Bioacoustics of Rhinella stanlaii

Description of the holotype . UFMT 7430 ( Figure 1 ). Snout vent length 50.4 mm ; head as wide ( 17.1 mm ) as long ( 17.2 mm ) at the angle of jaws. Other measurements are presented in Table 1 . Snout rounded, both in lateral and dorsal view, with a distinct fleshy vertical apical ridge that gives it a nearly mucronate aspect. Cephalic crests weakly developed, except from postorbital crests; paratoid glands small, elliptical; nostrils lateral, protuberant; distance from eye to nostril slightly larger than eye diameter and than internarial distance; upper eyelid width about 90% than eye diameter. Presence of a bony knob at the lower angle of jaws. Tympanum distinct, large, elliptical; its horizontal diameter representing 75% of the vertical diameter and of the upper eyelid width. A dorsolateral line of tubercles extend from the posterior border of the paratoid gland to the groin. Vertebral apophyses absent. Vocal slits present, lateral to the tongue. Skin on dorsal and ventral surfaces granulose, with scattered tubercles. Forearms and forelimbs robust, covered with small tubercles of the same size of those in the dorsum. A line of small tubercles along the external border of the forearms; fingers free, with tips slightly enlarged; length of fingers 4<2<1<3. Palmar tubercle oval, large, smooth; thenar tubercle oval, about one third of the palmar tubercle; subarticular and supranumerary tubercles present. Character SVL Holotype Male (n = 6) 50.4 47.5 ± 3.5 (42.3–52.6) Female (n = 5) 52.4 ± 1.0 (51–53.3) HL 17.2 16.6 ± 1.3 (14–18.1) 17.8 ± 0.9 (16.9–18.9) HW IND 17.1 18.0 ± 1.4 (16.6–20.1) 4.0 3.1 ± 0.5 (3.2–3.5) 17.8 ± 2.0 (15.5–20.2) 3.3 ± 0.2 (2.9–3.5) END 4.1 4.8 ± 0.6 (3.8–5.6) 4.7 ± 1.0 (3.5–6) ED UEW 4.0 4.6 ± 0.6 (4–5.4) 3.7 3.4 ± 0.2 (3–3.7) 4.7 ± 0.5 (4–5.3) 3.2 ± 0.6 (2.4–3.9) IOD 16 14.6 ± 1.1 (13–16.1) 15.5 ± 0.5 (15.2–16) POCL HTD 5.2 4.7 ± 0.5 (4.2–5.2) 2.8 2.4 ± 0.6 (1.6–3.1) 5.6 ± 0.3 (5.3–6) 2.7 ± 0.6 (1.9–3.3) VTD 3.7 3.1 ± 0.4 (2.5–3.7) 3.2 ± 0.4 (2.8–3.7) PGL HAL 9.2 7.8 ± 1.3 (6–9.2) 14.2 13.3 ± 1.0 (12–14.3) 7.9 ± 1.3 (6.8–9.9) 14.0 ± 0.5 (13.5–14.8) THL 13 11.1 ± 1.6 (8.8–13) 13.1 ± 2.0 (11–15.5) TL FL 18 16.3 ± 1.6 (14.7–19) 22.3 24.9 ± 2.0 (22.3–28) 17.9 ± 1.8 (16.5–20.3) 26.4 ± 2.9 (21.6–29.2) Hindlimbs robust, thigh length smaller than tibia length, thigh+tibia length approximately 62% of the SVL; tarsus+foot length longer than tibia and thigh length; a line of tubercles on external and internal borders of tarsus. Inner metatarsal tubercle oval, two times larger than the outer metatarsal tubercle; subarticular and supernumerary tubercles present, small, round. Length of toes 1<5<2<3<4, webbing formula: I 1-2 II 1-3 III 1-2 ½ IV 2 ½- 1V. Colour in preservative: dorsum light brown, dark brown on legs and head; a yellowish vertebral line from the snout to cloaca, surrounded by three black triangular spots at midbody and a pair of triangular black spots at the level of the parietal crest. Venter uniform brown; throat, hand, foot and legs dark brown; an irregular dark brown band in the forearm. Color in life ( Figure 2A ) similar to that in preservative. Iris gold, with black reticulations. Variation within type series. The type series include 20 specimens ( 6 adult males, 5 adult females and 9 juveniles ). Dorsal coloration varies a lot among individuals in the type series ( Figure 3 ), with the fine vertebral line present in 75% of the individuals and only one individual ( holotype ) have a wide vertebral line. Few specimens don’t have the triangular spots in the midbody. The ventral coloration is also variable, from the uniform brown to spotted bellies. Advertisement call. The call of the new species is composed by 18.4 ± 8.7 call series (9–30 calls, n=5), with duration of call series of 16.5 ± 7.2s (n=5), emitted at regular intervals of 10.4 ± 0.8s (n= 5). The call of R. paraguayensis sp. nov. is composed by mean 6.5 ± 0.65 notes (5–8 notes, n=77), with a mean duration of 0.3s ± 0.04s (0.2–0.42, n=82), followed by intercall intervals of 0.5s ± 0.015s (0.22–0.98, n=82). The average rate of call per minute was 39.3 ± 6.4. The mean duration of note was 0.03s ± 0.01s (0.01–0.07, n=163), followed by inter-notes intervals of 0.02s ± 0.01s (0.01–0.04, n=163). The average rate of notes per minute was 504.8 ± 113 (262.4–792.4). Frequency ranges from 618.9–1771.8 Hz (n=125); mean dominant frequency was 1438.7 ± 70.5 Hz (1113.7–1568.5, n=125), displayed between the fourth and fifth note ( Figure 4 ). Natural history. In the Upper Paraguay River Basin, individuals of Rhinella paraguayensis sp. nov. inhabit the leaf litter of gallery forests, and of forests established over paleo-levees throughout the Pantanal wetlands. Riparian systems are used as reproductive sites by individuals in this new species ( Figure 5 ), which apparently presents a unimodal pattern of reproduction, as shown below. During the peak of the floods, in April-May, which are coincident with the beginning of the dry season, the Paraguay River