Bioacoustics of Ameerega berohoka

Remarks. Souza et al. (2001) extended the distribution of the Allobates femoralis complex based on tadpoles and juveniles from the municipality of Piranhas, state of Goiás. We will not rule out that the tadpoles and juveniles found were those of A. berohoka , as we never recorded A. femoralis in the region. Silva Jr. et al. (2007) recorded what they called A. femoralis in the Basin of Caiapó River. Our comparison of voucher specimens confirmed that the specimens correspond to A. berohoka . The uncorrected p-distances analysis of the 16S mitochondrial rRNA fragment gene revealed higher degrees of divergence among the Ameerega species than previous studies (e.g. Lötters et al. 2009 ). Probably, this incongruence is related to difference in the matrix size of both works. We produced an alignment of 358 base pair (excluding indels from the alignment due to ambiguous alignments) that is smaller than the analysis performed by Lötters et al. (2009) that selected (564 bp). Probably, we selected a part of the 16S fragment relatively more variable than Lötters et al. (2009) , reflecting directly in the results. Ameerega berohoka is poison frog endemic to Brazilian Cerrado biome and distinguishable from other species in the Ameerega picta group by a combination of features such as: sized; snout shape; patterns of coloration in different parts of the body; patterns of tympanic annulus and supratympanic fold; development of foot and finger discs; pulses per note of the advertisement call; and the genetic divergence of the 16S rRNA fragment gene among other Ameerega species. The new species resembles A. picta , A. flavopicta , A. braccata , and A. boehmei by the features examined such as adult morphology, advertisement call parameters and genetic divergence. However, this arrangement may not reflect phylogenetic relationships. A phylogenetic study including all or at least most species in the A. picta group is necessary to test the monophyly and the relationships among the species in the A. picta group.

Diagnosis. A member of the Ameerega genus based on the combination of characters proposed by Grant et al. (2006) and Lötters et al. (2007) such as dorsal skin slightly granular; finger I> finger II when adpressed; toe and finger web absent and presence of bright flash marks; and absence of ventrolateral line. The new species is defined by the combination of: (1) a medium sized species (adult SVL 15.5–24.3 mm SVL, sexes grouped); (2) snout subelliptical in dorsal view ( Fig. 1 A); (3) snout rounded in lateral view ( Fig. 1 C); (4) hand small (HAL/SVL=0.2) ( Fig. 1 E); (5) dark dorsum with pale spots in life ( Fig. 2 A −B); (6) in life, bright-orange spots in axillae, on groin, and on medial surface of thighs ( Fig. 2 B); (7) dorsolateral stripes cream to pale yellow in life ( Fig. 2 A −B); (8) venter and flank with white and black reticulation ( Fig 1 B); (9) tympanic annulus well visible, supratympanic fold absent ( Figs. 1 C −2A); (10) finger discs well developed ( Fig. 1 E); (11) pulses per note of the advertisement call ( Fig. 3 A; Table 2 ); (12) genetic divergence of the 16S rRNA fragment gene among other Ameerega species ( HQ891922 ; Genbank accession number) (Table 3). Comparisons with related species. Ameerega berohoka can be distinguished from A. braccata , A. flavopicta and A. boehmei by dark dorsal coloration with irregular cream spots (versus dark brown dorsum with golden, yellow or white spots in A. braccata and bright yellow spots in the two other species mentioned). It further differs from A. braccata by larger adult size and by bluish marbling on venter (versus pattern absent in A. braccata ). Also differs from A. flavopicta by its smaller adult size (SVL= 21.9 mm in A. berohoka and SVL= 26.7 mm in A. flavopicta ). The new species is distinguished from A. picta by smaller size of hands (HAL/SVL = 0.2 versus 0.3 in A. picta ), snout sub-elliptical in dorsal view (versus truncate in A. picta ) and rounded in lateral view (versus protruding in A. picta ), dorsal irregular spots (versus spots absent in A. picta ). Ameerega berohoka differs from A. yungicola by the absence of maxillary teeth (versus teeth present in A. yungicola , Lötters et al. 2005 ) and from A. hahneli and A. altamazonica by the presence of bright-orange spots in axillae, in groin and on medial surface of thighs (versus yellow in A. hahlneli and A. altamazonica, Twomey & Brown 2008 ). The new species is distinguishable from A. boliviana , A. picta , A. petersi and A. pulchripectus by the presence of cream to pale yellow dorsolateral stripes (versus yellowish or green in the respective taxa mentioned). It differs from A. rubriventris by its white and blue ventral coloration (versus reddish-orange in A. rubriventris ).

