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Family Brachycephalidae Brachycephalus bufonoides and B.garbeanus were ressurrected from synonymy with B. ephippium and validated as full species (Pombal, 2010). The species listed as ��� Eleutherodactylus gr. lacteus (Miranda���Ribeiro, 1923)��� by Rocha et al. (2004) is probably meant to be Ischnocnema lactea. This species is problematic, however, and needs to be reviewed in order to define its actual status and geographic distribution (T. Silva���Soares, pers. comm.). As I. lactea was described from a site in the state of S��o Paulo and attribution of this name to other populations at present is considered problematic, we exclude this species from the list pending resolution of its taxonomic status. Ischnocnema nigriventris (Lutz, 1925) was originally described based on specimens from ���Serra de Cubat��o��� (state of S��o Paulo) and���Itatiaia��� (state of Rio de Janeiro) (Lutz, 1925). Presumably based on the latter record, the species was included in the list of Rocha et al. (2004). Since then, Berneck et al. (2013) have reviewed the species based on the existing syntypes and on newly collected material, concluding that I. nigriventris occurs only in the state of S��o Paulo. Berneck et al. (2013) argue that the record from Itatiaia was based on a specimen that is presently lost, and apparently not conspecific with the remaining syntypes (as previously suggested by Heyer, 1985). Thus, we herein remove this species from the list of amphibians occurring in the state of Rio de Janeiro., Published as part of Dorigo, Thiago Arnt, Vrcibradic, Davor & Rocha, Carlos Frederico Duarte, 2018, The amphibians of the state of Rio de Janeiro, Brazil: an updated and commented list, pp. 1-11 in Pap��is Avulsos de Zoologia 58 on pages 5-6, DOI: 10.11606/1807-0205/2018.58.05, http://zenodo.org/record/4614090, {"references":["Lutz, A. 1925. BatraciensduBresil. ComptesRendusetMemoiresHebdomadaires des Seances de la Societe de Biologie et des ses Filiales, Paris, 93: 137 - 139.","Heyer, W. R. 1985. New species of frogs from Boraceia, Sao Paulo, Brazil. Proceedings of the Biological Society of Washington, 98: 675 - 671."]}

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. 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. 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. 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 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). 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. 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 va

Brachycephalus is a frog genus endemic to the Brazilian Atlantic Forest and characterized by the bright yellow-orange aposematic colors and the high degree of miniaturization. Herein, we describe a new species of Brachycephalus from Serra do Brigadeiro, Municipality of Ervália, Minas Gerais State, southeastern Brazil. Specimens were collected at high altitudes (i.e., 1266–1498 m above sea level) amidst the leaf litter. The new species is characterized by the presence of black connective tissue covering all dorsal muscles, body completely yellow-orange in life, presence of skull and post-cranial plates, large size (SVL of adults: 14.8–18.5 mm), bufoniform body, absence of metacarpal and metatarsal tubercles, and presence of harmonics in its advertisement call.

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 Care��o, Municipality of Erv��lia, Minas Gerais State, Brazil, on 17 December 2015 by C.S. Guimar��es, 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. Guimar��es, P.C. Rocha and R.N. Feio. MZUFV 16491, 16579 (males), and 16780 (female) collected on 13 August 2015 by C.S. Guimar��es, 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. Guimar��es, 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. Guimar��es, C.L. Assis and R.N. Feio. Specimens collected at ��� Mata do Pai In��cio ���, 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. 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. 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, Ara��jo 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. 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 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). 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. 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 va, Published as part of Guimar��es, Carla Silva, Luz, Sofia, Rocha, Pedro Carvalho & Feio, Renato Neves, 2017, The dark side of pumpkin toadlet: a new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Brigadeiro, southeastern Brazil, pp. 327-344 in Zootaxa 4258 (4) on pages 329-341, DOI: 10.11646/zootaxa.4258.4.2, http://zenodo.org/record/570094, {"references":["Dayrell, J. S., Oliveira, E. F., Cassini, C. S. & Feio, R. N. (2006) Brachycephalus ephippium (Pumpkin Toadlet): geographic distribution. Herpetological Review, 37, 357.","Moura, M. R., Motta, A. P., Fernandes, V. D. & Feio, R. N. (2012) Herpetofauna da Serra do Brigadeiro, um remanescente de Mata Atlantica em Minas Gerais, Brasil. Biota Neotrop, 12 (1), 209 - 235.","Condez, T. H., Clemente-Carvalho, R. B. G., Haddad, C. F. B. & dos Reis, S. F. (2014) A new species of Brachycephalus (Anura: Brachycephalidae) from the highlands of the Atlantic Forest, Southeastern Brazil. Herpetologica, 70, 89 - 99. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 13 - 00044","Verdade, K. V., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. C. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta and Sawaya, 1998) (Anura, Brachycephalidae). Journal of Herpetology, 42, 542 - 549.","Araujo, C. B., Guerra, T. J., Amatuzzi, M. C. O. & Campo, L. A. (2012) Advertisement and territorial calls of Brachycephalus pitanga (Anura: Brachycephalidae). Zootaxa, 3302, 66 - 67.","Garey, M. V., Lima, A. M. X., Hartmann, M. T. & Haddad, C. F. B. (2012) A new species of miniaturized toadlet genus Brachycephalus (Anura: Brachycephalidae), from southern Brazil. Herpetologica, 68, 266 - 271. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00074.1","Campos, L. A., da Silva, H. R. & Sebben, A. (2010) Morphology and development of additional bony elements in the genus Brachycephalus (Anura: Brachycephalidae). Biological Journal of the Linnean Society, 99, 752 - 767. https: // doi. org / 10.1111 / j. 1095 - 8312.2010.01375. x","Toledo, L. F., Martins, I. A., Bruschi, D. P., Passos, M. A., Alexandre, C. & Haddad, C. F. (2015) The anuran calling repertoire in the light of social context. Acta Ethologica, 18, 87 - 89.","Clemente-Carvalho, R. B. G., Klaczko, J., Perez, S. I., Alves, A. C. R., Haddad, C. F. B. & dos Reis, S. F. (2011) Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae). Molecular Phylogenetics and Evolution, 61, 79 - 89.","Bornschein, M. R., Firkowski, C. R., Belmonte-Lopes, L. C., Ribeiro, L. F., Morato, S. A. A., Antoniazzi-Jr., R., Reinert, B. L., Meyer, A. L. S., Cini, F. A. & Pie, M. R. (2016 a) Geographical and altitudinal distribution of Brachycephalus (Anura: Brachycephalidae) endemic to the Brazilian Atlantic Rainforest. PeerJ, 4, e 2490,","Alves, A. C. R., Sawaya, R., Reis, S. F. & Haddad, C. B. F. (2009) A new species of Brachycephalus (Anura: Brachycephalidae) from the Atlantic rain forest in Sao Paulo state, southeastern Brazil. Journal of Herpetology, 43, 212 - 219. https: // doi. org / 10.1670 / 0022 - 1511 - 43.2.212","Clemente-Carvalho, R. B. G., Giaretta, A. A., Condez, T. H., Haddad, C. F. B. & dos Reis, S. F. (2012) A new species of miniaturized toadlet, genus Brachycephalus (Anura: Brachycephalidae), from the Atlantic Forest of southeastern Brazil. Herpetologica, 68, 365 - 374.","Haddad, C. F. B., Alves, A. C. R., Clemente-Carvalho, R. B. G. & Reis, S. F. (2010) A new species of Brachycephalus from Atlantic Rain Forest in Sao Paulo State, Southeastern Brazil (Amphibia: Anura: Brachycephalidae). Copeia, 2010, 410 - 420. https: // doi. org / 10.1643 / CH- 09 - 102","Pie, M. R. & Ribeiro, L. F. (2015) A new species of Brachycephalus (Anura: Brachycephalidae) from the Quiriri mountain range of southern Brazil. PeerJ, 3, e 1179."]}

