Bioacoustics of Pseudopaludicola murundu

Description of the holotype . Body elliptic and broad. Head triangular, longer than wide. Snout subelliptical in dorsal view and rounded in profile ( Figure 2 A, 2B). Nostrils slightly protuberant, directed anterolaterally. Mouth opening ventral. Canthus rostralis rounded. Loreal region slightly concave. Choanae rounded. Eye protuberant, its diameter larger than interorbital distance. Interorbital area flat. Tympanum indistinct. Vocal sac single, externally expanded, large, and with longitudinal folds; vocal slits present. Vomerine teeth absent. Tongue elliptical, longer than wide. Finger length I<II<IV<III. Toe length I<II<V<III<IV. Finger and toe tips without disks ( Figure 2 C, 2D). Thumb with keratinized beige nuptial pad. Finger webbing absent and toe webbing reduced I-II 2- 3 III 3-4 IV-V. Finger and toe subarticular tubercles conical and single. A large subconical ulnar tubercle. Few rounded supernumerary tubercles in the hand in the area delimited by the first subarticular tubercles, the elliptical internal metacarpal tubercle, and the ovoid external metacarpal tubercle. Hindlimbs very robust and long. Thigh shorter than tibia; foot slightly longer than thigh and slightly shorter than tibia. Supernumerary tubercles absent in the foot. Metatarsal tubercles present, elliptical; internal larger than the external; external more protuberant than the internal. A well developed fold from the internal metatarsal tubercle to the mid-ventral tarsus. Skin of belly smooth; ventral surface of thigh granular. Dorsum of head, body, and limbs smooth with scattered tubercles; the skin on the scapula region has two arc shaped granular folds. Flanks with scattered tubercles. Cloacal region granular. Measurements of the holotype are presented in table 1. Holotype Paratypes Measurement Male Males (N = 11) Females (N = 4) snout-vent length 13.7 14.7 ± 0.4 (14.0–15.4) 15.0 ± 1.0 (14.1–15.9) head length 5.5 5.7 ± 0.3 (5.1–6.1) 6.0 ± 0.4 (5.6–6.5) head width 5.2 5.5 ± 0.3 (5.1–5.8) 5.3 ± 0.2 (5.1–5.6) eye diameter 1.6 1.8 ± 0.3 (1.5–2.2) 1.9 ± 0.1 (1.8–2.1) interorbital distance 1.3 1.4 ± 0.2 (1.0–1.7) 1.2 ± 0.2 (1.0–1.4) internarial distance 1.4 1.5 ± 0.2 (1.4–1.9) 1.5 ± 0.1 (1.3–1.6) thigh length 8.1 8.6 ± 0.4 (7.9–9.2) 8.5 ± 0.4 (8.0–8.9) tibia length 9.5 9.9 ± 0.6 (8.8–10.6) 10.2 ± 0.6 (9.7–11.0) foot length 8.9 9.2 ± 0.6 (7.4–9.8) 9.0 ± 0.7 (8.2–9.6) In life, dorsum and limbs are brown with dark brown spots or stripes; vertebral line yellowish brown from the snout to vent; belly white; ventral view of legs yellowish white and vocal sac beige with dark dots; iris golden superiorly and brownish inferiorly (see Figure 1 ). In preservative, dorsum and dorsal part of limbs brown with dark brown spots or stripes; vertebral line beige from the snout to vent; belly light beige; legs yellowish white; vocal sac beige with dark dots and with dark longitudinal folds. Variation. Females slightly larger than males, lacking vocal sac and nuptial pads (present in males). The nuptial pad can be brownish to light cream color in males. The snout shape in lateral view varies from rounded to slightly truncate. In 75% of the individuals (12 out of 16 examined) the vertebral line is absent. In life this line can be white, yellow, or red. In life, the two arc shaped granular folds on the scapula region are generally darker than the general coloration of dorsum (see also Figure 1 ). Dorsal pigmentation varies among individuals, from brown to grayish. The number and form of the dorsal spots also varies, which can be small scattered brown dots up to large brown stripes over a lighter general dorsal coloration. Natural history notes. Males were observed calling in open areas of a private farm. The region was previously a mosaic composed of Cerrado (Brazilian savannah) and semidesciduous Atlantic forest. Males called from the ground near shallow (about 2–3 cm of depth) slow-flowing water bodies, on the “murundus”, which are small elevations on the ground (about 10 cm height), covered by grassy and/or arboreal vegetation. Calling males were spaced for at least 5 cm in distance, and it was possible to observe silent males even closer to the calling ones (however, we could not confirm if these males were performing satellite behavior). Males were observed calling at dawn and during the first hours of the night. One amplectant pair was observed and collected in the same microhabitat of the calling site. Egg laying was not observed, so we could not attribute any reproductive mode to P. murundu . Tadpoles were active at night in the shallow and slow-flowing water bodies, where males and the amplectant pair were observed. No other species was observed in syntopy. Geographic distribution. The new species is known only for the type locality, which is an open grassland field of anthropogene origin used for cattle ranching in a particular farm.

