The dark side of pumpkin toadlet: a new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Brigadeiro, southeastern Brazil Author Guimarães, Carla Silva Author Luz, Sofia Author Rocha, Pedro Carvalho Author Feio, Renato Neves text Zootaxa 2017 4258 4 327 344 journal article 33131 10.11646/zootaxa.4258.4.2 d07bb500-5b81-49ba-bce3-53dff7b6d337 1175-5326 570094 6918D108-AE71-4597-8144-950D995559DF Brachycephalus darkside sp. nov. Figures 1–4 and 7 . Brachycephalus ephippium —Pombal Jr. 2001 (part.) Brachycephalus ephippium — Dayrell et al. 2006 Brachycephalus ephippium —Pombal & Izecksohn 2011 (part.) Brachycephalus ephippium — Moura et al . 2012 . Holotype . MZUFV 16636 , adult male ( 17.9 mm SVL, Figure 1 ), collected at “Trilha do Cruzeiro” ( 20°52'40.7"S , 0 42°31'14.6"W ; 1266 m a.s.l. ), Parque Estadual da Serra do Brigadeiro ( PESB ), district of Careço , Municipality of Ervália , Minas Gerais State, Brazil , on 17 December 2015 by C.S. Guimarães , P.C. Rocha , C.L. Assis , R.N. Feio , and C. Novaes. Paratypes. Specimens collected at type locality: MZUFV 16634–35 (males) collected with holotype. MZUFV 16627, 16632–33 (males), 16628, 16631 (females), MNRJ 91327 and UFMG 19522 (males) collected on 20 November 2015 by C.S. Guimarães, P.C. Rocha and R.N. Feio. MZUFV 16491, 16579 (males), and 16780 (female) collected on 13 August 2015 by C.S. Guimarães, P.S. Hote and S. Luz. MZUFV 15716 (male), 15717, 15720–21 (adult specimens), and 15718–19 (juveniles) collected on 4 December 2014 by C.S. Guimarães, P.G. Taucce and B. Lisboa. MZUFV 15557–58, 15560–61 , 15566–15571, 15717 (adult specimens), and 15559, 15565 (males), collected on 18 September 2014 by C.S. Guimarães , C.L. Assis and R.N. Feio. Specimens collected at “ Mata do Pai Inácio ” , PESB , Municipality of Miradouro , Minas Gerais State, Brazil ( 20°46'42" S , 42°29' W ; 1340 m a.sl.) : MZUFV 6658–60 (adults) collected on 5 December 2005 by R.N. Feio, E.F. Oliveira and J.S. Dayrell. MZUFV 2897 (adult) collected on 5 September 1996 by R. Harvey. FIGURE 1. Brachycephalus darkside sp. nov. holotype (MZUFV 16636, SVL 17.9 mm). (A) Dorsal and (B) ventral views of the body; (C) lateral view of the head; (D) palmar view of the hand and (E) plantar view of the foot. Scale bar = 3mm. Diagnosis. The new species is characterized by the (1) presence of black connective tissue covering all dorsal muscles; (2) body completely yellow-orange in life; (3) presence of skull and post-cranial plates; (4) presence of paravertebral plates; (5) lack of ossified warts; (6) parotic plates laterally projected; (7) convex-shaped paravertebral plates with rounded edges; (8) dorsal shield not projected up over the vertebral spine; (9) first spinal plate with trapezium shape; (10) second spinal plate with rectangular shape; (11) large size (SVL of adults: 14.8– 18.5 mm ); (12) bufoniform body; (13) rounded snout; (14) absence of metacarpal and metatarsal tubercles; (15) absence of maxillae teeth; (16) large head; (17) presence of harmonic structures in the advertisement call. FIGURE 2. Cleared and double-stained specimen of Brachycephalus darkside sp. nov. (paratype MZUFV 15566, SVL 16.14 mm, adult). Dorsal (A) and lateral (B) view of the skull; dorsal (C) and ventral (D) views of the skull, vertebrae and para-axial bony plates. Legends: exoc (exoccipital), fp (frontoparietal), max (maxilla), nas (nasal), owin (oval window), pmax (premaxilla), ppl (parotic plate), prsph (parasphenoid), ptr (pterigoid), PV (pressacral vertebrae), pvpl (paravertebral plate), sac (sacrum), sq (squamosal), SPl (spinal plate), sphen (sphenethmoid), uro (urostyle), vm (vomer). FIGURE 3. Brachycephalus darkside sp. nov. , (A) dorsal