Description of holotype . Adult male, snout-vent length 19.7 mm ( Fig. 1 ). Dorsal skin of body and hindlimbs slightly granular and dorsal skin of head, both sides of head, forelimbs, and ventral surfaces smooth ( Fig. 1 A). Dorsal surface blackish ( Fig. 1 A). In life and preservative, cream dorsolateral stripe present on either side of the body from tip of snout, passing over eyelid continuing towards inguinal region and cream labial stripe from tip of lower jaw to forelimbs ( Fig. 1 A). Flanks black from groin to snout, ventrolaterally cream fading to white ventrally. In life and preservative, upper arm dorsally brown and ventrally cream ( Fig. 1 A −B); forearm dorsally brown and ventrally white with black reticulation ( Fig. 1 A −B). In life and preservative, hind limbs brown, dorsally with irregular black markings and ventrally white with black reticulation. In life, bright-orange spots present in axillae, groin and on medial surface of thighs, in preservative changed to white. In life and preservative, ventral surfaces of limbs, venter and head with coarse reticulation of irregular black lines. Gular region blackish ( Fig. 1 B). In life and preservative, hands and feet dorsally brown ( Fig. 1 A). In life, iris black. Oval tongue. Premaxillary and maxillary teeth absent. Vocal slits absent. Snout narrow sloping in lateral, sub-elliptical in dorsal view and truncate in ventral view. Nares situated and directed posterolaterally to tip of snout; nares visible from front and in ventral view but not in dorsal view. Canthus rostralis sloped, slightly concave; loreal region nearly vertical, slightly concave. Eye large and prominent with diameter 15 % of SVL; pupil rounded, horizontally elliptical. Tympanum circular, posterodorsally concealed, tympanic annulus well visible ( Fig. 1 C); diameter less than 50% of ED; supratympanic fold absent. Hands relatively small, length 30% of SVL ( Fig. 1 E). Relative length of adpressed fingers: II ≈ IV <I <III ( Fig. 1 E). Finger discs moderately expanded, most evident in finger III ( Fig. 1 E). A large, circular outer metacarpal tubercle on median base of palm ( Fig. 1 E); a smaller inner metacarpal tubercle on base of finger I ( Fig. 1 E); one prominent and well developed subarticular tubercle on fingers I and II, two well developed subarticular tubercles on fingers III and IV ( Fig. 1 E). Hind limbs relatively short with heel reaching shoulder when adpressed forward along body Fig. 1 A −B). Shank length 47% of SVL. Relative lengths of adpressed toes: I <II <V <III <IV ( Fig. 1 D); outer metatarsal tubercle rounded and well developed ( Fig. 1 D); outer metatarsal tubercle smaller than inner ( Fig. 1 D); inner metatarsal tubercle with ovoid shape ( Fig. 1 D). One subarticular tubercle on each toes I and II, two supranumerary subarticular tubercules on toes III and IV ( Fig. 1 D). Hands and feet lacking lateral fringes and webbing ( Fig. 1 D −E). No basal webbing on toes fringes. Tarsal tubercles absent ( Fig. 1 D). Measurements of holotype (in mm). SVL 19.7; HL 6.8; HW 6.1; IND 2.0; END 2.7; ED 2.9; TD 1.5; HAL 6.0; THL 9.7; TBL 9.3; FL 13.7. Variation. We found interpopulational coloration variation in the reticulated pattern of dorsum and venter and the transversal spots of hindlimbs. Variation of morphological measurements between males and females are shown in Table 1 . Male (N = 9) Female (N =4) Character Mean (± SD) Range Mean (± SD) Range SVL 20.8 (± 1.2) 19.2–23.4 23.0 (± 1.1) 21.9–24.3 HW 6.4 (± 0.3) 6.0–7.0 7.0 (± 0.4) 6.5–7.4 HEL 6.9 (± 0.5) 6.2–7.8 7.3 (± 0.4) 6.9–7.8 THL 9.8 (± 0.2) 9.6–10.2 11.0 (± 0.6) 10.2–11.5 TIL 9.9 (± 0.4) 9.3–10.6 10.6 (± 0.4) 10.0–11.1 FL 14.1 (± 0.6) 13.4–15.3 15.1 (± 0.8) 14.3–15.7 HAL 5.6 (± 0.5) 5.0–6.1 5.8 (± 1.0) 4.4–6.4 ED 2.8 (± 0.2) 2.6–3.1 2.7 (± 0.3) 2.5–3.1 TD 1.6 (± 0.1) 1.4–1.7 1.5 (± 0.2) 1.3–1.8 EM 2.4 (± 0.5) 2.0–3.1 2.4 (± 0.3) 2.1–2.9 ID 2.2 (± 0.4) 1.6–2.7 2.8 (± 0.2) 2.5–3.0 Coloration. Dorsum dark with pale reticulate spots, bright-orange spots present under axillae, dorsally over groin, and on medial surface of thighs. Dorsolateral stripes cream to pale yellow. Venter white or blueish and flanks with black reticulation. Dorsal hindlimbs light brown with dark spots ( Fig. 2 ). The coloration of preserved specimens resembles the coloration in life. The reticulate spots of dorsum are more in life. Dorsolateral stripes are cream and the venter white with black reticulation on flanks. Advertisement call. Oscillogram and spectrogram of the advertisement of Ameerega berohoka are shown in Figure 3 . Acoustic parameters of the call and comparisons to other species of the A. picta group are summarized in Table 2 . The advertisement call of the new species (n = 201 recorded from one specimen) is composed of a single note and pulsed with average duration of 82.5 ms (SD = 14.2) ( Fig. 3 A). The mean of the dominant frequency is 4.2 kHz ( Fig. 3 B) and frequency ranges from 4.0 to 4.3 kHz ( Fig. 3 B −C). The advertisement call of A. berohoka resembles those of other species in the A. picta group, mainly in dominant and frequency range ( Table 2 ). However, some features of advertisement call (note duration and frequency range) of the new species are more similar to closely geographically Ameerega species: A. flavopicta , A. braccata , and A. picta ( Table 2 ). The advertisement call is even more similar to A. flavopicta by a combination of parameters: note duration (82.5 ms in A. berohoka and 110 ms in A. flavopicta ), frequency range frequency (4.0−4.3 kHz in A. berohoka and 3.2−4.2 kHz in A. flavopicta ), and pulses per note range ( 10 in A. berohoka and 6 in A. flavopicta ) ( Table 2 ). notes (ms) (kHz)