Etymology. The specific name is a Tupi word that means small elevations on the ground, covered by grassy and/or arboreal vegetation. This is the most common calling site of this species. Advertisement call. The advertisement call of Pseudopaludicola murundu is composed of groups of notes (each with three to six pulses) that are emitted in mean intervals of 92 ms ( Figure 3 A and 3B). The calls are generally long, with more than 10 seconds of duration ( Figure 3 A) and high pitched, with most of the frequencies higher than 5 kHz. The mean rate of notes emitted per minute is 225. Males can call in antiphony as the interval between notes are similar to the duration of notes. The first pulse of the note generally occupies lower frequencies than the last one. The intermediary pulses occupy the intermediary frequencies between the first and the last pulses. Further information of the numerical characteristics of the advertisement call is provided in Table 2 . Parameter Values Dominant frequency (kHz) 5.72 ± 0.32 (n = 10) (5.17 – 6.37) Minimum frequency (kHz) 5.03 ± 0.20 (n = 10) (4.71 – 5.33) Maximum frequency(kHz) 6.82 ± 0.26 (n = 10) (6.44 – 7.25) Notes per call 45.60 ± 27.39 (n = 14) (15 – 82) Duration (n = 10) of the call (s) 12.79 ± 5.71 (4.55 – 24.85) Duration (n = 14) of the note (s) 0.10 ± 0.01 (0.09 – 0.11) Duration (n = 14) of the pulses (ms) 11.21 ± 2.29 (6.00 – 14.00) Pulses per note 5.07 ± 0.49 (n = 16) (3 – 6) Interval between notes of the same call (s) 0.09 ± 0.03 (n = 14) (0.03 – 0.13) Interval between pulses of the same note (ms) 3.36 ± 4.41 (n = 14) (00 – 12) Tadpoles. Larvae were obtained in shallow ponds at the type locality. As there was no other amphibian species in syntopy with P. murundu we assigned these tadpoles to this species (this tadpole confirm with general morphology of Pseudopaludicola tadpoles), however this identification is preliminary and not verified. The following description is based on seven tadpoles (CFBH 22670) in developmental stages 37-39 ( Gosner 1960 ). Body elliptical in dorsal and ventral views ( Figure 4 A,C), depressed/globular with flattened venter ( Figure 4 B); body wider than high; snout rounded; eyes medium-sized, dorsolateral; nostrils dorsal, small, and reniform; nostrils closer to the eyes than to the tip of snout; spiracle sinistral, its opening in the middle of body; cloacal tube large, medial; caudal musculature robust; dorsal fin originating on the anterior part of the tail; dorsal fin wider than ventral fin. Oral disc directed ventrally, emarginated, and bordered by one row of well developed marginal papillae, interrupted along a large area on the anterior labium; tooth row formula 2(2)/2 or 2(2)/2(1); upper and lower jaw sheathes strongly developed and serrate ( Figure 4 D). In preservative, dorsum pale brown; throat and belly transparent; caudal musculature with very scattered pale brown pigmentation; fins transparent with scattered pale brown pigmentation. Three tadpoles in developmental stage 37 measured (range, average, SD) total length 24.79–27.10 (25.65, 1.26); body length 8.05–8.90 (8.41, 0.44); maximum body height 3.67–4.36 (3.97, 0.36); maximum body width 5.02–5.68 (5.27, 0.36); internarial distance 1.24–1.27 (1.26, 0.02); interorbital distance 1.05–1.06 (1.06, 0.01); eye-nostril distance 0.39–0.49 (0.45, 0.05); eye diameter 1.26–1.33 (1.28, 0.04). Karyotype description. The karyotype of the P. murundu has 2n = 22 chromosomes. Pairs 1, 2, 5, 7, 9, 10, and 11 are metacentrics, pairs 3, 4, and 6 submetacentrics, and pair 8 telocentric ( Figure 5 A–C). Besides the centromeric heterochromatic blocks, positive C-bands were detected in the interstitial regions on the long arms of the pairs 1, 2, 3, 4, and 6, and on the short arms of the pairs 1 and 2 ( Figure 5 B). The nucleolar organizing region (NOR) was detected by silver staining (Ag-NOR) in the telomeric region on the long arm of the chromosome 8 ( Figure 5 C; Table 3 . The homologous of the pair 8, telocentric in both sexes, showed a notable size heteromorphism, and the Ag-NOR site was observed only in the larger homologous of this pair ( Figure 5 C) which were identified as 8 and 8’ ( Table 3 ). Number of chromosomes