Brachycephalus is a fascinating genus of miniaturized frogs endemic to the Brazilian Atlantic Forest (Bornschein et al. 2016a). Given the many new species that have been recently discovered (20 species over the past 10 years [Frost 2017]) and their often microendemic distribution (see Bornschein et al. 2016a), there has been an increasing awareness about the need for more extensive field work to locate additional new species, to describe their geographical distributions, and to devise comprehensive efforts to ensure their conservation. In a recent correspondence in Zootaxa, Condez et al. (2017) singled out nine of those new species that have been described by our research group (Ribeiro et al. 2015; Pie & Ribeiro 2015; Bornschein et al. 2016b); two additional species (Ribeiro et al. 2017) were described after their paper was published. According to Condez et al. (2017), our species descriptions included “inadequate diagnoses, which lacked indispensable information for any further comparisons among species” (p. 395). Herein, we explore the extent to which their arguments would undermine the validity of those species.

Made available in DSpace on 2020-12-10T16:58:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundacao Grupo Boticario de Protecao a Natureza Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The advent of next-generation sequencing allows researchers to use large-scale datasets for species delimitation analyses, yet one can envision an inflection point where the added accuracy of including more loci does not offset the increased computational burden. One alternative to including all loci could be to prioritize the analysis of loci for which there is an expectation of high informativeness. Here, we explore the issue of species delimitation and locus selection with montane species from two anuran genera that have been isolated in sky islands across the southern Brazilian Atlantic Forest: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). To delimit species, we obtained genetic data using target enrichment of ultraconserved elements from 32 populations (13 for Melanophryniscus and 19 for Brachycephalus), and we were able to create datasets that included over 800 loci with no missing data. We ranked loci according to their number of parsimony-informative sites, and we performed species delimitation analyses using BPP with the most informative 10, 20, 40, 80, 160, 320, and 640 loci. We identified three types of phylogenetic node: nodes with either consistently high or low support regardless of the number of loci or their informativeness and nodes that were initially poorly supported where support became stronger as we included more data. When viewed across all sensitivity analyses, our results suggest that the current species richness in both genera is likely underestimated. In addition, our results show the effects of different sampling strategies on species delimitation using phylogenomic datasets. Univ Fed Parana, Dept Zool, BR-81531980 Curitiba, Parana, Brazil Mater Nat Inst Estudos Ambientais, BR-80250020 Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No,Parque Bitaru, BR-11330900 Sao Paulo, Brazil Pontificia Univ Catolica Parana, Escola Ciencias Vida, BR-80215901 Curitiba, Parana, Brazil Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA Louisiana State Univ, Museum Nat Sci, Baton Rouge, LA 70803 USA Occidental Coll, Moore Lab Zool, 1600 Campus Rd, Los Angeles, CA 90041 USA Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No,Parque Bitaru, BR-11330900 Sao Paulo, Brazil Fundacao Grupo Boticario de Protecao a Natureza: 0895_20111 Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014 CNPq: 301636/2016-8

We present the first record of Ischnocnema octavioi from São Paulo state, Brazil. Until now, the species was thought to be endemic to Rio de Janeiro, where it was known from fewer than 10 localities. Based on recent data, we recommend that the IUCN Red List status for this species be re-evaluated.

Herein we provide new records of the leaf litter frogs Ischnocnema verrucosa and I. surda for the state of Minas Gerais, Southeast of Brazil. Our records fill the current gap in the geographic distribution of I. verrucosa in the east of Minas Gerais, and extend the distribution of I. surda in about 117 km straight southeast of its type locality. We also provide discussions regarding diagnose characters of I. surda.