view of right hand, dorsal view of right foot, and (C) ventral view of pectoral girdle with scapula and supra-scapula deflected. FIGURE 4. Histological analysis: (A) Dissected individual of Brachycephalus darkside sp. nov. (MZUFV 16579); (A1) closer view of the dark tissue; (A2) detail in the provision of pigmented tissue that follows adjacent muscle fibers. Transverse sections: (B) inclusion in resin; longitudinal section (C) inclusion in paraffin. Arrows indicate the dark pigments of connective tissue: epimysium (epm), perimysium (prm) and endomysium (edm). Muscle tissue (msc). Comparison with other species. Brachycephalus darkside sp. nov. differs from the remaining species of the genus by having black connective tissue (which can be seen as background coloration through the skin of specimens), covering all sides of dorsal muscles ( Fig. 4 ). There is no pigmentation in B. ephippium ( Fig. 5 ), B. izecksohni , B. pernix , and B. pitanga ; few scattered areas with pigmentation in the dorsolateral region adjacent to the dorsal musculature in some individuals of B. garbeanus and B. margaritatus ; and there is little dark pigmentation internally around the line of the spinal vertebrae in B. vertebralis . The skin of Brachycephalus darkside sp. nov. is orange in life in all parts of the body, which distinguishes it from all other species of the genus with colored stripes and/or spots and/or blotches (as B. auroguttatus , B. boticario , B. crispus , B. ferruginus , B. fuscolineatus , B. guarani , B. hermogenesi , B. izecksohni , B. leopardus , B. mariaeterezae , B. pernix , B. pitanga , B. pombali ), background greenish (as B. albolineatus , B. olivaceus , B. toby , B. verrucosus , B. tridactylus ), or brownish (as B. brunneus , B. didactylus , B. nodoterga , B. pulex , B. quiririensis , B. sulfuratus ). In preservative, specimens of the new species differ from those of B. alipioi , B. atelopoide , B. bufonoides , B. ephippium , B. garbeanus and B. margaritatus by the darkish background dorsum (pale cream in those species; see Fig. 1 and 5 ). The presence of skull and post-cranial plates of Brachycephalus darkside sp. nov. distinguishes it from B. albolineatus , B. auroguttatus , B. boticario , B. brunneus , B. didactylus , B. ferruginus , B. fuscolineatus , B. hermogenesi , B. izecksohni , B. leopardus , B. mariaeterezae , B. olivaceus , B. pernix , B. pombali , B. pulex , B. quiririensis , B. sulfuratus , B. tridactylus and B.verrucosus (absent in the referred species). The new species differs from the remaining species by the presence of paravertebral plates, except B. ephippium , B. garbeanus and B. margaritatus (paravertebral plates present in those species). The lack of osteoderms distinguishes B. darkside sp. nov. from B. atelopoide , B. crispus and B. margaritatus (present in those species). The presence of paravertebral plates in Brachycephalus darkside sp. nov. resembles the ones observed in B. ephippium , B. garbeanus and B. margaritatus . However, the new species differs from B. ephippium by the welldeveloped parotic plates, laterally projected (not projected in B. ephippium ), and by the convex paravertebral plates (flat in B. ephippium ). Brachycephalus darkside sp. nov. differs from B. garbeanus by having the dorsal shield not projected over the vertebral spine (projected over the borders of the vertebral spine in B. garbeanus ), and by the convex central shape and rounded edges of paravertebral plates (paravertebral plates flat with square edges in B. garbeanus ). It is distinguished from B. margaritatus by the first and second spinal plates with trapezium and rectangular shape, respectively (both triangular