Description of the advertisement call. The advertisement call of Pseudopaludicola serrana is composed of groups of pulsed notes (with one to four pulses) that are emitted in mean intervals of 0.18 s . Calls are generally long, varying from about 14 to 45 seconds in the analyzed series. Calls are high pitched, with frequencies varying between about 3.0 and 8.5 kHz. The mean rate of notes emitted per minute is 227. The pulses within a note generally present an ascending modulation, i.e., the first pulse presents lower frequencies than the second, which, in turn, presents lower frequencies than the third, and so on ( Figure 4 ). Further information of the physical characteristics of the advertisement call is in Table 2 . Comparisons with other species are presented in table 3. Pulse duration P. murundu P. saltica P. serrana P. murundu 0.00002 0.00008 P. saltica 0.00005 0.00005 P. serrana 0.00023 0.00015 Interval between notes P. murundu P. saltica P. serrana P. murundu 0.66200 0.00023 P. saltica 1.00000 0.00040 P. serrana 0.00068 0.00121 Dominant frequency P. murundu P. saltica P. serrana P. murundu 0.00009 0.21200 P. saltica 0.00026 0.00001 P. serrana 0.63600 0.00004 Interval between pulses P. murundu P. saltica P. serrana P. murundu 0.00109 0.00209 P. saltica 0.00327 0.03379 P. serrana 0.00628 0.10140 Tadpoles. Larvae were obtained from eggs laid in plastic bags after collection of adult specimens in type locality. The following description is based on 16 tadpoles (ZUEC 16457) in developmental stages 25 ( Gosner, 1960 ). Body elliptic in dorsal and ventral views ( Figure 5 ), depressed/globular with flattened venter; body wider than high; snout rounded; eyes medium-sized, dorsolateral; nostrils dorsal, small, and oval; nostrils closer to the eyes than to the tip of snout; spiracle sinistral, its opening in the middle of body; cloacal tube large, medial; caudal musculature robust; dorsal fin originating on the anterior part of the tail; dorsal fin wider than ventral fin ( Figure 5B ). Oral disc directed ventrally, emarginated, and bordered by one row of marginal papillae, interrupted along a large area on the anterior labium; tooth row formula 2(2)/2(1); upper and lower jaw sheathes strongly developed and serrate. Some of the tadpoles present unkeratinized mouthparts, probably due to Batrachochytrium dendrobatidis infection, acquired in the laboratory, as individuals collected in the same locality are apparently uninfected. In preservative and in life, dorsum pale brown; throat and belly transparent; caudal musculature with very scattered pale brown pigmentation; fins transparent with few scattered pale brown pigmentation ( Figure 5 ). Lateral lines were not observed. Three tadpoles at Gosner’s stage 25 measured [mean ± SD (range); size in mm] total length 9.34 ± 1.12 (8.05–10.05); body length 4.38 ± 0.56 (3.73–4.71); tail length 4.96 ± 0.56 (4.32–5.35); maximum body height 1.83 ± 0.31 (1.50–2.10); maximum body width 2.53 ± 0.39 (2.11–2.89); tail maximum width 1.61 ± 0.67 (1.00–2.32); internarial distance 0.48 ± 0.13 (0.34–0.60); interorbital distance 0.57 ± 0.07 (0.49–0.63); eyenostril distance 0.31 ± 0.08 (0.26–0.40); eye diameter 0.46 ± 0.06 (0.40–0.56).

