Brachycephalus sulfuratus, Condez & Monteiro & Comitti & Garcia & Amaral & Haddad, 2016

Condez, Thais Helena, Monteiro, Juliane Petry De Carli, Comitti, Estevão Jasper, Garcia, Paulo Christiano De Anchietta, Amaral, Ivan Borel & Haddad, Célio Fernando Baptista, 2016, A new species of flea-toad (Anura: Brachycephalidae) from southern Atlantic Forest, Brazil, Zootaxa 4083 (1), pp. 40-56 : 42-52

Brachycephalus sulfuratus sp. nov.

Figures 1–3

Holotype. CFBH 39137 , adult female, collected at Centro de Estudos e Pesquisas Ambientais da Univille , Vila da Glória , Distrito do Saí (26 ° 13'39"S, 48 ° 41'31"W, 123 m above sea level, Datum WGS 84), município de São Francisco do Sul, State of Santa Catarina, Brazil, on 14 November 2014, by T.H. Condez, J.P.C. Monteiro, and E.J. Comitti.

Paratypes. CFBH 39138, cleared and double-stained adult male, and CFBH 39139, adult male, both collected with the hotolype. CFBH 39140, adult female collected on 11 September 2014, CFBH 39329, adult female, and CFBH 39331, adult male, collected on 31 July 2015, CFBH 39330 and 39332, adult females, collected on 0 1 August 2015, at the same locality as the holotype, by J.P.C. Monteiro and E.C. Nardin. CFBH 39142, adult male, CFBH 39141 and 39144, adult females, CFBH 39143, cleared and double-stained adult male, collected on 0 4 December 2013, by T.H. Condez, J.P.C. Monteiro and E.J. Comitti, UFMG 17954 and 17955, adult females, CFBH 39407–39410 and UFMG 17956 and 17957, adult males, CFBH 39406, juvenile, collected on 28 August 2015, by J.P.C. Monteiro and E.C. Nardin, at Morro do Cantagalo, Vila da Glória, Distrito do Saí (26 ° 10'31"S, 48 ° 42'44"W, 161 m above sea level, Datum WGS 84), município de São Francisco do Sul, State of Santa Catarina, Brazil. CFBH 39145–39147, adult females, collected at Castelo dos Bugres, Área de Proteção Ambiental Dona Francisca (26 ° 13'43"S, 49 ° 03'27"W, 835 m above sea level, Datum WGS 84), município de Joinville, State of Santa Catarina, Brazil, on 29 November 2013, by T.H. Condez, J.P.C. Monteiro, E.J. Comitti, I. Borel, P.D. Pinheiro, and P.C.A. Garcia. CFBH 39148 and 39149, adult males, collected at Parque Estadual da Ilha do Cardoso (25 ° 06'53"S, 47 ° 55'40"W, 385 m above sea level, Datum WGS 84), município de Cananéia, State of São Paulo, on 19 December 2013, by T.H. Condez, L.N. Bandeira, and S. Pinheiro. CFBH 39150, adult female, collected at Morro do Anhangava (25 ° 22'51"S, 49 ° 01'26"W, 915 m above sea level, Datum WGS 84), município de Quatro Barras, State of Paraná, on 0 2 February 2012, by T.H. Condez, T.B. Rocha, and P.F. Colas-Rosas.

Referred specimens. MZUSP 129855 , juvenile, collected at Ilha do Cardoso (non-georeferenced data), município de Cananéia, State of São Paulo, on 22 January 1979, by an unknown collector . ZUEC 16602 , juvenile, collected at Área de Proteção Ambiental de Guaratuba (25°47’ S, 48°54’ W, 291 m above sea level), município de São José dos Pinhais, State of Paraná, on 18 January 2008, by A.K. Cunha and I. Soares.