Here we describe a new species of the Ischnocnema parva species series from the Parque Estadual do Desengano, in the northern part of the State of Rio de Janeiro, Brazil. We use morphology and mtDNA sequences to allocate the new species into the I. parva species series. The new species is closely related to I. parva genetically and morphologically, from which it is diagnosable by (1) the presence of a well-developed calcar tubercle, (2) a reduced Toe I, (3) a deep V-shaped median slit on the dorsal surface of toe discs, and (4) externally indistinct tympanum and tympanic annulus.

Made available in DSpace on 2018-12-11T17:11:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Deutscher Akademischer Austauschdienst Fundação de Amparo à Pesquisa do Estado da Bahia Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Katholischer Akademischer Ausländer-Dienst The Atlantic Forest (AF) of Brazil has long been recognized as a biodiversity conservation hotspot. Despite decades of studies the species inventory of this biome continues to increase with the discovery of cryptic diversity and the description of new species. Different diversification mechanisms have been proposed to explain the diversity in the region, including models of forest dynamics, barriers to gene flow and dispersal. Also, sea level change is thought to have influenced coastal diversification and isolated populations on continental islands. However, the timing and mode of diversification of insular populations in the AF region were rarely investigated. Here, we analyze the phylogeography and species diversity of the small-sized direct-developing frog Ischnocnema parva. These frogs are independent from water bodies but dependent on forest cover and high humidity, and provide good models to understand forest dynamics and insular diversification. Our analysis was based on DNA sequences for one mitochondrial and four nuclear genes of 71 samples from 18 localities including two islands, São Sebastião, municipality of Ilhabela, and Mar Virado, municipality of Ubatuba, both in the state of São Paulo. We use molecular taxonomic methods to show that I. parva is composed of six independently evolving lineages, with the nominal I. parva likely endemic to the type locality. The time-calibrated species tree shows that these lineages have diverged in the Pliocene and Pleistocene, suggesting the persistence of micro-refuges of forest in the AF. For the two insular populations we used approximate Bayesian computation to test different diversification hypotheses. Our findings support isolation with migration for São Sebastião population, with ∼1 Mya divergence time, and isolation without migration for Mar Virado population, with ∼13 Kya divergence time, suggesting a combination of different processes for diversification on AF islands. American Museum of Natural History Herpetology, Central Park West at 79th St. Zoological Institute Division of Evolutionary Biology University of Technology of Braunschweig, Mendelssohnstr. 4 Departamento de Ensino Instituto Federal de Educação Ciência e Tecnologia de Mato Grosso – IFMT Universidade Estadual de Santa Cruz Departamento de Ciências Biológicas Ilhéus Universidade Federal de Mato Grosso do Sul Centro de Ciências Biológicas e da Saúde Laboratório de Zoologia Cidade Universitária Universidade Estadual Paulista (UNESP) Departamento de Zoologia Instituto de Biociências and Centro de Aquicultura (CAUNESP), Campus Rio Claro Universidade Estadual Paulista (UNESP) Departamento de Zoologia Instituto de Biociências and Centro de Aquicultura (CAUNESP), Campus Rio Claro FAPESP: #2013/50741-7

