Bioacoustics of Odontophrynus juquinha

Odontophrynus juquinha sp. nov. is distinguished from O. cordobae ( Fig. 5C ) by having dorsal dark brown blotches of small to medium-size with low contrast on a light brown background (dorsal dark brown blotches with high contrast in all examined specimens of O. cordobae ) ( Fig. 1 , 4 , and 5C). It is also distinguished by the lack of a light mid-dorsal stripe, or greatly interrupted in most of the specimens (light mid-dorsal stripe slightly interrupted in O . cordobae ) ( Fig. 5C ). Whenever the mid-dorsal stripe is present, its yellowish coloration distinguishes the new species from O. cordobae (whitish or greyish coloration in O. cordobae ). Additionally, the new species is distinguished by the yellowish coloration in the background of both the head and the flanks of the body (whitish or greyish coloration in O. cordobae ). Moreover, the advertisement call of O. juquinha sp. nov. distinguishes it from O. cordobae by the lower pulse rate ( O. juquinha PR = 90.5–106.7 pulses/s, air temperature = 19.2°C; O. cordobae PR = 111–116 pulses/s, air temperature = 20°C) (see Table 2 for call comparisons, see also Discussion section for comments on the possible influence of temperature). The tadpoles of the new species are distinguished by being smaller than those of O. cordobae ( O. juquinha TL = 24.30–35.69 mm , stages 30–38, n = 6; O . cordobae TL = 40 mm , stage 37; Nascimento et al . 2013 and literature cited therein) and by having 4–8 submarginal papillae (single or paired in O. cordobae ).

Description of the holotype . Body stout; head wider than longer, its length 77% of its width, 33% of SVL ( Fig. 2 ). Snout rounded in dorsal profile, between sloping and truncate in lateral view ( Fig. 3C, D ). Canthus rostralis slightly distinct, concave in dorsal view; loreal region slightly concave. Nostrils elliptic elongated, directed dorsolaterally, and situated at the tip of the snout in lateral profile; internarial distance 44% of distance between the anterior margins of eyes, 77% of eye-nostril distance. Eye large, prominent, 32% of head length, its diameter 40% longer than eye-nostril distance. Upper eyelid with distinct warts varying from rounded to elongated, external border of the upper eyelid with a glandular ridge. Tympanum hidden, not visible externally ( Fig. 3C, D ). Premaxillary and maxillary teeth present; tongue round, approximately half free and notched posteriorly; vomerine teeth in two patches slightly posteromedian to choanae. Vocal sac median, subgular; vocal slits present, longitudinal. Skin of dorsum granular, consisting of glandular warts with variable size, distributed scattered throughout the dorsum, and a series of distinct glandular warts, with irregular size and shape, forming small ridges longitudinally oriented, on postorbital-parotoid region. No distinct temporal glandular wart. Venter uniformly granular. Forelimbs relatively short, stout; glandular longitudinal ridge in the external part of the forearm ( Fig. 3C ). Lengths of fingers: III <V <II <IV, with a deformation in the tip of left finger III; fringes absent, webbing absent. Inner and outer metacarpal tubercles well developed; inner oval; outer divided, with the inner oval and the outer elongated; subarticular tubercles well developed, oval, slightly bilobated on fingers III and IV; supernumerary tubercles rounded, scattered distributed in the palm. Nuptial pad present on thumb. Hind limbs relatively short, stout; lengths of toes: I <II <V <III <IV; tarsal fold present; subarticular tubercles present, rounded, oval and enlarged on finger I. Inner metatarsal tubercle strong, shovel-like, nonkeratinized; outer metatarsal tubercle slightly distinct, rounded; supernumerary tubercles rounded, scattered distributed in the foot palm. Webbing formula: I 1– 2 II 1 1/2– 3 III 2 1/2– 4- IV 4–2+ V, with dermal fringes developed ( Fig. 3B ). Measurements of the holotype (mm). SVL 44.3, HL 14.6, HW 18.9, IND 2.6, AMD 6.3, END 3.4, ED 4.8, AL 23.15, THL 17.2, TL 15.0, FL 27.9, MTL 5.1. Coloration of the holotype . Unknown in life. In preservative, dorsal color predominantly dark brown ( Fig. 2 ). Light brown stripe between the eyes, resembling a “U” shape. Head and extremities dark brown in dorsal view. Light brown stripe dorsolaterally, starting at the medial third of the body toward the hind limb. Upper lip light brown; two dark brown botches connecting each nostril to the upper lip; two dark brown blotches connecting the eyes to the upper lip; four dark brown small blotches in the upper lip. Light brown stripe from the posterior part of the eye towards the forelimb insertion. Venter yellowish-colored, with scattered dark brown blotches gradually increasing on the sides of the body. Gular area with dark grey pigmentation. Coloration in life. Description based on adult male paratypes UFMG 13901, 13903–4, Municipality of Santana do Riacho, Minas Gerais State, Brazil ( Fig. 1 ). Dorsal surface of head, body and extremities dark brown. Yellowish stripe between the eyes, resembling a “U” shape. Yellowish stripe dorsolaterally, starting at the medial third of the body toward the hindlimb. Nut-brown blotches distributed below the dorsolateral yellowish stripe. Upper lip yellowish; two dark brown blotches connecting each nostril to the upper lip; two dark brown blotches connecting the eyes to the upper lip; four dark brown small blotches in the upper lip. Yellowish stripe from the posterior part of the eye towards the forelimb insertion. Venter whitish, with scattered grey blotches gradually increasing on the side of the body. Gular area whitish, crowded with dark grey blotches. Pupil horizontally elliptical; iris golden marbled with dark stripes; dark edges of eyes. The size and shape of the yellowish blotches varied in the analyzed individuals. Dark brown blotch bellow the eye well defined in UFMG 13901 and 13903, whereas blurred in UFMG 13904. A yellow mid-dorsal stripe well defined in UFMG 13904, interrupted in UFMG 13901 and 13903. Dorsum predominantly light brown in UFMG 13901, dark brown blotches surrounding the longitudinal glandular ridges. Variation. Adult males can be distinguished from adult females by the presence of clearly developed nuptial pads on thumb, and a dark grey-pigmented gular region at the vocal sac. Adult females are usually about the same size as males (SVL = 44.3 ± 3.8, 38.4–52.1 mm in males, n = 16; SVL = 46.1 ± 1.5, 45.0– 47.1 mm in females, n = 2). Full mid-dorsal stripe was present in only one individual (i.e. UFMG 13904; Fig. 4L ); an interrupted mid-dorsal stripe was present in some individuals (e.g. UFMG 417, 5511–2, 5596, 5598, and 13901) whilst others presented the mid-dorsal stripe only in the supra-cloacal region (e.g. UFMG 419, 420–1, 5518, 5597, 11118, 11171, and 13902–3) (see Fig. 4 for dorsal color variation). Some individuals presented the dorsum light brown in preservative, with dark brown blotches surrounding the longitudinal glandular ridges (e.g. UFMG 417, 5511–2, 5518, 11171, 13901). Some variation was observed in the outer metacarpal tubercle division, with individuals presenting it only partially divided (e.g. UFMG 5512, 5517), and others with a merely distinguishable division (e.g. UFMG 5518, 11171). Although the level of keratinization varied, all paratypes presented the inner metatarsal tubercle keratinized. Karyotype. All examined specimens of Odontophrynus juquinha sp. nov. presented a diploid number of 2n = 2x = 22 chromosomes. All chromosomes were biarmed (fundamental number FN = 44) and the karyotype consisted of four large (1–4), three medium (5–7), and four small pairs (8–11) ( Fig. 6A ). Chromosome pairs 1, 5–7, and 11 were metacentric and chromosome pairs 2–4 and 8–10 were submetacentric. Pair 4 had an evident secondary constriction (SC) in the interstitial region of its short arm, which probably bears the nucleolus organizer region (NOR) as it has been observed in the other diploid species of the genus Odontophrynus (Barrio & Pistol de Rubel 1972 ; Ruiz et al. 1981 ; Rosset et al. 2006 ; Borteiro et al. 2010 ). Meiotic analysis revealed 11 ring bivalents with terminal chiasmata in diplotene, diakinesis, and metaphase I; and 11 dyads in metaphase II ( Fig. 6B–D ). Vocalizations. Advertisement call of Odontophrynus