Diagnosis. Brachycephalus sulfuratus sp. nov. is a new species of flea-toad, distinguished from all its congeners by the following combination of characters: (1) small body size (SVL of adults: 7.4–8.5 mm for males and 9.0– 10.8 mm for females); (2) “leptodactyliform” body; (3) pectoral girdle arciferal and less robust compared to the Brachycephalus species with “bufoniform” body; (4) procoracoid and epicoracoid fused with coracoid but separated from the clavicle by a large fenestrae; (5) toe I externally absent; toes II, III, IV, and V distinct; phalanges of toes II and V reduced; (6) skin smooth with no dermal ossifications; (7) in life, general background color brown with small dark-brown spots; skin of throat, chest, arms, and forearms with irregular yellow blotches; in ventral view, cloacal region of alive and preserved specimens surrounded by a dark-brown inverted v-shaped mark outlined with white; (8) advertisement call long, composed of a set of 4–7 high-frequency notes (6.2–7.2 kHz) repeated regularly.

Description of holotype. The holotype of Brachycephalus sulfuratus sp. nov. has a “leptodactyliform” body ( Figure 1 and 2 ); head wider than long, narrower than body; head length approximately 26% of SVL; snout long, with length slightly short than the eye diameter, rounded in lateral and dorsal views ( Figure 3A and 3B ); nostrils protuberant, directed anterolaterally; canthus rostralis distinct and straight; loreal region weakly concave; mouth nearly sigmoid; eye slightly protruding in dorsal and lateral views, eye diameter 48% of HL; tympanum absent; tongue longer than wide, posterior half not adherent to floor of mouth; choanae relatively small and round; vomerine odontophores absent. Upper arm slightly shorter than forearm; length of upper arm plus forearm 43% of SVL; hands approximately as long as upper arm; fingers II and III thin and distinct; finger I and IV very small, vestigial; tip of fingers II and III slightly pointed; relative lengths of fingers II <III; metacarpal tubercles present, outer large and rounded, inner thin and elongated; subarticular tubercles undefined in hand ( Figure 3C ). Tibia shorter than thigh; total leg length 85% of SVL; foot length larger than thigh length; toe I externally absent; toes II, III, IV, and V distinct; toes II and V reduced; tip of toes II and V slightly rounded, tip of toes III and IV pointed; relative length of toes V<II <III <IV; metatarsal tubercles present, both oval, outer slightly larger than inner; subarticular tubercles undefined in foot ( Figure 3D ). Skin smooth without dermal ossifications ( Figure 2 ).

Measurements of holotype (in mm). SVL 9.8; HL 2.5; HW 2.7; ND 0.4; IND 1.0; ED 1.2; IOD 1.9; END 0.8; THL 4.3; TBL 4.1; FL 6.3; AL 2.0; FAL 2.3; HAL 1.8.

Color of holotype in life. Dorsal coloration grey to brown covered with dark brown and red spots ( Figure 1A ). The dark brown spots are concentrated on the head and medial portion of the dorsum, in which a large irregular mark is distinguishable. The interorbital area exhibits a dark brown nearly v-shaped mark. Dorsal surfaces of arms and legs exhibit dark brown blotches, which on the thigh and tibia resemble incomplete stripes. Above the cloacal region, in dorsal view, there is a dark brown inverted m-shaped mark. A dark brown stripe extends laterally from the tip of the snout to the flanks and the surface of thigh ( Figure 1A ). In lateral view, the tip of the snout and posterior regions of maxilla and ocular globe present undefined yellow spots. The maxilla is dark brown with distinct white spots. Ventral surface of body pale-brown, slightly transparent, with small dark brown spots and white blotches ( Figure 1B ). Some yellow blotches are also spread among the gular region, arms, forearms, and disposed in an inverted v-shape on the chest. In ventral view, the cloacal region is surrounded by an inverted vshaped dark brown mark with white borders. The pupil is black and the iris is golden.

Color of holotype in preservative. General background color is pale-brown covered with small dark brown dots ( Figure 2 ); dorsum with dark brown marks on the ocular region, knees, and cloacal region; dark brown stripes on dorsal surfaces of thigh, tibia, and foot; ventral surfaces of hands and feet with dark brown blotches; in ventral view, cloacal region surrounded by an inverted v-shaped dark-brown mark.