Brachycephalus sulfuratus sp. nov. Figures 1���3 Holotype. CFBH 39137, adult female, collected at Centro de Estudos e Pesquisas Ambientais da Univille, Vila da Gl��ria, Distrito do Sa�� (26 �� 13'39"S, 48 �� 41'31"W, 123 m above sea level, Datum WGS 84), munic��pio de S��o 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 Gl��ria, Distrito do Sa�� (26 �� 10'31"S, 48 �� 42'44"W, 161 m above sea level, Datum WGS 84), munic��pio de S��o Francisco do Sul, State of Santa Catarina, Brazil. CFBH 39145���39147, adult females, collected at Castelo dos Bugres, ��rea de Prote����o Ambiental Dona Francisca (26 �� 13'43"S, 49 �� 03'27"W, 835 m above sea level, Datum WGS 84), munic��pio 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), munic��pio de Canan��ia, State of S��o 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), munic��pio de Quatro Barras, State of Paran��, 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), munic��pio de Canan��ia, State of S��o Paulo, on 22 January 1979, by an unknown collector. ZUEC 16602, juvenile, collected at ��rea de Prote����o Ambiental de Guaratuba (25��47��� S, 48��54��� W, 291 m above sea level), munic��pio de S��o Jos�� dos Pinhais, State of Paran��, 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 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 (x��=1.8��0.2) and the interval between calls is 3.1���7.4 seconds (x��=5.1��1.4). The call is composed of 4���7 notes (x��=5.3��0.9), repeated in a rate of 0.1���0.3 notes/second (x��=0.2��0.0). Notes last 131���233 milliseconds (x��=195��13) and present 7���11 pulses (x��=8.8��1.3). Pulses last 20���30 milliseconds (x��=23.6��4.8) and are repeated at a rate of 6.1���12.3 pulses/second (x��=9.3��1.8). The minimum frequency is 4.5���5.5 kHz (x��=4.9��0.3), the maximum frequency is 8.2���10.3 kHz (x��=9.3��0.3), and the dominant frequency is 6.2���7.2 kHz (x��=6.7��0.3). Variation. Morphometric variation is given in Table 1. In our sample, females are larger than males (SVL of females x��= 9.9 mm ��0.4; SVL of males x��=8.0 mm��0.4, Welch���s t-test t = 10.2, df = 9.6, p p F = 12.86, TukeyHSD tests p = 0.02 and p p F = 31, TukeyHSD tests p 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-fr, Published as part of Condez, Thais Helena, Monteiro, Juliane Petry De Carli, Comitti, Estev��o Jasper, Garcia, Paulo Christiano De Anchietta, Amaral, Ivan Borel & Haddad, C��lio Fernando Baptista, 2016, A new species of flea-toad (Anura: Brachycephalidae) from southern Atlantic Forest, Brazil, pp. 40-56 in Zootaxa 4083 (1) on pages 42-52, DOI: 10.11646/zootaxa.4083.1.2, http://zenodo.org/record/1050887, {"references":["Clemente-Carvalho, R. B. G., Antoniazzi, M. M., Jared, C., Haddad, C. F. B, Alves, A. C. R., Rocha, H. S., Pereira, G. R., Oliveira, D. F., Lopes, R. T. & Reis, S. F. (2009) Hyperossification in miniaturized toadlets of the genus Brachycephalus (Amphibia: Anura: Brachycephalidae): microscopic structure and macroscopic patterns of variation. Journal of Morphology, 270 (11), 1285 - 1295. https: // dx. doi. org / 10.1002 / jmor. 10755","Haddad, C. F. 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(2014) A new species of Brachycephalus (Anura: Brachycephalidae) from the highlands of the Atlantic Forest, southeastern Brazil. Herpetologica, 70 (1), 89 - 99. https: // dx. doi. org / 10.1655 / HERPETOLOGICA-D- 13 - 00044","Napoli, M. F., Caramaschi, U., Cruz, C. A. G. & Dias, I. R. (2011) A new species of flea-toad, genus Brachycephalus Fitzinger (Amphibia: Anura: Brachycephalidae), from the Atlantic rainforest of southern Bahia, Brazil. Zootaxa, 2739, 33 - 40.","Izecksohn, E. (1971) Novo genero e nova especie de Brachycephalidae do estado do Rio de Janeiro, Brasil. Boletim do Museu Nacional, Rio de Janeiro, nova serie, Zoologia, 280, 1 - 12.","Giaretta, A. A. & Sawaya, R. J. (1998) Second species of Psyllophryne (Anura: Brachycephalidae). Copeia, 1998, 985 - 987. https: // dx. doi. org / 10.2307 / 1447345","Pombal, J. P. Jr., Sazima, I. & Haddad, C. F. B. (1994) Breeding behavior of the pumpkin toadlet, Brachycephalus ephippium (Brachycephalidae). 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(2011) Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae). Molecular Phylogenetics and Evolution, 61, 79 - 89. https: // dx. doi. org / 10.1016 / j. ympev. 2011.05.017","Padial, J. M., Grant, T. & Frost, D. R. (2014) Molecular systematics of terraranas (Anura: Brachycephaloidea) with an assessment of the effects of alignment and optimality criteria. Zootaxa, 3825 (1), 1 - 132. https: // dx. doi. org / 10.11646 / zootaxa. 3825.1.1","Pimenta, B. V. S, Bernils, R. S. & Pombal, J. P. Jr. (2007) Amphibia, Anura, Brachycephalidae, Brachycephalus hermogenesi: Filling gap and geographic distribution map. Notes on Geographic Distribution. Checklist, 3 (3), 277 - 279.","Oliveira, J. C. F., Coco, L., Pagotto, R. V., Pralon, E., Vrcibradic, D., Pombal, J. P. Jr. & Rocha, C. F. D. (2012) Amphibia, Anura, Brachycephalus didactylus (Izecksohn, 1971) and Zachaenus parvulus (Girard, 1853): Distribution extension. Checklist, 8 (2), 242 - 244.","Clemente-Carvalho, R. B. G., Monteiro, L. R., Bonato, V., Rocha, H. S., Pereira, G. R., Oliveira, D. F., Lopes, R. T., Haddad, C. F. B., Martins, E. G. & Reis, S. F. (2008) Geographic variation in cranial shape in the pumpkin toadlet (Brachycephalus ephippium): a geometric analysis. Journal of Herpetology, 42 (1), 176 - 185.","Clemente-Carvalho, R. B. G., Perez, S. I., Tonhati, C. H., Condez, T. H., Sawaya, R. J., Haddad, C. F. B. & Reis, S. F. (2015) Boundaries of morphological and molecular variation and the distribution range of a miniaturized froglet, Brachycephalus nodoterga (Anura: Brachycephalidae). Journal of Herpetology. [in press]"]}

We describe a new species of Ischnocnema from the municipality of Arcos, in the karst region of the upper Rio Sao Francisco basin, State of Minas Gerais, Brazil. The new species is diagnosed by the possession of a W-shaped mark on the back, at the level of the shoulder, with one tubercle at each posterior point, red iris in life, and moderate-sized discs on fingers III and IV. These characteristics resemble those of Ischnocnema manezinho and I. sambaqui (from the I. lactea series) but also that from the species in the I. verrucosa series.

We describe a new species of brachycephalid frog from Atlantic forest of Serra do Morato, Municipality of Guaraquecaba, state of Parana, Brazil. Brachycephalus tridactylus sp. nov. is characterized by the absence of external trace of finger IV, orange color in life, dorsolateral and ventrolateral regions with regular small olive-gray spots, and ventral region and thighs orange with olive-grayish irregular coalescent spots and small dots; skin on the dorsum of head and central body dorsum smooth with no dermal co-ossification. The advertisement call of the new species has a single short note that decreases in dominant frequency from beginning to end.

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18. mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate., Facultad de Ciencias Naturales y Museo

A recent study demonstrated marked variation in cranial shape between a population of Brachycephalus ephippium from Jundiai in Sao Paulo state and populations from Atibaia and Sao Francisco Xavier (Sao Paulo) and Macae de Cima in the state of Rio de Janeiro in southeastern Brazil. This result contrasts with earlier work describing differences in cranial shape between the population from Rio de Janeiro and those from Sao Paulo. Here, we investigate the nature and extent of variation between populations of B. ephippium using two lines of evidence. First, we reevaluate patterns of morphological variation by incorporating semi-landmarks into the quantitative description of cranial shape and by using scanning electron microscopy (SEM) to describe qualitative skeletal features. Second, we assess molecular variation in the mitochondrial genes cytochrome b, 12S rRNA, and 16S rRNA, and the nuclear Rag-1 gene, totaling 3,697 base pairs. Geometric analyses of cranial shape and SEM images of skeletal traits ...