Diagnosis. Pseudopaludicola serrana is assigned to the genus Pseudopaludicola due to the presence of hypertrophied antebrachial tubercle (see Lynch, 1989 ). Pseudopaludicola serrana is an intermediate size species for the genus and is characterized by the following combination of characters: (1) long hindlimbs and absence of T-shaped terminal phalanges, (2) single, dark, and subgular vocal sac with dark longitudinal folds in males; (3) immaculate to light brown, nuptial pads in males that cover the external part of finger I; (4) SVL/ HL greater than 2.7; and (5) advertisement call with dominant frequency above 5 kHz, pulse duration range between 13 and 23 ms, and mean interval between notes of 177 ms. Comparison with other species. Pseudopaludicola serrana is distinguished from the species of the P. pusilla group ( P. canga , P. ceratophryes , P. llanera , and P. pusilla ) by the absence of T-shaped terminal phalanges (the paratype ZUEC 16396 had its fingers dissected). From the species of the P. falcipes group, except from P. murundu and P. saltica , it distinguishes by having very long hindlimbs, with tibio-tarsal articulation reaching beyond the end of the snout. From P. saltica it is distinguished by having larger and clearer nuptial pads (smaller and darker in P. saltica ), longitudinal folds in the vocal sac (absent in P. saltica ), and a darker vocal sac (immaculate in P. saltica ). From P. saltica and P. murundu it distinguishes by having the head not as long (SVL/HL = 2.99 ± 0.30; 2.71–3.34 / HL/HW = 0.97 ± 0.06; 0.86–1.02) as in P. saltica (SVL/HL = 2.28 ± 0.11; 2.00–2.44 / HL/HW = 1.15 ± 0.08; 1.01–1.33) and P. murundu (SVL/HL = 2.42 ± 0.12; 2.25–2.62 / HL/HW = 1.11 ± 0.07; 1.01–1.24) ( Table 1 ). The physical characteristics of the advertisement calls also distinguish P. serrana From P. saltica and P. murundu . Significant differences can be observed in pulse duration (between the three species), interval between pulses (between the three species, although the ranges of variation are overlapped), interval between notes (significant difference between P. serrana and the two other species), and dominant frequency (significant difference between P. saltica and the two other species) (see Tables 2 , 3 , and 4 ).

Description of the holotype . Body elliptic and broad. Head triangular, slightly longer than wider. Snout sub-elliptical in dorsal view and rounded in profile ( Figure 2A, 2B ). Nostrils slightly protuberant, directed anterolaterally. Mouth opening ventral. Canthus rostralis rounded. Loreal region slightly concave. Choanae rounded. Eye protuberant, its diameter almost the same size of the interorbital distance. Interorbital area flat. Tympanum indistinct. Vocal sac single, externally expanded, large, and with longitudinal folds; vocal slits present. Vomerine teeth absent. Tongue elliptical, longer than wide. Finger length I<IV<II<III. Toe length I<II<III=V<IV. Finger and toe tips without disks ( Figure 2C, 2D ). Thumb with keratinized pale beige nuptial pad. Finger webbing absent and toe webbing reduced I-II 2-3 III 3-4 IV-V. Finger and toe subarticular tubercles conical and single. A large subconical ulnar tubercle. Few rounded supernumerary tubercles in the hand in the area delimited by the first subarticular tubercles, the elliptical internal metacarpal tubercle, and the ovoid external metacarpal tubercle. Hindlimbs robust and long. Thigh shorter than tibia; foot longer than thigh and slightly shorter than tibia. Supernumerary tubercles absent in the foot. Metatarsal tubercles present, elliptical; internal larger than the external; external more protuberant than the internal. A well developed fold from the internal metatarsal tubercle to the mid-ventral tarsus. Skin of belly smooth; ventral surface of thigh granular. Dorsum of head, body, and limbs smooth with scattered tubercles; the skin on the scapula region has two arc shaped granular folds. Flanks without tubercles. Cloacal region not granular. Measurements