Osteology. The cleared and double-stained material revealed the following characters in Brachycephalus sulfuratus sp. nov. No hyperossification of the skull or skeleton. Nasals, sphenethmoid, frontoparietals, prootics, and exoccipitals fused. Premaxillae broad, not fused medially; odontoids absent; alary process of premaxillae distinct and separated from the nasal. Maxillae arched in ventral view; odontoids present. Quadratojugal and pterygoid present. Vomer not fused medially, vomerine odontophores absent. Palatine absent. Parasphenoid and sphenethmoid fused and robust. Squamosal elongated in lateral view, zygomatic ramus short and not ornamented. Tympanic annulus absent. Mandible edentate. Pectoral girdle arciferal and less robust than in other Brachycephalus; clavicle, coracoid, and scapula fused and completely ossified; procoracoid and epicoracoid fused with coracoid, but separated from the clavicle by a large fenestrae; suprascapula expanded, anterior half ossified as cleithrum; omosternum present and cartilaginous; sternum absent. Vertebral column composed of eight presacral, non-imbricate vertebrae; hyperossification absent in the spinal processes of vertebrae; all presacral vertebrae with transverse processes; transverse process of sixth presacral vertebra elongated but not ornamented. Humerus slightly shorter than forearm; radius and ulna fused but distinguishable. Manus with distal carpals (I–IV) fused with centrale; radiale and ulnare about the same size; one prepollical element; phalangeal formula 1–2–3–1; tips of terminal phalangeal element of fingers I and IV falciform and tip of finger II and III pointed. Hindlimbs with tibia and fibula fused but distinguishable, forming the tibiafibula; tibiafibula slightly shorter than femur; tibiale and fibulare fused at their distal and proximal ends (medially not fused). Pes with distal tarsal element I, II, III present and IV–V absent; centrale present. One very reduced prehallical element present; phalangeal formula 1–2–3–4–1; tips of terminal phalangeal elements of toes I and V rounded; tips of the terminal phalangeal elements of toes II, III, and IV arrow-shaped.

Advertisement call. The advertisement call of the new species is long, composed by the regular repetition of five or six high-frequency pulsed notes ( Figure 4 ). The call lasts 1.5–2.3 seconds (x̄=1.8±0.2) and the interval between calls is 3.1–7.4 seconds (x̄=5.1±1.4). The call is composed of 4–7 notes (x̄=5.3±0.9), repeated in a rate of 0.1–0.3 notes/second (x̄=0.2±0.0). Notes last 131–233 milliseconds (x̄=195±13) and present 7–11 pulses (x̄=8.8±1.3). Pulses last 20–30 milliseconds (x̄=23.6±4.8) and are repeated at a rate of 6.1–12.3 pulses/second (x̄=9.3±1.8). The minimum frequency is 4.5–5.5 kHz (x̄=4.9±0.3), the maximum frequency is 8.2–10.3 kHz (x̄=9.3±0.3), and the dominant frequency is 6.2–7.2 kHz (x̄=6.7±0.3).

Variation. Morphometric variation is given in Table 1. In our sample, females are larger than males (SVL of females x̄= 9.9 mm ±0.4; SVL of males x̄=8.0 mm±0.4, Welch’s t-test t = 10.2, df = 9.6, p <0.01).

