Bioacoustics of Rhinella mirandaribeiroi

Morphologic and Morphometric Morphometric variable values can be found in Table 1 . The topotypes of R. mirandaribeiroi are morphologically similar to B. g. lutzi and other specimens from the Cerrado , and they can be diagnosed from most species in the group by: granular or continuous cranial crests, low supraorbital crest, infraorbital crest extending usually beyond the postorbital crest, generally discontinuous reduced maxillary crest, and absence of a parietal crest ( Figures 1–2 ). Loreal region clearly visible in dorsal view, tympanum very close to the postorbital crest (DTP 0.4–1.0% CRC), a usually present clear longitudinal vertebral line, and a light cream or yellowish ventral side, with or without pigmentation. Neither morphological nor morphometric characters allowed for the diagnosis between populations. Population-Variables Topotypes Soure PA Cerrado populations Topotypes B. g. lutzi N = 23 N = 45 N = 2 Snout-vent length 44.4 ± 2.5 (38.3–49.9) 48 ± 3.8 (41.2–59.4) 43.5 ± 7.5 (43.5–53.3) Thigh length 15.7 ± 1.1 (13.8–7.9) 16.8 ± 2 (12.2–20.8) 17.1 ± 5.5 (15.8–18.3) Tibia length 15.1 ± 0.9 (13.8–17) 16.5 ± 1.5 (13.0–19.9) 16.4 ± 5.1 (15.2–17.5) Tarsus length 9.4 ± 0.7 (8.2–10.8) 10.2 ± 1 (8.09–12.0) 11.3 ± 4.8 (10.6–12) Hand length 9.8 ± 0.6 (8.8–11) 10 ± 0.9 (8.44–12.2) 11 ± 4.7 (10.4–11.6) Foot length 15.8 ± 1.4 (12.9–18.3) 16.5 ± 1.5 (13.4–19.8) 17.8 ± 4.7 (17.2–18.4) Head height 6.4 ± 0.4 (5.8–7.2) 6.5 ± 0.5 (5.5–8.2) 6.3 ± 3.9 (5.9–6.7) Head width 14.3 ± 0.59 (12.8–15.7) 15.2 ± 0.9 (13.4–17.7) 15.4 ± 3.9 (14.5–16.3) Head length 12.8 ± 0.8 (11.4–14.3) 13.3 ± 0.9 (11.3–15) 13.4 ± 3.1 (12.8–14.1) Eye diameter 3.6 ± 0.4 (2.9–4.5) 4.2 ± 0.5 (3–5.1) 4.2 ± 2.1 (3.7–4.7) Tympanum diameter 2.3 ± 0.3 (1.6–2.8) 2.7 ± 0.37 (2–3.4) 2.3 ± 2.1 (2.3–2.4) Tympanum height 2.9 ± 0.4 (2.3–3.7) 3.4 ± 0.3 (2.8–4) 3 ± 2.2 (3–3.1) Snout width 5.2 ± 0.4 (4.5–5.7) 5.5 ± 0.4 (4.2–6.3) 5.1 ± 2.3 (5–5.3) Parotoid gland width 7.4 ± 1.2 (4.9–9.8) 8.2 ± 1.4 (5.2–10.9) 43.5 ± 7.5 (43.5–53.3) Parotoid gland height 5.6 ± 0.4 (4.83–6.59) 6.3 ± 0.9 (4.7–9.4) 5.7 ± 2 (5.4–6.0) Internarial distance 2.43 ± 0.2 (1.9–2.7) 2.59 ± 0.19 (2.2–3.1) 2.45 ± 1.9 (2.1–2.8) Eye-snout distance 2.32 ± 0.4 (1.6–3) 2.85 ± 0.36 (2–3.6) 3.2 ± 2 (2.8–3.6) Eye–nostril distance 4.28 ± 0.5 (3.5–5.3) 5.02 ± 0.57 (3.9–6) 5.4 ± 2.1 (4.9–5.9) Interorbital distance 4.96 ± 0.4 (4.1–5.8) 5.65 ± 0.47 (4.9–6.6) 5.35 ± 2 (5.1–5.6) Interorbital distance II 4.81 ± 0.4 (4–5.6) 4.94 ± 0.33 (4.2–5.8) 4.55 ± 1.8 (4.4–4.7) Eyelid width 3.69 ± 0.2 (3.3–4.1) 4.38 ± 0.35 (3.5–5) 4.25 ± 1.5 (3.7–4.8) ......continued on the next page Population-Variables Topotypes Soure PA Cerrado populations Topotypes B. g. lutzi N = 23 N = 45 N = 2 Supratympanic crest length 1.93 ± 0.4 1.98 ± 0.55 2 ± 1.1 (1–2.5) (0.8–3.2) (1.5–2.5) Postorbital space 3.06 ± 0.3 3.46 ± 0.66 2.85 ± 1.1 (2.3–3.6) (0.3–4.3) (2.6–3.1) Distance from tympanum to 0.27 ± 0.1 0.26 ± 0.12 0.3 ± 0.3 postorbital crest (0.2–0.4) (0.1–0.5) (0.1–0.5) Distance from tympanum to 0.97 ± 0.2 0.8 ± 0.18 0.55 ± 0.2 supra tympanic crest (0.5–1.4) (0.4–1.2) (0.3–0.8) Distance from tympanum to 0.71 ± 0.3 0.86 ± 0.34 0.6 ± 0.1 parotoid gland (0.2–1.25) (0.3–1.5) (0.5–0.7) Acoustics We found conserved parameters of call features among samples. All evaluated specimens have advertisement calls consisting of regular series of pulsed notes (trills) that can last up to 11 seconds. The call has ascending amplitude modulation, with the initial notes generally having low amplitude and then quickly reaching the maximum, which is maintained up the end. The notes consist of three to five concatenated pulses (without intervals of silence). No acoustic feature was different among the evaluated populations. Topotypical population of R. mirandaribeiroi (Soure, Marajó Island) In the type locality, males vocalize after rains at night in shallow puddles formed along dirt roads on the outskirts of the city. The immediate surroundings of these vocalization sites were open anthropogenic environments with pasture or cultivated areas. Syntopic anurans include Leptodactylus fuscus , Scinax x-signatus , Boana raniceps , S. fuscomarginatus, Physalaemus ephippifer, and Adenomera hylaedactyla . The advertisement call consists of a sequence of approximately 126 notes and lasts around 4 seconds, emitted at a rate of 8 calls per minute with 33 notes per second. The calls reach maximum amplitude rapidly and remain constant until the end ( Figure 3 ). The notes have a duration of about 20 ms and contain 3−4 pulses per note. The dominant frequency is around 2800 Hz. Further details in Table 2 .