in B. margaritatus ) and by the dorsal plate with rounded edges (nearly square and curved down in B. margaritatus ). The new species differs by its larger SVL ( 14.8 – 18.5 mm ) from Brachycephalus albolineatus , B. auroguttatus , B. boticario , B. brunneus , B. didactylus , B. fuscolineatus , B. guarani , B. hermogenesi , B. izecksohni , B. leopardus , B. mariaeterezae , B. nodoterga , B. olivaceus , B. pitanga , B. pulex , B. quiririensis and B. verrucosus (combined SVL in those species: 7.0–14.0 mm). Brachycephalus darkside sp. nov. differs from B. didactylus , B. hermogenesi and B. pulex by the bufoniform body and snout rounded in dorsal view (body leptodactyliform and snout pointed in those species). The absence of metacarpal and metatarsal tubercles distinguish the new species from Brachycephalus hermogenesi (metacarpal and out metatarsal present in this species), B. auroguttatus , B. boticario , B. brunneus , B. fuscolineatus , B. hermogenesi , B. izecksohni , B. leopardus , B. mariaeterezae , B. olivaceus , B. pitanga , B. pulex , B. quiririensis , B. toby and B. verrucosus (outer metatarsal present in those species). Brahycephalus darkside sp. nov. differs from B. brunneus , B. bufonoides , B . ferruginus , B. izecksohni and B. pombali by the absence of maxillae teeth (present in those species). The large head (wider than long) distinguishes B. darkside sp. nov. from B. ephippium and B. garbeanus (head longer than wide in those species). The advertisement call of Brachycephalus darkside sp. nov. distinguishes it from all its congeneric species by the presence of harmonic structures (absent in the other species of Brachycephalus ; see Table 3 for complete comparison). It is distinguished from B. tridactylu s by the pulsed notes (not pulsed in this species), from B. pernix by having five to eight pulses per note (three in B. pernix ), and from B. pitanga by having less pulses per note (mean of 11.1± 1.2 pulses per note in B. pitanga ). It is distinguished from B. crispus , B. hermogenesi and B. pitanga by the shorter notes (ND = 83–163 ms in B. darkside sp. nov. ; ND = 280 ± 20 ms in B. crispus ; ND = 200 ms in B. hermogenesi ; ND = 170 ± 13 ms in B. pitanga ) and by the higher note rate (NR = 186.4–243.4 notes/s in B. darkside sp. nov. ; NR = 1.67 ± 0.09 notes/s in B. crispus ; NR = 1.09 notes/s in B. hermogenesi ; NR = 2.65 ± 0.18 notes/s in B. pitanga ). Brachycephalus darkside sp. nov. has the lowest peak frequency within the genus (PF = 2.8–3.8 kHz in B. darkside sp. nov. ; combined dominant frequency in the other species = 4.6–6.8 in other species; Condez et al. 2014 , Verdade et al. 2008 , Araújo et al. 2012 and Garey et al. 2012 ). Description of holotype . Body robust and bufoniform; head wider than long, 32.9% (HL/SVL) and 34.4% (HW/SVL) ( Fig. 1 A); snout short, rounded in dorsal and lateral views; nostrils slit-shaped, slightly protuberant, anterolaterally directed; canthus rostralis indistinct; loreal region weakly concave; lips sigmoid; eyes slightly protruding, anterolaterally directed, 8.7% (ED/HW) and 30% (ED/HL); tympanum indistinct; vocal sac not externally expanded; vocal slit presents; tongue longer than wide, posterior half not adhered to the mouth floor; choanae small and round; vomerine odontophores absent. FIGURE 5. Ventral and dorsal view of dissected preserved specimens of (A) Brachycephalus darkside sp. nov. , showing the presence of dark tissue in the dorsal musculature, and (B) the absence of dark tissue in B. ephippium . FIGURE 6. Spectrogram (above) and oscillogram (bellow) of the calls of Brachycephalus darkside sp. nov. (A) Sequence of four notes of the advertisement call, recorded on 20 