The dorsal pattern in life varies from gray or pale cream to dark and reddish-brown ( Figure 5 ). Some specimens have an irregular dark-brown mark on the middle of the dorsum ( Figure 5C, E , and G). Some specimens have two circular dark brown blotches on the dorsal region of the pelvic girdle ( Figure 5E ), sometimes continuing the dark stripes of the thigh, tibia, and tarsus. The dark brown inverted m-shaped mark above the cloacal region varies in size and darkness from individual to individual. The lateral dark-brown stripe is always present but can be very noticeable ( Figure 5C ) or slightly distinct ( Figure 5E ). In addition to yellow blotches in lateral view, some specimens also have red blotches on the dorsal surfaces of limbs ( Figure 5C and 5G ). The general ventral pattern is brown, varying in the amount of white, yellow, and red blotches. The inverted v-shaped mark in the chest can be discrete ( Figure 5H ) or dark brown accompanied by yellow and white blotches ( Figure 5B, 5D, and 5F ). In ventral view, some individuals present the inverted v-shaped mark around the cloacal region, clearly distinguishable as dark brown with white outline ( Figures 5B and 5D ), while in other specimens this mark is just slightly distinct ( Figure 5H ).

We found slight structural and temporal differences in the advertisement call parameters among the three analyzed localities, which are most probably related to intraspecific variation ( Table 2). On average, the advertisement calls of males from Castelo dos Bugres are longer when compared to the other two populations (p <0.01, F = 12.86, TukeyHSD tests p = 0.02 and p <0.01, respectively) and males from Parque Estadual da Ilha do Cardoso had higher values on dominant and maximum frequencies (p <0.01, F = 31, TukeyHSD tests p <0.01). In spite of these differences, the main call structure of B. sulfuratus sp. nov. is preserved and the range of divergent call parameters overlapped among the analyzed populations ( Table 2).

Call (s) Interval (s) Notes/call Notes/s Note (ms) Pulses/ note A 1.7±0.1 4.5±1.7 4.9±0.4 0.2±0.1 177±12 8.25±0.8 (1.5–1.8) (3.1–7.4) (4–6) (0.2–0.3) (131–205) (7–9)

B 2.2±0.1 5.9±0.8 5.6±0.6 0.2±0.1 202±14 8.5±1.5 (2.1–2.3) (5.2–6.7) (5–7) (0.1–0.2) (180–233) (7–10) C 1.8±0.1 5.2±1.2 5.1±0.5 0.2±0.1 206±10 9.4±1.2 (1.7–2.1) (3.7–7.0) (4–6) (0.1–0.2) (171–228) (8–11)

continued.

Pulses/s Pulse (ms) Min Freq (Hz) Max Freq (Hz) Dom Freq (Hz) A 9.1±0.9 25±5 4.9±0.3 9.1±0.2 6.6±0.1 (8.5–10.7) (20–30) (4.5–5.2) (9.0–9.4) (6.5–6.7) B 7.6±1.4 20±0 5.1±0.4 8.2±0.0 6.4±0.2 (6.1–9.0) (20–20) (4.7–5.5) (8.2–8.2) (6.2–6.6) C 10.1±1.9 24±5 5.0±0.3 9.9±0.3 6.9±0.2 (7.7–12.3) (20–30) (4.5–5.4) (9.4–10.3) (6.7–7.2) Comparisons with other species. Brachycephalus sulfuratus sp. nov. shares the diminutive size and reduced number of functional toes with other species of Brachycephalus. The texture of the skin of the head and dorsum of B. sulfuratus sp. nov. is smooth, and without dermal ossification. This characteristic distinguishes the new species from B. ephippium, B. garbeanus, and B. margaritatus, all of which have the largest SVLs in the genus (pers. observation) and the most extreme condition of hyperossification, as revealed by the presence of a dorsal bony shield ( Clemente-Carvalho et al. 2009). Brachycephalus sulfuratus sp. nov. also differs from B. alipioi, B. atelopoide, B. bufonoides, B. crispus, B. guarani, B. nodoterga, B. pitanga, B. vertebralis and B. toby, species with intermediate condition of hyperossification of the skull and skeleton ( Clemente-Carvalho et al. 2009; Haddad et al. 2010; Pombal 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014). The lack of hyperossification is shared among the new species and B. auroguttatus, B. boticario, B. brunneus, B. didactylus , B. ferruginus, B. fuscolineatus, B. hermogenesi , B. izecksohni, B. leopardus, B. mariaterezae, B. olivaceus, B. pernix, B. pombali, B. pulex , B. quiririensis, B. tridactylus, and B. verrucosus ( Clemente-Carvalho et al. 2009; Napoli et al. 2011; Ribeiro et al. 2015; Pie & Ribeiro 2015). Except for B. didactylus , B. hermogenesi , B. pulex , and B. sulfuratus sp. nov., all of these species have a “bufoniform” body and in most of them the body background color is orange. The fleatoads, B. didactylus , B. hermogenesi , B. pulex , and B. sulfuratus sp. nov., have the smallest SVLs within the genus, a “leptodactyliform” body, and a brown general background color (pers. observation). The main morphological differences among these species are related to the loss of phalangeal elements and the reduced number of toes. Brachycephalus sulfuratus sp. nov. has the toe I externally absent and toes II, III, IV, and V distinct and functional. This condition is very distinct from B. pulex , which presents toes I, II, and V absent, and toes III and IV distinct and functional ( Napoli et al. 2011). Brachycephalus didactylus is easily distinguished from B. sulfuratus sp. nov. by having toe I absent, toes II, III, and IV distinct and functional, and toe V vestigial ( Izecksohn 1971). Toes of B. hermogenesi have the same configuration as B. sulfuratus sp. nov. (see Giaretta & Sawaya 1998). According to the original description of B. hermogenesi the tip of toe II is pointed, which could be considered distinct from the new species. However, after analyzing several individuals of both species we conclude that this character is variable (being rounded or slightly pointed) and not informative.