Field activities in several localities within the basin of the Doce River, state of Minas Gerais, Brazil, resulted in the collection of a new Ischnocnema that we assign to the I. verrucosa species series. The new species resembles I. verrucosa and I. octavioi by its possession of small digital discs, short legs, conspicuously tuberculate dorsal surfaces, a W-shaped mark between the shoulders with two large tubercles at the bases, and a red iris with a vertical black bar in life. It differs from I. verrucosa and I. octavioi by its possession of externally indistinct tympanum and tympanic annulus and visible white glandular-appearing nuptial pads in males. We also assign I. octavioi and I. penaxavantinho to the I. verrucosa species series.

We describe a new species of Ischnocnema from Santa Teresa, in the Atlantic Rainforest of the State of Espirito Santo, Brazil. Ischnocnema abdita sp. nov. is a member of the Ischnocnema lactea species series; it differs from other Ischnocnema mainly by presenting small size (adult male SVL 15.7 ±0.49 mm; range 15.0–16.8 mm); small discs on fingers I and II, and elliptical, wider than long, moderate sized discs on fingers III and IV; and finger I slightly smaller than finger II. The new species closely resembles Ischnocnema bolbodactyla, differing from it by presenting upper and lower margins of iris red in life; inguinal region with many yellow blotches in life; posterior region of belly and ventral surfaces of thighs, shanks, and feet roughly marbled with large pale yellow blotches; and hidden dorsal area of thighs dark brown with few large pale yellow blotches.

We describe a new toadlet species of the genus Brachycephalus from the Atlantic rain forest in the State of Sao Paulo, in southeastern Brazil. Specimens were collected from the leaf litter at approximately 750 m above sea level. The new species differs from its congers by the following combination of characteristics: SVL in adult males 11.3–12.1 mm and SVL in adult females 13.4–14.2 mm, skin on head and dorsum with co-ossification, general color in life orange with dorsal greenish irregular markings covered with small dark spots and dots, skull hyperossified, spinal processes of sacral and pre-sacral vertebrae hyperossified, and hyperossification of the distal end of the process of the fourth vertebra. The new species shares with most other Brachycephalus species remarkable traits, such as miniaturization and hyperossification of the skeleton. The new species probably occurs in isolation in highlands of the Atlantic Forest.

We studied the anuran amphibians from RPPN Campo Escoteiro Geraldo Hugo Nunes. The region is located in the Municipality of Guapimirim, State of Rio de Janeiro, in Southeastern Brazil and represents a lowland Atlantic Rainforest remaining, which has 45.2 ha. The hydromorphic soil is conducive to the occurrence of permanent and temporary wetlands. For the study, we carried out field expeditions since the 1980's, being the collected specimens housed at the zoological collection of the Instituto de Biologia, Universidade Federal do Rio de Janeiro. A total of 40 amphibian species of the order Anura have been found at the study site. The species are distributed in 10 families: Hylidae (N = 23), Bufonidae (4), Leptodactylidae (4), Cycloramphidae (2), Microhylidae (2), and Brachycephalidae, Craugastoridae, Hemiphractidae, Leiuperidae and Strabomantidae with one species each. Data on local reproductive environments of the recorded species are provided.

We describe the extra bony elements, plates, and osteoderms present in species of the genus Brachycephalus. Samples of eight species of Brachycephalus, including seven populations of Brachycephalus ephippium, were examined. The large additional elements associated with the skull (parotic plate) and vertebrae (vertebral and paravertebral plates) all comprise intramembranous bone, similar to that of the frontoparietal or nasal bones of the skull of most of frogs. Additionally, in the dermis of one unnamed species, we discovered and described true osteoderms. We discuss the morphological nature and diversity of theses elements and their importance as evidence of phylogenetic relationship within Brachycephalus. In summary, three distinct conditions of extra bony elements occur in the genus Brachycephalus: (1) bony plates may be present or absent in species of the genus; (2) a few, small bony plates may be developed and these may be represented by (a) paravertebral plates small and restricted to the distal ends of the transverse processes of the presacral IV, (b) parotic plates small and not covering the tops of the squamosals, and (c) ornamented spinal plates on all vertebrae; and (3) well-developed bony plates may be present as (a) paravertebral plates forming a ‘bone-shield’ on the dorsal surface of the trunk, ornamented, and visible through the integument, (b) parotic plates covering the tops of the squamosals, and (c) spinal plates associated with all vertebrae, and ornamented on vertebrate I–VI. Although the phenomenon of miniaturization may be associated with the appearance of new elements in at least some of the species in the genus, the traditional rule may not be universally applicable. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 752–767. ADDITIONAL KEYWORDS: plates – Brachycephalus ephippium – miniaturization – osteoderm – osteology.

Herein we describe two new species of the Ischnocnema lactea species series. The new species were collected in Itatiaia highlands, located in Itatiaia National Park, southeastern Brazil. Ischnocnema melanopygia sp. nov. is characterized by dorsal stripes generally present; dark stripe on the cloacal area, tarsus and feet; calcar well developed; green yellowish and dark coloration on the inguinal region and posterior area of thigh in life. Ischnocnema concolor sp. nov. is characterized by absence of calcar; presence of well developed supernumerary tubercles on hands and feet; dorsum smooth, brown in preservative, without any observable pattern. These species are allocated in the Ischnocnema lactea species series and are compared to all other species of the group.

