5–3.9); femur III length, 8.8 ± 0.61 (7.6–10.3); tibia III length, 5.9 ± 0.41 (5.2–6.9). Female (n = 9): body length, 16.5 ± 2.64 (13.6–20.1); pronotum width, 4.8 ± 0.36 (4.2–5.2); pronotum length, 2.7 ± 0.26 (2.4–3.1); head width, 44.6 ± 0.24 (4.2–4.8); interocular distance, 3.4 ± 0.3 (3.0–3.8); femur III length, 9.7 ± 0.5 (9.1–10.6); tibia III length, 6.4 ± 0.38 (5.6–7.0); dorsal field length, 3.3 ± 0.6 (2.1–4.2); dorsal field width, 2.0 ± 0.3 (1.4–2.6); ovipositor length, 11.5 ± 1.6 (8.8–13.6). Karyotype ( Fig. 36 ) . Diploid chromosome number of 2n = 24 + X ♂ = 25, and 2n = 24 + XX ♀ = 26; three pairs of metacentric chromosomes (pair 1, ci = 43.1, with secondary constriction in p arm, which left a small satellite at the chromosome tip; pair 2, ci = 44.7; pair 4, ci = 44.4), five pairs of submetacentric (pair 3, ci = 34.2; pair 5, ci = 15.1; pair 6, ci = 29; pair 7, ci = 14.8; pair 10, ci = 17.1) and four pairs of acrocentric (pair 8, ci = 10.3; pair 9, ci = 11.5; pair 11, ci = 11.1; pair 12, ci = 10). The X chromosome is metacentric (ci = 43.4), and the largest of the complement.