Morphometrics and Morphology Both species share the following morphological features: 1) granular or continuous cranial crests; 2) low supraorbital crest; 3) infraorbital extending usually beyond the postorbital crest; 4) typically reduced and discontinuous maxillary crest; 5) absent parietal crest; 6) visible loreal region in dorsal view; and 7) light cream or yellowish ventral coloration with or without pigmentation. However, a clear dorsal vertebral line is present in 95% of the individuals from the Cerrado ( R. mirandaribeiroi ) and only in 5% from the Caatinga and Mata Atlântica ( R. granulosa ) ( Figure 1 , 2 ). Overall, the morphometric variables were similar between the two species ( Table 3 ). Species -Variables R. mirandaribeiroi R. granulosa N =70 N = 61 Snout–vent length 47.0 ± 3.9 (38.3–59.3) 48.7 ± 4.8 (37–59.4) Thigh length 16.5 ± 1.8 (12.2–20.8) 17.4 ± 2.1 (13.3–22.3) Tibia length 16.1 ± 1.5 (13–19.9) 17.8 ± 1.9 (14.5–21.8) Tarsus length 10 ± 1 (8.1–12) 11.1 ± 1.3 (8.4–13.9) Hand length 10 ± 0.8 (8.4–12.2) 11.3 ± 1.2 (8.5–14.4) Foot length 16.4 ± 1.5 (12.9–19.8) 18.1 ± 2.0 (13.6–22.2) Head height 6.5 ± 0.5 (5.5–8.2) 6.7 ± 0.6 (5.5–8.2) ......continued on the next page Species -Variables R. mirandaribeiroi R. granulosa N =70 N = 61 Head width 14.9 ± 0.9 (12.8–17.7) 15.6 ± 1.3 (13–18.3) Head length 13.2 ± 0.9 (11.3–15) 13.8 ± 1.2 (11.4–16.4) Eye diameter 4 ± 0.5 (2.9–5.1) 4.6 ± 0.5 (3.5–5.8) Tympanum diameter 2.6 ± 0.4 (1.6–3.4) 2.7 ± 0.4 (1.9–3.8) Tympanum height 3.3 ± 0.4 (2.3–4) 3.3 ± 0.4 (2.4–4.1) Snout width 5.4 ± 0.4 (4.2–6.3) 5.3 ± 0.5 (4.1–6.5) Parotid gland width 8.0 ± 1.4 (4.9–10.9) 7.2 ± 1.1 (5–10.9) Parotid gland height 6.1 ± 0.9 (4.7–9.4) 6.1 ± 0.9 (4.3–8.8) Internarial distance 2.53 ± 0.2 (1.9–3.1) 2.6 ± 0.3 (2–3.5) Eye–snout distance 2.7 ± 0.5 (1.6–3.6) 3.1 ± 0.5 (2.4–4.4) Eye–nostril distance 4.8 ± 0.7 (3.5–6) 5.3 ± 0.8 (4–7.5) Interorbital distance 5.4 ± 0.6 (4.1–6.6) 5.3 ± 0.6 (4.5–7.1) Interorbital distance II 4.9 ± 0.3 (4–5.8) 4.9 ± 0.7 (3.8–7.1) Eyelid width 4.1 ± 0.5 (3.3–5) 4.4 ± 0.5 (3.5–5.8) Supratympanic crest length 2 ± 0.5 (0.8–3.2) 2.3 ± 0.3 (1.6–3) Postorbital space 3.3 ± 0.5 (2.3–4.3) 4.2 ± 0.6 (3.2–5.8) Distance from tympanum to postorbital crest 0.3 ± 0.1 (0.1–0.5) 0.4 ± 0.2 (0.1–0.8) Distance from tympanum to supra tympanic crest 0.9 ± 0.2 (0.4–1.4) 1 ± 0.2 (0.4–1.5) Distance from tympanum to parotoid gland 0.8 ± 0.3 (0.2–1.5) 1.3 ± 0.3 (0.7–2.2) Molecular Analyses Both species showed expressive genetic divergences (3.8–6.1%) in the mitochondrial fragment. The intraspecific genetic distance was on average 0.09 for R. mirandaribeiroi and 0.06 for R. granulosa . Acoustics We found conserved call parameter features between the two species. The advertisement call of R. granulosa consists of a sequence of approximately 170 notes and lasts about 5 seconds, emitted at a rate of 5 calls per minute with 32 notes per second. The notes had a duration of approximately 22 ms and with 4 to 5 pulses per note. The dominant frequency is around 2700 Hz. The calls reach maximum amplitude quickly and remain constant until the end ( Figure 6 ). Further details can be found in Table 4 . Our results are consistent with the published analysis of these data (Giarretta et al. 2018).