discharges increased ( Barros et al. 2004 ), facilitating the movements of dense stands of macrophytes (particularly the water hyacinth Eichhornia azurea ; Figure 5 ). These macrophyte stands offer both vocalization perches for calling males and lentic habitats for the deposition of egg clutches, as well as protection for the tadpoles. Additionally, the displacement of macrophyte stands downriver may facilitate the transport and dispersal of tadpoles, juveniles and also adults of R. paraguayensis sp. nov. , as already reported for several amphibian species using macrophytes as rafts in others river systems ( Hoogmoed, 1993 ; Schiesari et al. 2003 ). Observations of calling males occurred on seven occasions. In 10 June 1989 a male calling from nude ground at “Rodovia Transpantaneira”, Poconé, Mato Grosso. In April and May 2001 , males of the new species were observed calling on floating stems and leaves of E. azurea at “Parque Nacional do Pantanal”, Poconé municipality, Mato Grosso. From 4 March 2002 males were observed calling (a male was recorded and used in the call description) in a flooded swampy areas in the left margin of the Sepotuba River, Cáceres municipality, Mato Grosso. Between 14–17 May 2002 several males of R. paraguayensis sp. nov. were observed calling on nude ground in the Paraguay River banks, approximately 0.5 m from the river margin, in sites where the availability of floating mats of E. azurea was low, in the Serra do Amolar, Corumbá municipality, Mato Grosso do Sul. In October 2002 , males were observed calling from riparian systems of the Paraguay River, in the Serra do Amolar, Corumbá municipality, Mato Grosso do Sul. In 11 and 12 August 2003 , during the dry season, several males of the new species were observed calling on floating stems and leaves of E. azurea , as far as 2 m from the Paraguay River bank. River width in this locality, near Acurizal ranch ( 17º49'51”S ; 57º32'53”W ), is near 100 m . Vocalizations were heard from around 15:00 h, in full sun, to 21:00h, with full moon. Therefore, calling activities of Rhinella paraguayensis sp. nov. in the Pantanal were recorded from March to October and seems to be correlated with the Paraguay River flooding cycle. Rhinella paraguayensis sp. nov. is usually abundant in the localities where the species was already recorded. In a field study in the Parque Nacional do Pantanal, it was the third most abundant among the 13 anuran species recorded by pitfall trapping (http://www.ibama.gov.br/siucweb/unidades/parna/planos_de_manejo/78/index.htm).

Moreover, there are clear ecological differences between some of the species in the Rhinella margaritifera species group, including geographic distribution, reproductive habitat, and vocalization microhabitat ( Lima et al . 2007 ). Species in the R. margaritifera complex are distributed mainly in the Amazonia Biome, with a few species occurring in other forested (e.g. Atlantic Forest – R. hoogmoedi Caramaschi & Pombal 2006 ) or opened ecosystems (e.g. Cerrado (Brazilian savanna) – R. scitula Caramaschi & Niemeyer 2003 ; Pantanal – R. paraguayensis sp. nov. , present study; Figure 7 ). The calling and reproductive site of R . paraguayensis sp. nov. (Leaves of floating macrophyte or in the ground of river margins) contrasts with several species in the R. margaritifera complex. For instance, males of R. magnussoni call from leaf litter or bellow fallen leaves distant from streams or terrestrial ponds, with egg deposition in fallen tree trunks ( Lima et al . 2007 ); in R. castaneotica , calling and egg deposition site occurs in the empty fruit capsules of Brazilian nut trees ( Caldwell 1991 ); males of R. proboscidea use shallow puddles bordering small streams or temporary pools ( Zimmerman & Bogart 1988 , Menin et al . 2006 ); in R. margaritifera , males call from the leaf litter surrounding permanent pools, swamps and artificial ponds ( Wells 1979 ), while males of Rhinella sp. use shallow pools on top of large flat boulder with concave surface in the middle of creek beds ( Hoogmoed and Ávila-Pires 1991 ). The calling site of the new species bears resemblance with that of R. sclerocephala , whose males were observed calling perched on leaf axis of vegetation (Mijares-Urrutia & Arends 2001 ); of R. hoogmoedi , observed perched in tree trunks at 60 cm from the ground ( Pombal & Gordo 2004 ), and of R. lescurei , which calls on dead trunks, vines and roots distant about 10 m from slowly running water ( Fouquet et al. 2007b ). Males calling on the ground near river margins were also observed in R. scitula and R. hoogmoedi , although these species were recorded using small streams inside riparian forest until now ( Caramaschi & Niemeyer 2003 ; Pombal & Gordo 2004 ), instead of the margins of larger rivers, as observed in R. paraguayensis sp. nov. In spite of several recent contributions, much work is still needed to evaluate species composition, as well as understanding the geographical distribution and ecological diversity of R. margaritifera complex.