Bioacoustics of Pseudopaludicola atragula

Description of holotype . Small size (SVL 14.07 mm ). Canthus rostralis indistinct, rounded, smooth; vocal sac single (subgular), with areolate, whitish dark reticulations ( Figure 2 A); vocal slits present; choanae well-separated from each other. Surfaces of dorsum, venter, upper eyelids, belly and throat smooth; H-shaped glandular ridge on shoulder region. Snout rounded to subovoid in dorsal view, and acuminate in profile; head wider than long; sides of head (upper lips, under the eyes, and tympanum area) and flanks with white granules ( Figure 2 B). Nostrils slightly protuberant, directed anterolaterally, closer to tip of snout than to eyes; pupil rounded; upper eyelids; tympanum indistinct; vomerine teeth absent; tongue ovoid, posteriorly free. Cloaca smooth. Inner metatarsal tubercle oval; outer metatarsal tubercle conical; toe tips slightly pointed, not webbed and extensively fringed to almost their tips ( Figure 2 C); relative length of toes I<II<III~V<IV; subarticular tubercles large, rounded; supernumerary tubercles absent. Hand with fingers free ( Figure 2 D), with tips rounded, not expanded; relative length of fingers I<II~IV<III; thumb thickened, with keratinized gray nuptial pad on internal surface; two subconical antebrachial tubercles, the distal one scarcely perceptible; outer metacarpal tubercle round; inner metacarpal tubercle elongated; subarticular finger tubercles rounded; supernumerary finger tubercles indistinct. Color. In life ( Figure 3 A–B), dorsum dark gray; flanks pale gray; no vertebral line from the snout to vent; belly white; upper lips with white spots which extend to the insertion of the forelimbs; vocal sac with large white areolations (nearly circular protuberances), interspersed among dark reticulation. Dark reticulation is particularly evident in the anterior half of the vocal sac. In preservative, dorsum grayish brown, with darker brown blotches; belly whitish; ventral surface of hands and feet dark gray, mottled; white spots on upper lips, extending to the dorsal insertion of the forelimbs and to the flanks; vocal sac with large white areolations over a grey, nearly translucent skin, peppered with black melanophores. Dark transverse stripes in thigh and shank, tarsus and foot, and forearm. Variation. Dorsal coloration is highly variable, with nearly 25% of the examined specimens presenting a light, thin vertebral stripe, from the tip of the snout to vent. In preserved specimens, the gular region may vary from light to darker grey, always peppered with black melanophores. Females have a more robust body and their gular region, although areolate, is always unpigmented, clear white ( Table 1 ). Holotype Males Females (N=22) (N=7) Description of the advertisement call. We analyzed 41 calls of four specimens of Pseudopaludicola atragula ( Table 2 ). A typical advertisement call of the new species ( Figure 4 ) consists of a single pulsed note, with 9–36 (mean 22.8±5.4) pulses emitted in a very short period of time, during a single airflow ( McLister et al . 1995 ). The call (= one note) resembles the sound of clockwork motors used in wind-up toys. Mean duration of each note is 0.45± 0.07 s (0.3– 0.7 s). Inter-notes interval ranges from 0.5± 0.2 s (0.04– 0.9 s) and average rate of notes per minute is 65.3±15.1 notes/min (42–98). Mean frequency ranged from 2500.01 (±341.1) Hz to 5088.5 (±249.2) Hz, and mean dominant frequency from 3981.5±156.9 Hz (3617.6–4263.6 Hz). Mean duration of each pulse is 0.003± 0.001 s (0.001– 0.007 s). Inter-pulses interval ranges from 0.01± 0.008 s (0.002– 0.064 s). Average rate of pulses per minute is 2935.5±549.8 pulses/min (1436–3957). SVL 14.07 13.6±0.9 (12.2–15.1) 15.9±0.5 (15.5–16.7) HL 5.47 5.3±0.3 (4.7–5.9) 6.1±0.3 (5.7–6.5) HW 4.69 4.6±0.2 (4.1–5.1) 5.2±0.5 (4.6–5.7) IOD 1.18 1.1±0.08 (1–1.3) 1.2±0.1 (1–1.4) ED 1.35 1.4±0.1 (1.2–1.7) 1.6±0.1 (1.5–1.9) END 1.2 1.3±0.1 (1–1.6) 1.5±0.2 (1.3–1.8) IND 1.1 1.1±0.1 (0.9–1.5) 1.3±0.1 (1.1–1.4) HAL 3.93 3.9±0.3 (3.3–4.4) 4.5±0.3 (4–4.8) THL 6.43 6.2±0.3 (5.7–6.8) 7.2±0.4 (6.7–7.9) TL 7.09 6.7±0.5 (5.9–7.7) 7.9±0.3 (7.4–8.3) TAL 4.02 3.8±0.3 (3.3–4.2) 4.6±0.2 (4.1–4.9) FL 8.3 7.7±0.6 (6.5–8.7) 9.2±0.5 (8.5–9.6) Comparison with other species. A combination of external and/or internal morphological characters, bioacoustic properties of the call, and/or karyotype allows to distinguish the new species from all other 17 Pseudopaludicola species (from now on, characters of compared species in parentheses). From eight out of the 13 Pseudopaludicola species not belonging to P . pusilla group ( P. ameghini , P. c a ng a , P. f a cu re a e , P. falcipes , P. giarettai , P. hyleaustralis , P. mineira , P. m y s t ac a l i s , P. murundu , P. parnaiba , P. pocoto , P. saltica , and P. ternetzi ) the new species differs: from P. falcipes , by the presence of an abdominal fold (abdominal fold interrupted or absent; Lobo 1996 ; Lavilla & Cei 2001 ); from P. murundu , and P. saltica , by the shorter hindlimbs, with tibio-tarsal articulation reaching the nostrils (presence of very long hindlimbs, with tibio-tarsal articulation reaching beyond the end of the snout; Toledo et al . 