juquinha sp. nov. is characterized by a single pulsed note (n = 10 individuals), without frequency modulation and with variable duration (ND = 446.8 ± 61.0, 322–610 ms, n = 61 calls) and number of pulses (PN = 44.5 ± 5.7, 32–62 pulses/note, n = 61 calls) ( Fig. 7A , Table 3 ). Pulses had mean duration of 6 ± 0.8 ms (PD = 5–8 ms, n = 92 pulses), at regular intervals and constant rate (IPD = 4.3 ± 0.9, 3–6 ms, n = 85 intervals; PR = 99.4 ± 4.1, 90.5–106.7 pulses/s, n = 58 notes). The calls from Serra do Cabral and Diamantina had a slightly higher dominant frequency then the one from Serra do Cipó. Other variables presented small variations that overlapped. We observed an affix in 18% of the analyzed notes (n = 11: six calls from two individuals recorded at Serra do Cipó, and five calls from one individual recorded at Serra do Cabral) ( Fig. 7B, C ). This affix was always part of the same note described as the advertisement call. Males displayed it either after, as a suffix (45.5%, n = 5; Fig. 6B ), or before, as prefix (54.5%, n = 6; Fig. 7C ). The affix presented short duration (Affix Duration = 99.8 ± 29.7, 58–149 ms, n = 11; Table 3 ) and less pulses than the advertisement call (PN = 10.1 ± 3.0, 6–16 pulses/note, n = 11). Affix’ pulses presented the same characteristics of the advertisement call (PD = 6.4 ± 0.9, 5–8 ms, n = 63; IPD = 4.3 ± 0.9, 3–6 ms, n = 55; PR = 101.7 ± 3.6, 97.3–107.3 pulses/s, n = 11). Dominant frequency is slightly lower than the advertisement call (DF = 890 ± 94, 796–984 Hz, n = 11). Despite the similarity between the affix and the advertisement call, they are clearly distinguishable in the spectrogram ( Fig. 7B, C ). Moreover, calls with an affix are audibly distinct than the common advertisement call. Although the affix seems to be related to the presence of other calling males, we did not test its social context. Note duration (ms) Pulses per note Dominant frequency (Hz) Mean ± SD Range Mean ± SD Range Mean ± SD Range All localities 446.8 ± 61.0 322–610 44.5 ± 5.7 32–62 941.1 ± 71.8 843.8–1078.1 Type locality (n = 35 calls; 19.2°C) 460.6 ± 50 360–610 45.5 ± 5.8 34–62 887.8 ± 38.7 843.8–937.5 Serra do Cabral (n = 20 calls) 396.5 ± 39.1 322–443 41.3 ± 4.5 32–47 1007.8 ± 35 984.4–1078.1 Diamantina (n = 6 calls) 533.5 ± 28.7 492–567 49.3 ± 2.5 46–52 1012.1 ± 52.7 947.5–1076.7 Affix (all localities) 99.8 ± 29.7 58–149 10.1 ± 3.0 6–16 890 ± 94 796–984 Pulse duration (ms) Interpulse interval (ms) Pulse rate Mean ± SD Range Mean ± SD Range Mean ± SD Range All localities 6.0 ± 0.8 5–8 4.3 ± 0.9 3–6 99.4 ± 4.1 90.5–106.7 Type locality (n = 35 calls; 19.2°C) 6.0 ± 0.7 5–8 4.3 ± 0.8 3–6 98.6 ± 2.8 92.1–102.5 Serra do Cabral (n = 20 calls) 5.8 ± 0.7 5–8 4.1 ± 0.9 3–6 103.5 ± 1.9 99.3–106.7 Diamantina (n = 6 calls) 6.5 ± 0.9 5–8 4.3 ± 0.9 3–6 92.5 ± 1.6 90.5–94.9 Affix (all localities) 6.4 ± 0.9 5–8 4.3 ± 0.9 3–6 101.7 ± 3.6 97.3–107.3 Tadpole description. Tadpoles in stage 30–38 have 24.3–35.7 mm of TL (n = 6 individuals). Body depressed (BH/BW = 0.74–0.78) ( Fig. 8A, B ), longer than one third of total length (BL/TL = 0.40–0.46); oval in dorsal view and globular depressed in lateral view. Snout ovoid in dorsal view (BWN/BWE = 0.69–0.75) and rounded in lateral view. Nostrils elliptic (only one individual has shown rounded nostrils), dorsally located (IND/BWN = 0.28–0.32), with small medial apophysis; closer to snout than to eyes (NSD/END = 0.55–0.65). Eyes large (ED/BWE = 0.25– 0.29), dorsally located (IOD/BWE = 0.55–0.61), dorsolaterally directed. Spiracle sinistral, and lateroventral (SDEH/BH = 0.47–0.51); posterodorsally directed, visible in lateral and dorsal view; its inner wall fused to the body wall, with posterior portion free and longer than external wall (one individual has the inner and external edge of spiracle of same size); opening located approximately at half of the body (SED/BL = 0.51–0.57). Intestinal switchback point located at the center of the abdominal region ( Fig. 8B ). Vent tube long, dextral, with ventral wall fused to bottom edge of ventral fin. Tail and body with approximately same height (MTH/BH = 0.94–1.02); tail musculature slightly developed (TMH/MTH = 0.36–0.38); caudal end acute. Dorsal fin low (DFH/TMH = 0.39– 0.43), with margin slightly convex and maximum height nearly half of the tail; emerges on posterior third of the body. Ventral fin with margin slightly convex; origin concealed by vent tube; lower than dorsal fin (DFH/VFH = 1.35–1.55). Oral disc ( Fig. 8C ) anteroventrally positioned, small (ODW/BW = 0.28–0.32), laterally emarginated; one row of marginal papillae in alternate disposition throughout the oral disc; wide anterior gap present; four to eight submarginal papillae present in both sides of oral disc. Labial tooth row formula (LTRF) 2(2)/3(1); A-1 and A-2 of the same length; P-1 and P-2 of the same length, longer than P-3. Jaw sheaths wide, finely serrated on the margins; upper jaw sheath M-shaped and lower V-shaped. Natural History. Adult males of Odontophrynus juquinha sp. nov. can be found calling with the body partially submersed in the shallow water of temporary ponds or streamlets within campo rupestre (rupestrian grasslands) vegetation. They often call at the beginning of the raining season (i.e. from September to January) after heavy rains. Males usually prefer to call hidden beneath grasses and bushes, and promptly cease calling when approached. Chorus can be heard from long distances. We also witnessed O. juquinha sp. nov. calling during daylight after heavy rains. Tadpoles are exotrophic, benthic, inhabiting mainly lentic environments such as streamlets backwater, swamps and temporary ponds (ecomorphological guild II: A:1; sensu McDiarmid & Altig 1999). In addition, species such as Scinax curicica Pugliese et al., 2004 , S . pinima ( Bokermann and Sazima, 1973 ) , S. squalirostris ( Lutz, 1925 ) , Boana cipoensis ( Lutz, 1968 ) , Leptodactylus camaquara Sazima and Bokermann, 1978 , L. cunicularius Sazima and Bokermann, 1978 , L. furnarius Sazima and Bokermann, 1978 , L. fuscus ( Schneider, 1799 ) , L. jolyi Sazima and Bokermann, 1978 , L. syphax Bokermann, 1969 , and Physalaemus cuvieri Fitzinger, 1826 , can also be found calling at the same streamlets and ponds that the new species inhabit ( Eterovick & Fernandes, 2002 ; Eterovick & Barros 2003 ).

Diagnosis. Odontophrynus juquinha sp. nov. is a medium-sized species belonging to the genus Odontophrynus based on a combination of morphological characters (i.e. head wider than long, snout truncate in profile, tympanum hidden, first subarticular tubercle on toe I enlarged, inner metatarsal tubercle large, tarsal fold short, granular skin on the dorsum and venter) ( Savage & Cei 1965 ; Cei 1987 ; Caramaschi 1996 ; Caramaschi & Napoli 2012 ). The new species belong to the Odontophrynus americanus species group based on the absence of large dorsal, tibial and forearm glandular warts, with postorbital, temporal, and parotoid glandular warts not distinctly developed but with a series of small glandular warts of irregular size and shape, forming glandular ridges longitudinally oriented, on postorbital-parotoid regions ( Fig. 2A , 5 ). Odontophrynus juquinha sp. nov. is distinguished from the remaining species belonging to the O. americanus species group by the following combination of characters: (1) medium sized (SVL = 38.45–52.09 mm in males in O . juquinha ); (2) head wider than long (HL/HW = 0.7–0.8); (3) dorsal dark brown blotches of small to medium-size with low contrast on a light brown background ( Fig. 5 ); (4) light mid-dorsal stripe absent or greatly interrupted in most of the specimens; (5) yellowish coloration of mid-dorsal stripe and background of both the head and the flanks of the body; (6) moderate number of glandular warts on dorsum; (7) diploid complement of 2n = 2x = 22 chromosomes with fundamental number FN = 44; (8) secondary constriction on the interstitial region of the short arm of the pair 4; (9) advertisement call with dominant frequency of 840–1080 Hz; (10) pulse rate of 90.5–106.7 pulses/s; (11) small tadpoles (mean TL = 24.30–35.69 mm ); (12) single lateral emargination on each side of oral disc; (13) caudal end acute; (14) 4–8 submarginal papillae in both sides of lip; and (15) internal wall of spiracle with a small free portion.