4 Corresponding author. abstract. In this study we describe the morphological variation of Ischnocnema ramagii from a population sample within the Municipality of Salvador, Bahia State, Brazil, and characterize two extremely distinct morphotypes within this taxon (striped vs. non-striped dorsum); we describe its karyotype, and compare the karyotypic structure of the distinct morphotypes recognized herein. Specimens with striped dorsa were less common (13-18%) than those with non-striped dorsa (81-86%), the latter group comprising a mix of discrete color pattern states, whose frequencies were not different among juveniles, males, and females. The PCAs on morphometric data resulted in a strong degree of superposition between striped and non-striped dorsa, for both genders. We encountered a diploid number of 30 chromosomes for both males and females, with chromosome pairs 1 to 15 being telocentric. The karyotypic comparison between specimens with striped and non-striped dorsa did not show any noticeable difference. Nevertheless the karyotype of I. ramagii from Salvador, Bahia, differed from the karyotype described for I. paulodutrai from Ilheus, Bahia, which showed a submetacentric chromosome pair. This result supports the presence of two full "sibling" species in Bahia State, one from the south (I. paulodutrai) and another from the north (I. ramagii), validating the identity of I. paulodutrai as a full species, and refuting the recognition of Salvador samples as I. paulodutrai. The high number of telocentric chromosomes suggests a closer relationship between I. ramagii and "Eleutherodactylinae" species from Central America and the northern region of Brazil.

A new species of brachycephalid frog is described from Sao Luis do Paraitinga, in the Atlantic Rain Forest of Sao Paulo State, southeastern Brazil. The new species is characterized by male SVL = 10.8–12.1 and female SVL = 12.6–14.0 mm; and general color orange with dorsal reddish irregular markings, lateral surfaces with small dark brown spots, and belly with brownish spots and small dots. Comparisons with other brachycephalid species and osteological data are provided.

Brachycephalus hermogenesi is an endemic leaf litter inhabitant of the Atlantic forest of southeastern Brazil, whose original distribution included a restricted area near the boundaries of the States of Sao Paulo and Rio de Janeiro. We were surprised to find out, while conducting herpetofaunal surveys at Estacao Biologica de Boraceia (EBB), that the background forest insect–like sound we have been searching for corresponded to calling individuals of the species. Males call during the day at high densities, hidden under the leaf litter. Individuals do not answer playback, seem to move very infrequently, and seem to ignore nearby calling activity. We gathered data on annual and daily vocal activity of the species at EBB, observing a total of 1,549 calls given by 31 focal individuals in November 2003 and 2005. The call varies from short single note calls to calls composed of groups of two to seven similar notes emitted at regular intervals. We also extend the known distribution of the species southw...

[Cryptic prey may increase likelihood of being detected if they move. When approached by predators, they must assess probabilities of survival if they remain immobile or attempt escape. Probability of survival is the joint probability of being detected, attacked if detected, and captured if attacked. We studied escape responses of several species of cryptic Craugastor (= Eleutherodactylus ) frogs to simulated ground and aerial predators by varying elevation angle of attack. Frogs were predicted to escape more frequently when attacked from higher angles because a predator attacking from above on a trajectory leading directly to the prey is more likely to have detected and be attacking than a predator approaching directly, but horizontally. Frogs approached horizontally rarely jumped. The proportion of frogs that jumped increased as attack angle increased, and the proportion that turned or twitched was greatest for 45° approaches. Distance jumped did not vary among attack angles. Escape was directed away from the object approaching at 45°, but was random with respect to the frog's orientation during vertical approaches. Variation in escape tendency may occur because risk increases as attack angle increases, possibly because risk of being detected from above is greater and/or aerial predators are more efficient., Cryptic prey may increase likelihood of being detected if they move. When approached by predators, they must assess probabilities of survival if they remain immobile or attempt escape. Probability of survival is the joint probability of being detected, attacked if detected, and captured if attacked. We studied escape responses of several species of cryptic Craugastor (= Eleutherodactylus ) frogs to simulated ground and aerial predators by varying elevation angle of attack. Frogs were predicted to escape more frequently when attacked from higher angles because a predator attacking from above on a trajectory leading directly to the prey is more likely to have detected and be attacking than a predator approaching directly, but horizontally. Frogs approached horizontally rarely jumped. The proportion of frogs that jumped increased as attack angle increased, and the proportion that turned or twitched was greatest for 45° approaches. Distance jumped did not vary among attack angles. Escape was directed away from the object approaching at 45°, but was random with respect to the frog's orientation during vertical approaches. Variation in escape tendency may occur because risk increases as attack angle increases, possibly because risk of being detected from above is greater and/or aerial predators are more efficient.]

Three new species of rainfrogs of the genus Pristimantis are described from a large mesa (tepui), the Wokomung Massif, of the Pakaraima Mountains in west-central Guyana. Pristimantis dendrobatoides n. sp. is known from 1385–1411 m, P. jester n. sp. from 1411–1650 m, and P. saltissimus n. sp. from 698–1560 m elevation. The three species are syntopic at elevations around 1400 m in cloud forest. All three taxa are unusual among species of Pristimantis in the production of malodorous and distasteful skin secretions when handled, conditions that are atypical for the genus. Two of the new species (P. dendrobatoides, P. jester) also have bright, red skin coloration, and the third (P. saltissimus) is either cryptically colored or brightly colored.

We describe a new species of Phrynopus (Anura: Brachycephalidae) from two close localities at the upper limits of cloud forest in the southern Peruvian Departamento Cusco, between 3555–3950 m a.s.l. The new species is characterized by having medium size (maximum snout-vent length 23.4 mm), dentigerous processes of vomers absent, tympanic membrane inconspicuous, dorsal skin coarsely shagreen in life, dorsolateral folds, ventral skin areolate, dorsum tan, venter bold black with conspicuous bluish-gray spots, and a bluish-white iris.

We surveyed the morphology of the auditory region of members of the anuran genus Brachycephalus. The sample included seven of the eleven known species; in addition, Brachycephalus ephippium and B . vertebralis were each represented by several specimens from different localities. All species lack a tympanum, tympanic annulus, stapes, and Eustachian tube. However, the operculum and the m . opercularis remain. The large operculum bears a robust process for the m . opercularis and covers most of the fenestra ovalis. The auditory apparatus of Brachycephalus is distinguished by the large size and the orientation of the fenestra ovalis, which faces posterior, rather than lateral. The morphology of these structures is compared to those of other minute brachycephalid frogs considered to be close relatives of Brachycephalus, and the importance of these characters as evidence of the monophyly of the genus is discussed. We also discuss the importance of other morphological features cited to support close relationships among small-sized brachycephalids, and consider the role of miniaturization in generating convergent pattern of loss of morphological features.