The general color of Brachycephalus sulfuratus sp. nov. in life and preservative is very distinct from all currently known species of Brachycephalus, except for the flea-toads B. didactylus , B. hermogenesi , and B. pulex . These species exhibit a brown general body color with variable dorsal and ventral patterns of dark ornamentation ( Izecksohn 1971; Giaretta & Sawaya 1998; Napoli et al. 2011). The m-shaped mark around the cloacal opening in dorsal view, the ventral inverted v-shaped mark in the chest, and variable patterns of stripes on the legs are shared among the four species.

The new species differs from B. didactylus , B. hermogenesi , and B. pulex by having (in life) yellow blotches on the ventral surfaces of the throat, chest, arms, and forearms. Another small differences among the flea-toads relate to the x-shaped dorsal mark, which is diagnostic for B. pulex ( Napoli et al. 2011) and can be slightly visible in B. didactylus ( Izecksohn 1971) and some B. hermogenesi specimens ( Giaretta & Sawaya 1998). In the specimens of B. sulfuratus sp. nov. the irregular mark on the middle of the dorsum and the two circular dark blotches on the dorsal view of pelvic girdle are coincident with the x-shaped dorsal mark, though in this species this mark is not clearly distinguished. Another remarkable feature of the new species is the singular inverted v-shaped mark around the cloacal region in ventral view ( Figure 6A ), which is not clearly distinguishable in B. pulex ( Napoli et al. 2011) and generally rounded and not ornamented in B. didactylus ( Izecksohn 1971) and B. hermogenesi ( Giaretta & Sawaya 1998; Figure 6C ). The ornamented marks around the cloacal region of B. sulfuratus sp. nov. are usually sharper when compared to B. hermogenesi ( Figure 6 ). The m-shaped mark around the cloacal opening, which is dark and defined in B. sulfuratus sp. nov. ( Figure 6B ), is present but not clearly defined in B. hermogenesi ( Figure 6D ).