November 2015, 12h40, air temperature 22°C, paratype MZUFV 16629. (B) Sequence of six notes of the aggressive call, recorded on 17 December 2015, 19h00, air temperature 19°C, holotype MZUFV 16636. Arm and forearm relatively slender; arm slightly shorter than forearm (AL 22.3% and FAL 22.3% of SVL); forearm slightly hypertrophied; hands shorter than arm. All fingers are distinct; fingers II and III robust; I and IV very small, vestigial; tip of finger I rounded and the others pointed; relative lengths of fingers IV <I <II <III; subarticular, inner and outer metacarpal tubercles absent ( Fig. 1 E). Shank as long as thigh (SHL/SVL = 37.2% and THL/SVL = 39.5%); foot longer than tarsus and shorter than shank; toes II, III e IV relatively distinct with pointed tip, I and V vestigial; relative length of toes I <V <II <III <IV; subarticular, inner and outer metatarsal tubercles absent. Skin on head and dorsum rough, due to surface ornamentation of the skull and post-cranial plates; posterior region of the body with few scattered warts; skin of the body granular dorsolaterally and around cloacal opening; skin on belly and limbs smooth ( Figs. 1 and 7 ). Measurements of holotype (in mm). SVL 17.9, HL 5.9, HW 6.2, ED 1.8, IOD 2.2, END 1.0, ND 0.4, IND 2.2, UAL 4.0, FAL 4.3, HAL 3.3, THL 7.1, SHL 6.7, TAL 4.1, FL 5.6. Osteology. ( Figure 2 ) The cleared and double stained specimens revealed a well-developed parotic plate and ornamented dermal roofing bones in the skull of Brachycephalus darkside sp. nov. Dorsomedially, the parotic plate articulates with the frontoparietal. Laterally, the parotic plate is expanded, making the posterior region of head wide and, consequently, the head wider than long. Nasal, sphenethmoid, frontoparietals, prootics, and exoccipital fused, forming a dorsal cranial plate. Premaxillae broad, not fused medially; pars dentalis present, but odontoids absent. Alary process of premaxillae distinct, narrowly separated from the nasal and widely separated from each other, with length approximately twice of height. In ventral view, maxillae arched, odontoids absent. Quadratojugal absent. Pterygoid slender, anterior ramus long, articulating with maxillary arch; posterior ramus short, articulating with ventral ramus of squamosal; medial ramus short, articulating with the prootic. Dentigerous process of vomer absent, prechoanal and postchoanal ramus reduced but distinct. Palatine absent. Parasphenoid robust. Squamosal broad in lateral view; anterior zygmatic ramus extremely reduced, not ornamented; posterior otic ramus short, allocated below the parotic plate and may or not present ornamentations on the anterior portion not covered by the parotic plate. Anterior zygmatic ramus short and not ornamented. Tympanic annulus absent. Mandible edentate. Pectoral girdle arciferal and robust ( Fig. 3 C). Procoracoid and epicoracoid fused and completely ossified. Procoracoid and epicoracoid synostotically united with clavicle, coracoid, and scapula. Suprascapula expanded, with anterior half ossified as cleithrum. Omosternum and sternum absent. Ventral column composed of eight nonimbricate vertebrae and hyperossified spinal process of vertebrae. Atlas (first pressacral vertebra) lacks transverse process. Presence of paravertebral and spinal plates well developed and ornamented. Spinal plates associated with the spinal process of all presacral and sacral vertebrae. First spinal plate (vertebra II) broad; trapezium convex-shaped; second spinal plate (vertebra III) thin, almost rectangle convex-shaped and nearly joint with principal group of dorsal plates. Dorsally, the paravertebral plate completely conceals the transverse process of