We describe a new species of the Eleutherodactylus discoidalis group from cloud forests of the Apurimac and Kosnipata valleys, southern Peru. The new species differs from other species of the group mainly by having a coarsely shagreen dorsum, long and slender hind legs, very long feet, Finger I equal in length to Finger II and fingertips not expanded. The species also lacks supernumerary tubercles on the feet.

A new miniaturized toadled of the genus Brachycephalus (Anura: Brachycephalidae) is described from Serra do Quiriri in the municipality of Campo Alegre, Santa Catarina, southern Brazil. Specimens were collected from the leaf litter between from 1,263 and 1,318 m above sea level. The new species is distinguished from all its congeners by the combination of the following characters: snout–vent length 9.9–13.1 mm; skin on head and dorsum without dermal co-ossification; snout mucronate in dorsal view; dorsum rugose; general color brown, with a narrow orange vertebral stripe. The region where the new species is located is also shared with other endemic anuran species and has experienced strong anthropogenic impacts,suggesting that immediate actions should be taken to ensure their long-term preservation.

Brachycephalus (Anura: Brachycephalidae) is a remarkable genus of miniaturized frogs of the Brazilian Atlantic Rainforest. Many of its species are highly endemic to cloud forests, being found only on one or a few mountaintops. Such level of microendemism might be caused by their climatic tolerance to a narrow set of environmental conditions found only in montane regions. This restriction severely limits the chance of discovery of new species, given the difficulty of exploring these inaccessible habitats. Following extensive fieldwork in montane areas of the southern portion of the Atlantic Rainforest, in this study we describe seven new species of Brachycephalus from the states of Parana and Santa Catarina, southern Brazil. These species can be distinguished from one another based on coloration and the level of rugosity of the skin in different parts of their body. These discoveries increase considerably the number of described species of Brachycephalus in southern Brazil.

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases. Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330 Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues.

11-oxoTTX is an analogue 4–5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H+), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.

Made available in DSpace on 2018-11-26T17:39:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-27 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundacao Grupo Boticario de Protecao a Natureza We describe two new species of miniaturized toadlet in the B. pernix group of Brachycephalus (Anura: Brachycephalidae) from the Atlantic Forest of the state of Parana, southern Brazil. The first new species is distinguished from all congeners by the pale red coloration from the head to the pelvic region, with sides of the body and thighs dorsally yellowish green. It is known only from the type locality in a cloud forest at altitudes ranging between 1,144-1,228 m a.s.l. The second species, although more closely related to B. izecksohni, is morphologically similar to B. brunneus in its overall brown coloration, but distinct from that species in the color of the iris (black with conspicuous golden spots, instead of entirely black). It was found on three mountains, at altitudes between 1,095-1,320 m a.s.l., and in vegetation types including cloud forest, montane forest, and secondary forest. The two new species exhibit neither vertebral fusions nor osteoderms, but one has both a distinct neopalatine and well-developed odontoids on the maxillae. We discuss the conservation status of both species. Pontificia Univ Catolica Parana, Escola Ciencias Vida, Curitiba, Parana, Brazil Mater Natura Inst Estudos Ambientais, Curitiba, Parana, Brazil Univ Florida, Florida Museum Nat Hist, Gainesville, FL 32611 USA Univ Fed Parana, Dept Zool, Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Sao Vicente, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Sao Vicente, SP, Brazil CNPq: 571334/2008-3 CNPq: 301636/2016-8 Fundacao Grupo Boticario de Protecao a Natureza: 0895_20111 Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014

Since the first, and simplest, description of a species of the genus Brachycephalus Fitzinger 1826, by Spix (1824), an increasing number of informative traits has been investigated to facilitate species diagnoses, including those dealing with osteology (Izecksohn 1971), sexual dimorphism in body size (Alves et al. 2006), hyperossification of the skull and skeleton (Haddad et al. 2010) and advertisement call (Garey et al. 2012), plus traits from molecular data (Condez et al. 2016). However, the recent descriptions of nine species of Brachycephalus (see Pie & Ribeiro 2015; Ribeiro et al. 2015; Bornschein et al. 2016) included inadequate diagnoses, which lacked indispensable information for any further comparisons among species. Herein, we intend to address the main flaws of these species descriptions and highlight the urgent need for a rigorous review of the genus Brachycephalus in order to assess the validity of these taxonomic proposals.