The main structure of the advertisement call of B. sulfuratus sp. nov. is exceptional within Brachycephalus. It differs greatly from B. ephippium, B. pitanga, and B. crispus because it is composed of a set of 4–7 high-frequency notes (dominant frequency = 6.7 kHz), repeated regularly. In B. ephippium, B. pitanga, and B. crispus, the advertisement call is short and simple, characterized by one low-frequency note (dominant frequencies around 5.0 kHz) with 5–20 pulses ( Pombal et al. 1994; Araújo et al. 2012; Condez et al. 2014). In the same way, the advertisement call of B. sulfuratus sp. nov. also differs from that of B. tridactylus, which is composed of a set of 1– 3 lower frequency notes (dominant frequency = 4.8 kHz) with 1–3 pulses ( Garey et al. 2012). The advertisement call of B. hermogenesi is the most similar to the new species, being quite similar in frequency (dominant frequency = 6.8 kHz), which are the highest recorded for the genus. However, the advertisement call of B. hermogenesi can be simple or composed of 2–7 shorter notes with 1–3 pulses ( Verdade et al. 2008).

Natural history. Specimens were found amidst the leaf-litter in ombrophilous forests. In spite of the wide geographic and elevational distribution, specimens were found in very similar environmental conditions. All localities are remnants of primary forests in coastal mountain ranges and a dense layer of roots and leaf-litter generally covers the forest floor. All captured specimens were found in the rainy season, during the day, at air temperatures of 20–26 ° C. Males were found calling under the leaf-litter on cloudy and rainy days. The reproductive activity seems to occur throughout the year.

Distribution. The new species occurs in the States of São Paulo, Paraná, and Santa Catarina and it is apparently widely distributed, occurring from the sea level up to 1000 m ( Figure 7 ).

Etymology. The specific name sulfuratus refers to the adjective derived from the Latin word sulfur, for the chemical element. The adjective is used in reference to the yellow-lemon blotches on the pectoral skin of the new species.

Molecular analysis. The implicit enumeration search resulted in two most parsimonious trees of 842 steps, conflicting only in the internal relationships among B. sulfuratus sp. nov. specimens and the position of B. alipioi within the group of species that present the intermediate condition of hyperossification (B. alipioi, B. pitanga, B. vertebralis, B. nodoterga, and B. toby). Our topology is congruent with the previous phylogenetic hypotheses in recovering two major sister clades ( Figure 8 ; see Clemente-Carvalho et al. 2011; Padial et al. 2014). One clade is composed of species distributed in southern Atlantic Forest (B. izecksohni, B. brunneus, B. ferruginus, B. pombali, and B. pernix), and the other is composed of species from northern Atlantic Forest ( B. didactylus , B. ephippium, B. garbeanus, B. alipioi, B. nodoterga, B. pitanga, B. vertebralis, and B. toby). The flea-toads B. didactylus , B.

hermogenesi , and B. sulfuratus sp. nov. were not recovered as a clade. Brachycephalus didactylus is nested among the species from the second major clade, while the position of B. hermogenesi and B. sulfuratus sp. nov. was not resolved. The populations of the new species are clustered in a monophyletic group. Despite of the uncertainty of the position of B. hermogenesi , the genetic distances between this species and B. sulfuratus sp. nov. range from 0.169–0.170 (average = 0.170, SD = 0), corroborating the distinction between these two species. We found also low genetic distances among the analyzed specimens of B. sulfuratus sp. nov., with maximum values corresponding to 0.001 between the specimen from Parque Estadual da Ilha do Cardoso and the other three specimens.

Remarks. Brachycephalus has been a target species for herpetological surveys of mountainous areas in the Atlantic Forest due to its restricted geographic distribution and the extraordinary number of species that are expected to be discovered (Ribeiro et al. 2015; Pie & Ribeiro 2015). Even so, the diminutive size, inconspicuous color and discrete habits of flea-toads, seem to have hampered a better understanding of their species diversity. Interestingly, and unlike most other known species within this genus, flea-toads can be widely distributed. Brachycephalus didactylus and B. hermogenesi are known from more than five localities (see Appendix 1), and cover a wide elevational gradient ranging from the sea level up to 1000 m ( Izecksohn 1971; Pimenta et al. 2007; Verdade et al. 2008; Oliveira et al. 2012). In the same way, B. sulfuratus sp. nov. is known from six localities and its distribution also covers a wide elevational gradient. Brachycephalus pulex , the northernmost species, is an exception among flea-toads, being known only from type locality at 840 meters above sea level ( Napoli et al. 2011).