2010 ; Pansonato et al . 2013 ); from P. giarettai , by the smaller body size (16.2–18.0 mm SVL in males) and by the relative length of fingers (finger I shorter than finger IV; Carvalho 2012 ); from P. ameghini , by the smaller body size ( 14.1–19.3 mm SVL in males and 17.3–22.7 mm SVL in females; Haddad & Cardoso 1987 ; Pansonato et al . 2013 ); from P. mineira , by head longer than wide (head as long as wide; Pereira & Nascimento 2004 ); from P. ternetzi , by the smaller body size (SVL of males 16.0– 18.6 mm ; SVL of females 16.0– 22.2 mm ; Lobo 1996 ). Advertisement call parameters Pseudopaludicola atragula sp. nov. N=41 (4) Number of pulses per note 22.8±5.4 (9–36) Note duration (s) 0.45±0.07 (0.3–0.7) Inter-notes interval (s) 0.5±0.2 (0.04–0.9) Pulse duration (s) 0.003±0.001 (0.001–0.007) Inter-pulses interval (s) 0.01±0.008 (0.002–0.064) Minimum frequency (Hz) 2500.01±341.1 (1932.7–3331.3) Maximum frequency (Hz) 5088.5±249.2 (4322.8–5578.4) Dominant frequency (Hz) 3981.5±156.9 (3617.6–4263.6) Average rate of notes/min 65.3±15.1 (42–98) Average rate of pulses/min 2935.5±549.8 (1436–3957) The appearance of the vocal sac of male Pseudopaludicola atragula —areolate, whitish, with dark reticulation—distinguishes the new species from P. ameghini , P. falcipes , P. g i are t t ai , P. hyleaustralis , and P.

Diagnosis. The new species is assigned to the genus Pseudopaludicola based on the presence of hypertrophied antebrachial tubercles ( Lynch 1989 ). Pseudopaludicola atragula sp. nov. is diagnosed by the following combination of characters: (1) small size (SVL 12.2–15 mm in males and 15.5–16.7 mm in females); (2) absence of either T-shaped terminal phalanges or expanded toe tips (disks or pads); (3) short hindlimbs, with tibiotarsal articulation only reaching the eye; (4) areolate vocal sac, with dark reticulation; (5) karyotype with 2n=18 chromosomes; (6) advertisement call composed of a single note with 9–36 nonconcatenated pulses (resembling the sound of clockwork motors used in wind-up toys); (7) note duration of 0.3– 0.7 s; (8) note repetition rate of 42–98 notes/min; (9) absence of harmonics; (10) dominant frequency of advertisement call 3617.6–4263.6 Hz.

Etymology. The specific epithet is derived from the Latin words “ atra ”, meaning dark, black, and “ gula ”, meaning throat or gullet. It is used in reference to the dark gular region of most males (in life) – unusual within the genus Pseudopaludicola – with white areolations interspersed among dark reticulation. Concluding remarks. Morphologically and cytogenetically, Pseudopaludicola atragula most closely resembles P. facureae . Indeed, based on similarities in the chromosome numbers of the two species (2n=18), Duarte et al . (2010) suggested that the populations from Icém (São Paulo, Brazil ) and Uberlândia (Minas Gerais, Brazil ), both treated by those authors as “ Pseudopaludicola aff. canga ”, could belong to the same taxon. Visual examination of qualitative morphological characters of males and females in our sample of Pseudopaludicola atragula did not evidenced any diagnostic character to distinguish this taxon from P. f ac u re ae . Comparative analyses of morphometric data through a PCA, however, clearly allowed distinguishing the two taxa ( Figure 7 ). The first two axes of the PCA explained 87% of the total variability in males ( Figure 7 A) and higher loadings corresponded to snout-vent length, head length, and thigh length. The PCA explained 85% of the total variability in females ( Figure 7 B) and higher loadings corresponded to snout-vent length, head length, and foot length. The DFA with morphometric measurements, using all specimens, assigned 100% of the individuals of Pseudopaludicola atragula and 100% of the individuals of P. f acureae to the correct species ( Table 4 ). Males Females True group True group Assigned to P. atragula P. facureae P. atragula P. facureae P. atragula 22 0 7 0 P. facureae 0 13 0 9 Number correct 22 13 7 9 % 100 100 100 100 Bioacoustic parameters and spectrograms presented herein also evidenced the taxonomic validity of the species from northwestern state of São Paulo. As here shown, with a single contraction of the trunk muscles, males of P. atragula emit