vertebra IV-VII, and partially covers the posterior tip of transverse process of vertebra III. Lateral edge of the paravertebral plates almost rounded; anterior and posterior corners reach the level of transverse process of vertebra II and almost the sacral diapophysis, respectively. Paravertebral plates fused medially to the block consisting of the fusion of spinal plates IV–VII (VIII in some individuals). Ventrally, transverse process of vertebrae IV and V fused to the paravertebral plates. Dorsally, the central of each paravertebral plate has an elevation that provides a convex shape. Parotic, cranial, spinal and paravertebral plates ornamented with dermal roofing bones. Forearm slightly shorter than humerus. Radius and ulna fused and distinguishable. Manus with distal carpals (I–V) fused, with centrale, radiale and ulnare about the same size. Prepollex very reduced, with one element. Phalangeal formula 1-2-3-1 ( Fig. 3 A). Tips of the terminal phalangeal elements of fingers arrow-shaped. Tibia and fibula fused, distinguishable, forming tibiafibula. Tibiafibula and femur of approximately the same length. Tibiale and fibulare fused at their distal and proximal ends, not fused medially. Hindlimbs with tarsal elements I, II, III present, and IV–V absent; centrale present. One very-reduced prehallical element. Phalangeal formula 1-2-3-4-1 ( Fig. 3 B). Tips of terminal phalangeal elements of toes II, III and IV arrow shaped. Toes I and V reduced with terminal phalangeal element rounded in shape. Histology. Histological analysis showed a connective tissue covering the dorsal musculature (epimysium), and between muscle fibers (perimysium and endomysium) ( Fig. 4 A-D). This tissue presented spots of extracellular matrix containing dark pigments, which were reflected in the macroscopy of fixed specimens as well ( Fig. 1 A). FIGURE 7. Photos of Brachycephalus darkside sp. nov. in life: (A) female, (B) male, (C) same male hidden beneath roots, (D) roots where the male was found. Coloration of Holotype . In life, body bright yellow-orange. Venter yellow-orange, slightly pale compared to remaining of body. Dark background at central region of dorsum, from posterior region of skull to inguinal region, beneath the yellow-orange skin. Dorsal plates slightly dark. Eyes completely black ( Fig. 7 ). Coloration in preservative. General body color cream. Dark background beneath the cream skin from central region of dorsum to inguinal region, including posterior region of skull. Ossified regions such as skull and dorsal plates grayish. Eyes completely black ( Fig. 1 ). Variation. Measurements of adults and juveniles are given in Table 1 . The density of warts along the posterior region of the body and granular regions vary between individuals (i.e., in some specimens the body is entirely smooth). Skull and post-cranial plates varies ontogenetically; juveniles may lack hyperossification or present skull and post-cranial skeleton less ornamented than adults, as previously described for Brachycephalus ephippium ( Campos et al. 2010 ) . Among adult individuals the paravertebral plates are proportionally different in size; they can be bigger or smaller in individuals of same size. The second spinal plate may contact spinal plates fused with paravertebral plates. The spinal plate of presacral VIII and sacral vertebra may be separated from the dorsal shield. The top of the squamosal may present ornamented dermal ossification. Some specimens presented 1/3 of tong adhered to floor of mouth, and oval choanae. TABLE 1. Measurements in millimeters of Brachycephalus darkside sp. nov. type series. Character abbreviations are listed in Material and Methods section.