Despite recent advances in the taxonomy of Brachycephalus, the knowledge of the natural history and distribution of most species is still rudimentary. Advertisement calls are available for only six species ( Condez et al. 2014), to which we added the distinctive call of B. sulfuratus sp. nov. We also analyzed variation in temporal and spectral parameters among populations of the new species, which will provide useful comparative data for future taxonomic work. Besides the data provided herein for the new species, analyses of intraspecific variation in osteology, external morphology, and gene sequences, in species of Brachycephalus with spatially discontinuous distribution had only been provided for B. ephippium ( Clemente-Carvalho et al. 2008) and B. nodoterga ( Clemente-Carvalho et al. 2015).

Figures

FIGURE 1. Adult female of Brachycephalus sulfuratus sp. nov. in life. (A) Lateral and (B) ventral views. Holotype, CFBH 39137, from Centro de Estudos e Pesquisas Ambientais da Univille, Vila da Glória, distrito do Saí, município de São Francisco do Sul, State of Santa Catarina, Brazil. SVL 9.8 mm. (Photo by T. H. Condez, 14 November 2014).

FIGURE 2. Preserved adult female of Brachycephalus sulfuratus sp. nov. in (A) dorsal and (B) ventral views. Holotype, CFBH 39137. Scale bar 2 mm.

FIGURE 3. Adult female of Brachycephalus sulfuratus sp. nov. Holotype, CFBH 39137. (A) Dorsal and (B) lateral views of head, (C) ventral view of left hand and (D) ventral view of left foot.

FIGURE 4. Advertisement call of Brachycephalus sulfuratus sp. nov, voucher specimen CFBH 39148, recorded at Parque Estadual da Ilha do Cardoso, município de Cananéia, State of São Paulo, 19 December 2013, 14: 25 p. m., air temperature 25 ° C, air relative humidity 87 %. (A) Oscillogram (above) and spectrogram (below) of three consecutive calls, (B) oscillogram (above) and spectrogram (below) of one call composed by the repetition of six notes, and (C) oscillogram (above) and spectrogram (below) of one pulsed note.

FIGURE 5. Color variation in Brachycephalus sulfuratus sp. nov. (A) Dorsolateral and (B) ventral views of CFBH 39148, adult male, from município de Cananéia, State of São Paulo. (C) Dorsolateral and (D) ventral views of CFBH 39142, adult male, from município de São Francisco do Sul, State of Santa Catarina. (E) Dorsolateral and (F) ventral views of CFBH 39138, adult male, from município de São Francisco do Sul, State of Santa Catarina. (G) Dorsolateral and (H) ventral views of CFBH 39145, adult female, from município de Joinville, State of Santa Catarina.

FIGURE 6. Ornamented marks in the cloacal region of Brachycephalus sulfuratus sp. nov. and the morphologically related species Brachycephalus hermogenesi. (A) Ventral view highlighting the inverted v-shaped mark and (B) detail of the cloacal opening in B. sulfuratus sp. nov. (CFBH 39332). (C) Ventral view and (D) detail of the cloacal opening in B. hermogenesi (AAGUFU 0181). Scale bar 2 mm.

FIGURE 7. Distribution of Brachycephalus sulfuratus sp. nov. along the States of São Paulo (SP), Paraná (PR), and Santa Catarina (SC), Brazil. The star represents the type-locality at município de São Francisco do Sul (SC) and circles represent the paratypes occurrence. The referred specimen from São José dos Pinhais (PR), not included as paratype, is represented by the triangle.

FIGURE 8. Topology of one of the two most parsimonious trees (of 842 steps) for the genus Brachycephalus. Branch lengths correspond to the number of unambiguous parsimony transformations. Numbers close to nodes are Jackknife absolute frequencies. Nodes without values indicate frequencies above 70 %. See Appendix 2 for DNA sequences included in this analysis.