an advertisement call composed of a single pulsed note with 9–36 nonconcatenated pulses, without harmonics, resembling the sound of clockwork motors used in wind-up toys. Calls are also composed of single notes and present harmonics only in P. giarettai , but in this species notes are non-pulsed, there are four harmonics, and the call resembles a whistle. In the remaining species of Pseudopaludicola where bioacoustic parameters were already analyzed, the call consists in a succession of series of notes. In particular, we call attention to the fact that calls already described within the genus Pseudopaludicola might be divided in three distinct groups according to type of notes: non-pulsed notes; notes with concatenated pulses, and notes with nonconcatenated pulses ( Figure 5–6 ). Bioacoustic data, therefore, are here confirmed to represent a reliable tool in the recognition of cryptic species within the genus Pseudopaludicola . Acoustic parameters of calls and the spectrogram of topotypes of Pseudopaludicola atragula are readily distinguished from topotypical advertisement calls of P . facureae , composed of series of 2–55 (mean 15.8±10.1) non-pulsed notes, with series duration of 0.1– 4.3 s, emitted at intervals of 0.2– 1.7 s ( Table 5 ; Figure 4 ). Mean note duration in this species is 0.02±0.005 (0.01–0.04), emitted on average at intervals of 0.03±0.03 (0.01–0.4) with average rate of notes per minute of 1249.1±388.5 (646–2000). Mean frequency ranges from 2688.5 (±573.9) Hz to 5947.9 (±336.8) Hz, and mean dominant frequency is 4559.4±212.2 (4306.6–4995.7), usually concentrated in the first harmonic ( Figure 4 B). Therefore, the call of P. atragula differs from P. f ac u re ae by having pulsed notes, higher note duration, lower note repetition rate and dominant frequency, and especially in the absence of harmonics ( Figure 8 ). Evaluation of uncorrected pairwise distances revealed high genetic divergences between Pseudopaludicola atragula and its congeners (9.4–17.9%), except for species that share with P. atragula the diploid number of 2n=18 chromosomes. These findings seem to be in agreement to its phylogenetic relationships: P. atragula + P. f ac u re ae (genetic divergence of 3.2%) and P. canga + Pseudopaludicola sp. ( aff. canga ), showing genetic divergences of 7.2 and 7.9%, respectively, to P. atragula , were recovered as sister groups ( Veiga-Menoncello et al. 2014 ). Although our results revealed an overall high genetic distance among species of Pseudopaludicola , a lower genetic divergence was revealed when closely related species are compared (e.g., 1.6% between P. ameghini and P. ternetzi , and 2.5% between P. saltica and P. murundu ). This scenario is similar to that reported for certain species in the dendrobatid genus Ameerega ( Lötters et al. 2009 ) and in the hylid genus Osteocephalus ( Jungfer et al. 2013 ) . The uncorrected p-distance of 3.2% found between P. atragula and P. f a c ure a e is very similar to values regarded by some authors as a “strong molecular” and “substantial genetic” differentiation between related taxa, even though they could represent conspecific lineages, or cryptic species instead (e.g., Vences et al. 2011 ). By integrating morphological, bioacoustic and molecular data, evidence presented herein support the recognition of P. atragula as a new, valid species of the genus Pseudopaludicola . As here reported, Pseudopaludicola atragula occurs syntopically with other two congeners at the type-locality: P. mystacalis and P. ternetzi . Although not yet studied in detail, co-occurrence is a rather frequent situation throughout the range of the genus ( e.g. , Silva et al. 2008 ; Pansonato et al. 2012b ; Pansonato et al. 2013 ). In general, individuals of Pseudopaludicola spp. seem to occupy habitats with vegetation consisting only in sparse, tall grasses, in seasonally flooded shallow areas or in permanent wet areas with a thin water film flow ( Santos et al . 2007 ; Silva et al . 2008 ; Giaretta & Facure 2009 ). Geographical, morphological, ecological, bioacoustic, and genetic patterns of co-occurring species of Pseudopaludicola should be evaluated using integrative approaches, for a better understanding of habitat use, geographic distribution, and evolution in the genus. Advertisement call parameters Pseudopaludicola facureae N=280 (8) Number of notes per series (s) 15.8±10.1 (2–55) Series duration (s) 0.8±0.6 (0.1–4.3) Inter-series interval (s) 0.5±0.2 (0.2–1.7) Note duration (s) 0.02±0.005 (0.01–0.04) Inter-notes interval (s) 0.03±0.03 (0.01–0.4) Minimum frequency (Hz) 2688.5±573.9 (1114.3–3992.6) Maximum frequency (Hz) 5947.9±336.8 (5372.1–6771.4) Average rate of notes/min 1249.1±388.5 (646–2000)