of the holotype are presented in table 1. Holotype Paratypes Measurement Male Males (N = 5) Female Snout-vent length 15.2 15.22 ± 0.31 (15.0–15.8) 18.9 Axial-groin length 4.8 5.07 ± 0.36 (4.7–5.6) 6.7 Head length 5.5 5.13 ± 0.57 (–) 6.7 Head width 5.4 5.30 ± 0.54 (4.5–6.0) 7.3 Eye diameter 1.3 1.34 ± 0.18 (1.2–1.6) 1.9 Upper eyelid width 1.2 1.13 ± 0.11 (1.0–1.2) 1.5 Interorbital distance 1.2 1.43 ± 0.19 (1.2–1.7) 1.7 Internarial distance 1.5 1.55 ± 0.12 (1.4–1.7) 2.1 Eye-nostril distance 1.3 1.42 ± 0.11 (1.3–1.6) 1.7 Snout-eye distance 3.0 2.88 ± 0.11 (2.8–3.0) 3.4 Humerus length 2.3 2.47 ± 0.27 (2.1–2.8) 2.3 Radio-ulna length 2.3 2.37 ± 0.22 (2.1–2.6) 3.2 Hand length 4.6 4.44 ± 0.20 (4.2–4.7) 4.6 Thigh length 8.5 8.85 ± 0.42 (8.4–9.4) 10.9 Tibia length 10.3 10.25 ± 0.39 (9.9–10.9) 12.9 Foot length 9.6 9.73 ± 0.23 (9.6–10.1) 12.5 Color of the holotype . In life, dorsum and limbs are brown with green and brown spots or stripes; vertebral line from the snout to vent absent; belly white; ventral view of legs yellowish white and vocal sac beige with dark dots; iris reddish-brown superiorly and grayish-brown inferiorly; superior labium with vertical wide green stripes (see Figure 1A ). In preservative, dorsum and dorsal part of limbs grayish brown with dark brown spots or stripes; belly and ventral surface of legs light beige; vocal sac grayish. Variation. Females larger than males, lacking vocal sac and nuptial pads (present in males). Males present arms proportionally thicker than females. Nuptial pad can be brownish to light cream color. In 76.5 % of the individuals (13 out of 17 examined, including material not included in type series) the vertebral line is absent. In life this line can be yellow or red (see Figure 1B and C ). Dorsal pigmentation varies considerably among individuals in the number and form of the dorsal spots, and in the general pattern, from brown to grayish, but always with some green blotches ( Figure 1A, B, and C ). Natural history notes. Males were observed calling in open fields in the domain of Cerrado (Brazilian savannah). The species was observed calling in riparian fields (Campo Rupestre). Males called from the sandy, muddy or rocky ground, near shallow (from 2 to 5 cm of depth) slow-flow or still water bodies. Males were observed calling in the sundown and during the first hours of the night. Two defensive behaviors were observed: ceasing the calling activity and flee against human approach. Gravid females present ovules with black animal pole and yellowish-beige vegetative pole. A couple entered in axillary amplexus inside a plastic bag where they laid 32 eggs , which developed into tadpoles in laboratory until Gosner´s stage 25. Species Frequency (kHz) Duration (ms) Dominant Minimum Maximum Note Pulse P. saltica 4.60 ± 0.18 (4.39–4.91) 3.05 ± 0.24 (2.52–3.34) 6.35 ± 0.35 (5.92–6.93) 76.33 ± 18.59 (31.0–97.0) 33.0 ± 4.43 (24.0–38.0) P. murundu 5.72 ± 0.32 (5.17–6.37) 5.03 ± 0.20 (4.71–5.33) 6.82 ± 0.26 (6.44–7.25) 98.86 ± 8.57 (87.0–112.0) 11.21 ± 2.29 (6.0–14.0) P. serrana 5.75 ± 0.11 (5.51–6.03) 3.56 ± 0.44 (3.12–4.50) 7.98 ± 0.41 (7.01–8.49) 85.33 ± 22.00 (25.0–115.0) 18.87 ± 3.04 (13.0–23.0) continued. Species Interval between (ms) Pulses / note (n) Notes Pulses P. saltica 99.33 ± 15.99 (80.0–140.0) 19.25 ± 10.48 (0.0–32.0) 2.08 ± 0.51 (1–3) P. murundu 92.50 ± 26.0 (31.0–132.0) 3.36 ± 4.41 (0.0–12.0) 4.93 ± 0.70 (3–6) P. serrana 177.13 ± 64.00 (96.0–283.0) 11.53 ± 7.33 (0.0–26.0) 3.07 ± 0.80 (1–4) Geographic distribution. The new species is known from three localities at the Espinhaço mountain range: Serra do Cipó, municipality of Santana do Riacho (approximately 19°08’- 43°40’; 1071 m ; ZUEC 2323), Serra da Moeda, municipality of Brumadinho (type locality), and Serra do Lenheiro, municipality of São João del Rei (approximately 21°08’ – 44°17’; 1081 m ; ZUEC 16445–53; 16455–56), all higher than 1,000 m above sea leavel and in the state of Minas Gerais, Southeastern Brazil ( Figure 3 ).