Traits SVL	Adults (n = 31) Mean ± SD Range 16.7 ± 1.3	14.8–18.5	Juveniles (n = 3) Mean ± SD Range 13 ± 2.0	11.3–15.2
HL HW ED	5.5 ± 0.4 6.3 ± 0.5 1.8 ± 0.4	4.8–6.3 5.4–7.3 1.6–3.4	4.5 ± 0.5 5.2 ± 0.6 1.5 ± 0.1	3.9–5.0 4.6–5.8 1.4–1.6
IOD END ND	3.7 ± 0.4 1.0 ± 0.1 0.5 ± 0.03	2.5–4.1 0.9–1.2 0.4–0.6	3.0 ± 0.4 0.8 ± 0.1 0.4 ± 0.1	2.7–3.4 0.7–1.0 0.4–0.4
IND UAL FAL	2.2 ± 0.1 3.4 ± 0.3 3.8 ± 0.5	1.9–2.5 2.8–3.9 2.8–4.5	1.8 ± 0.3 2.8 ± 0.7 3.1 ± 0.6	1.6–2.1 2.1–3.6 2.8–3.8
HAL THL SHL	3.2 ± 0.4 7.0 ± 0.7 6.5 ± 0.3	2.0–3.6 6.3–9.3 5.9–7.0	3.1 ± 0.5 4.7 ± 2.1 4.2 ± 1.9	2.4–3.4 2.3–6.3 2.1–5.9
TAL FL	4.2 ± 0.4 5.4 ± 0.4	3.5–5.0 4.8–6.2	3.9 ± 0.8 4.3 ± 0.6	3.0–4.7 4.0–5.0

Call description. We observed two distinct calls of Brachycephalus darkside sp. nov. herein referred as the advertisement call and an aggressive call ( sensu Toledo et al. 2015 ). The advertisement call is the most common type of call (76% of all calls, n = 790 notes; Fig. 6 A, Table 2 ) and is characterized by pulsed notes emitted in extremely long sequences. The longest call recorded had more 250 notes and was not fully recorded (NN = 114 ± 97.1, 9–253 notes per call, n = 7; CD = 30.4 ± 25.3, 2.9– 66.2 s , n = 7 sequences). We were only able to record two intervals between calls, which ranged from 6.2 to 11.2 s , hampering the calculation of call rate. Each note had six pulses on average (PN = 6.3 ± 0.7, 5–8 pulses, n = 790 notes) emitted at constant rate ( PR = 56.9 ± 4.9, 36.8–78.4 pulses/s). The first pulse is often the most energetic one ( Fig. 6 A), followed by a decrease in amplitude in the second pulse and a new rise in the next one or two pulses, then the amplitude fades until the last pulse. Note duration was relatively stereotyped (ND = 111.5 ± 13.7, 83–163 ms, n = 790 notes), and so was the interval between notes (NI = 159.5 ± 14.5, 122–215 ms, n = 783 intervals), and the note rate (NR = 211.4 ± 25.6, 186.4– 243.4 notes/min, n = 5 recordings). Dominant frequency (DF) ranged from 2484.4 to 5765.6 Hz with peak of energy around 3.3 kHz (PF = 3382.1 ± 184.6, 2856.4–3796.9 Hz). Up to three harmonics could be observed in the advertisement call, with only two of them visible in all recordings. A second type of call was recorded whilst a male Brachycephalus darkside sp. nov. vocalized within 20 cm of a conspecific male in an aggressive social context ( sensu Toledo et al. 2015 ). The aggressive call (23%, n = 245 calls; Fig. 6 B, Table 2 ) is also characterized by pulsed notes emitted in sequences shorter than the ones from the advertisement call (CD = 4.1 ± 1.3, 2.4– 6.9 s , n = 10 calls), with variable number of notes (NN = 24.5 ± 7.9, 15–41 notes/call, n = 10 calls) and emitted about eight times per minute (CR = 8.46 calls/min, n = 1) with brief intervals (CI = 2.8 ± 0.7, 2.0– 3.8 s , n = 9 intervals). Each note was shorter than the ones from the advertisement call (ND = 31.1 ± 5.7, 18–44 ms, n = 245 notes), emitted at higher rate (NR = 356.6 ± 6.5, 343.7–364.2 notes/min, n = 10 calls) and variable intervals (143.3 ± 16.7, 96–334 ms, n = 235 intervals). Notes had average of 2.5 pulses (PN = 2.5 ± 0.5, 2–3 pulses, n = 245 notes) emitted at higher rate ( PR = 79.9 ± 9.6, 57.1–111.1 pulses/s, n = 245 notes) than observed in the advertisement call. There is an increase in amplitude from the first to the second pulse, followed by a decline in amplitude from the second to the third pulse. Sometimes the second and third pulses were juxtaposed. Dominant frequency (DF) ranged from 2906.2 to 4406.2 Hz with peak of energy around 3.4 kHz (PF = 3429.7 ± 146.5, 3046.9– 3984.4 Hz). Notes of the aggressive call presented a downward frequency modulation: the first pulse presented the highest values of dominant frequency (DF1 = 3421.9–4218.8) and peak of energy (PF1 = 3895.5 ± 73.4, 3703.1–3984.4 Hz); the second pulse had intermediate values (DF2 = 3046.9–3937.5 Hz; PF2 = 3481.6 ± 109.8, 3328.1–3656.2 Hz) and the third pulse had the lowest values (DF3 = 3281.2–3515.6 Hz; PF3 = 3066.4 ± 71.7, 2953.1–3328.1 Hz). TABLE 2. Parameters of the advertisement and aggressive calls of Brachycephalus darkside sp. nov. Descriptions based on six individuals, recorded at Parque Estadual da Serra do Brigadeiro.

Parameters	Advertisement call (n = 5 individuals)	Aggressive call (n = 1 individual)
Number of notes per call	114 ± 97.1 (9–253) n = 7	24.5 ± 7.9 (15–41) n = 10
Call duration (s)	30.4 ± 25.3 (2.9–66.2) n = 7	4.1 ± 1.3 (2.4–6.9) n = 10
Interval between calls (s)	6.2–11.2 n = 2	2.8 ± 0.7 (2.0–3.8) n = 9
Call rate (calls/min)	–	8.46; n =1
Note duration (ms)	111.5 ± 13.7 (83–163) n = 790	31.1 ± 5.7 (18–44) n = 245
Interval between notes (ms)	159.5 ± 14.5 (122–215) n = 783	143.3 ± 16.7 (96–334) n = 235
Note rate (notes/min)	211.4 ± 25.6 (186.4–243.4) n = 5	356.6 ± 6.5 (343.7–364.2) n = 10
Number of pulses per note	6.3 ± 0.7 (5–8) n = 790	2.5 ± 0.5 (2–3) n = 245
Pulse rate (pulses/s)	56.9 ± 4.9 (36.8–78.4) n = 790	79.9 ± 9.6 (57.1–111.1) n = 245
Peak frequency (Hz)	3382.1 ± 184.6 (2856.4–3796.9) n = 790	3429.7 ± 146.5 (3046.9–3984.4) n = 245
Dominant frequency range (Hz)	2484.4–5765.6	2906.2–4406.2

TABLE 3. Call parameters in the genus Brachycephalus . Note duration in ms (ND); Interval between notes in ms (NI); Note rate in notes/s (NR); Number of pulses per note (PN); Pulse rate in pulses/s (PR); Peak frequency in kHz (PF); Dominant frequency range in kHz (DF). 1: Peak frequency in this work. 2: Emphasized frequencies in Pombal et al. (1994). 3: Notes/min in Araújo et al. (2012). 4: Calls/s in Garey et al. (2012). 5: Range of values not made available in the call description. Range (mean ± SD).

B. crispus	B. darkside sp. nov.	B. ephippium	B. hermogenesi	B. pernix	B. pitanga	B. tridactylus
ND	(280 ± 20)	83–163 (111.5 ± 13.7)	93–149 (112 ± 6.5)	(200 ± 10)	(60)	(170 ± 13)	(110 ± 20)
NI	(350 ± 20) 122–215	(159.5 ± 14.5) 123–149	(134 ± 6.8)	–	(140)	–	–
NR	(1.67 ± 0.09)	186.4–243.4 (3.5 ± 0.4)	–	1.09	–	(2.65 ± 0.2)³	(0.16 ± 0.03)4
PN	7–12	5–8 (6.3 ± 0.7)	5–15 (12 ± 1.96)	–	3	(11.1 ± 1.2)	Unpulsed
PR	(17.4 ± 2.12)	36.8–78.4 (57.0 ± 4.9)	–	–	–	(62 ± 8)	–
PF	(4.6 ± 0.19)	2.8–3.8 (3.4 ± 0.2)¹	–	(6.8 ± 0.8)	–	(4.9 ± 0.2)	(4.8 ± 0.2)
DF	3.5–5.7	2.4–5.7	3.4–5.3²	–	4.5–6.7 (5.6)	–	3.2–6.4 (4.8 ± 0.2)
Ref.	Condez et al. 2014 5	This work	Pombal et al. 1994	Verdade et al. 2008 5	Wistuba 1998	Araújo et al. 2012 5	Garey et al. 2012 5

Etymology. The epithet " darkside " is a noun in apposition, derivative of the English language. It refers to the dark side of the body of Brachycephalus darkside sp. nov. , which corresponds to the dark tissue surrounding the dorsal musculature, creating a dark background to the bright yellow-orange dorsum ( Fig. 4 ). It is also a reference to the album "The Dark Side of the Moon" by the British rock band Pink Floyd. Natural history. Specimens of Brachycephalus darkside sp. nov. were found amidst leaf litter of “Trilha do Cruzeiro”, at the habitat Floresta Estacional Semidecidual Montana , between 1266 and 1498 m a.s.l. We observed active individuals from October to December, between 12h and 19h. Males were found calling exposed over leafs or beneath them, and females walking through the leaf litter. During the dry months (from July until September), individuals of B. darkside sp. nov. were found hidden deep into the layers of leaf litter, buried and within underground roots of trees ( Fig. 7 ). The new species is abundant in the area where it occurs and reproduces syntopicaly with species of the genus Ischnocnema . Distribution. Brachycephalus darkside sp. nov. is only known for the Municipalities of Ervália and Miradouro within the Serra do Brigadeiro mountain range, at the northern portion of the Mantiqueira mountain range, Minas Gerais State, southeastern Brazil ( Fig. 8 ). Considering the distances between locations, vegetation and altitude range ( 1266–1498 m a.s.l.), it is likely that the new species also inhabit more locations within the Serra do Brigadeiro. Comments. Brachycephalus darkside sp. nov. is described based on its external morphology, osteology, histology and vocalizations. The new species is assigned to the B. ephippium group based on morphological features and on its geographic distribution ( Clemente-Carvalho et al. 2011 , Ribeiro et al. 2015). Moreover, the strict altitudinal record of B. darkside sp. nov. corroborates the hypothesis that the B. pernix and B. ephippium groups are indeed “montane groups” ( Bornschein et al. 2016a ). In addition, we agree with Bornschein et al. (2016a) that populations under the name of B. ephippium should be subject of scrutiny as they are likely cryptic species waiting to be unveiled. FIGURE 8. Geographical distribution of Brachycephalus darkside sp. nov. , and of Brachycephalus didactylus , B. pernix and B. ephippium species groups. BA (Bahia State), MG (Minas Gerais State), ES (Espírito Santo State), RJ (Rio de Janeiro State), SP (São Paulo State), PR (Paraná State), SC (Santa Catarina State). As mentioned in the Materials and Methods section, it was not always possible to determine the sex of all individuals of Brachycephalus darkside sp. nov. The small size of them hampered the accurate visualization of vocal slits without causing permanent damage to topotype specimens. On the other hand, several works mentioned the presence of vocal slits in females of Brachycephalus (e.g. Alves et al. 2009 ; Clemente-Carvalho et al . 2012 ; Haddad et al. 2010 ; Pie & Ribeiro 2015 ; Ribeiro et al. 2015), although these authors do not specify how sex was determined. Given this difficulty, we believe further studies should evaluate carefully the sex of specimens of Brachycephalus as the misidentification might hamper the appropriate diagnose and comparison between and within species.