Brachycephalidae This taxon includes the sister taxa Brachycephalus and Ischnocnema, a relationship first discovered by Heinicke et al. (2007), who placed most southeastern Brazilian taxa then referred to “ Eleutherodactylus ” (Frost et al., 2006) in a redelimited Ischnocnema. Previously, Caramaschi & Canedo (2006) had restricted Ischnocnema to Ischnocnema verrucosa, and placed it in the synonymy of Eleutherodactylus. A new test of the sister relationship of Ischnocnema and Brachycephalus was recently provided by Canedo & Haddad (2012), who found the taxa to be reciprocally monophyletic in both maximum likelihood and Bayesian analyses. Interestingly, their parsimony analysis of a similarity alignment placed Brachycephalus as the sister of Haddadus binotatus, another taxon from southeastern Brazil. Such a relationship had never been proposed, and although it is not corroborated in our analyses, it surely deserves future attention, particularly with regard to the effects of alignment and optimality criterion. Brachycephalus.—This genus of small, diurnal frogs that usually are brightly colored and contain tetrodotoxin (Pires et al. 2005) is endemic to southeastern Brazil. Its unique pectoral girdle has long been recognized as a synapomorphy of the genus (Izecksohn, 1971; Pombal & Gasparini, 2006) and was used to synonymize Psyllophryne (i.e., P. didactyla and P. hermogenesi) with Brachycephalus by Kaplan (2002), a synonymy subsequently validated by analyses of DNA sequences that found the two species to be nested among other species of Brachycephalus (Clemente-Carvalho et al., 2011; current results). Frost et al. (2006), Heinicke et al. (2007), Hedges et al. (2008a), and Pyron & Wiens (2011) only included B. ephippium in their analyses, and Canedo & Haddad (2012) added B. cf. didactylus. Clemente-Carvalho et al. (2011) provided the first extensive molecular phylogenetic analysis of the genus by including 14 of the 18 currently recognized species (Pombal & Izecksohn, 2011). Inadequate outgroup sampling (the tree was rooted on a single terminal of Ischnocnema) prevented the monophyly of Brachycephalus, the position of Psyllophryne within Brachycephaloidea, or the sister relationships with Ischnocnema from being tested in that study. Nevertheless, our results agree with Clemente-Carvalho et al. ’s (2011) Bayesian analysis of the concatenated nuclear and mitochondrial data in recovering two major sister clades, a southern clade formed by locally endemic species restricted to the high and humid Atlantic Forest of Paraná (B. brunneus, B. ferruginus, B. izecksohni, B. pernix, and B. pombali) and the northern clade, allopatric to the first, distributed from farther north in Paraná to Espírito Santo (B. alipioi, B. didactylus, B. ephippium, B. garbeanus, B. hermogenesi, B. nodoterga, B. pitanga, B. toby, and B. vertebralis), with the two species of the former Psyllophryne placed separately in the second clade. Ischnocnema.— This genus is distributed within the Atlantic Forest of south, southeastern, and northeastern Brazil and Misiones, Argentina (Canedo & Haddad, 2012; Frost, 2014). The monophyly of the genus was hypothesized by Heinicke et al. (2007) and Hedges et al. (2008a), who analyzed 5 of the 33 species. A more complete test of generic monophyly was recently provided by Canedo & Haddad (2012) in a study that was published while the present manuscript was being written, which unfortunately precluded the use of their data in our analyses. Nevertheless, their taxon sampling of other brachycephalids was large enough (214 species) to provide a strong test of the monophyly of Ischnocnema. Their major finding was a polyphyletic Ischnocnema sensu Hedges et al. (2008a) with three named species (I. paulodutrai, I. ramagii, and I. vinhai) and two unnamed species recovered as part of the Pristimantis conspicillatus species group. Also, Ischnocnema bilineata was found to be the sister of a clade composed of the genera Noblella and Barycholos in Holoadeninae. Accordingly, Canedo & Haddad (2012) placed three nominal species of Ischnocnema in Pristimantis, but left " Eleutherodactylus " bilineatus Bokermann, 1975 "1974" as incertae sedis within Holoadeninae 6 (of Craugastoridae, below). For the remaining species of Ischnocnema, they recognized four monophyletic species series and left two species unassigned to any group, although five species were only tentatively assigned to groups based on their morphology. We follow their assignment of species to species series (see Appendix 2)., Published as part of Padial, José M., Grant, Taran & Frost, Darrel R., 2014, Molecular systematics of terraranas (Anura: Brachycephaloidea) with an assessment of the effects of alignment and optimality criteria, pp. 1-132 in Zootaxa 3825 (1) on pages 50-51, DOI: 10.11646/zootaxa.3825.1.1, http://zenodo.org/record/4920347, {"references":["Heinicke, M. P., Duellman, W. E. & Hedges, S. B. (2007) Major Caribbean and Central American frog faunas originated by oceanic dispersal. Proceedings of the National Academy of Sciences, USA, 104, 10092 - 10097. http: // dx. doi. org / 10.1073 / pnas. 0611051104","Frost, D. R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C. F. B., De Sa, R. O., Channing, A., Wilkinson, M., Donnellan, S. C., Raxworthy, C. J., Campbell, J. A., Blotto, B. L., Moler, P., Drewes, R. C., Nussbaum, R. A., Lynch, J. D., Green, D. M. & Wheeler, W. C. (2006) The amphibian tree of life. Bulletin of the American Museum of Natural History, 297, 1 - 370. http: // dx. doi. org / 10.1206 / 0003 - 0090 (2006) 297 [0001: tatol] 2.0. co; 2","Caramaschi, U. & Canedo, C. (2006) Reassessment of the taxonomic status of the genera Ischnocnema Reinhardt and Lutken, 1862, and Oreobates Jimenez-de-la-Espada, 1872, with notes on the synonymy of Leiuperus verrucosus Reinhardt and Lutken, 1862 (Anura: Leptodactylidae). Zootaxa, 1116, 43 - 54.","Canedo, C. & Haddad, C. F. B. (2012) Phylogenetic relationships within anuran clade Terrarana, with emphasis on the placement of Brazilian Atlantic rainforest frogs genus Ischnocnema (Anura: Brachycephalidae). Molecular Phylogenetics and Evolution, 65, 610 - 620. http: // dx. doi. org / 10.1016 / j. ympev. 2012.07.016","Pires, Jr. O. R., Sebben, A., Schwartz, E. N. F., Morales, R. A. V., Bloch Jr, C. & Schwartz, C. A. 2005. Further report of the occurrence of tetrodotoxin and new analogues in the anuran family Brachycephalidae. Toxicon, 45, 73 - 79. http: // dx. doi. org / 10.1016 / j. toxicon. 2004.09.016","Izecksohn, E. (1971) Novo genero e nova especie de Brachycephalidae do Estado do Rio do Janeiro, Brazil. (Amphibia, Anura). Boletim do Museu Nacional, Nova Serie, Zoologia, 280, 1 - 12.","Pombal, J. P. Jr. & Gasparini, J. L. (2006) A new Brachycephalus (Anura, Brachycephalidae) from the Atlantic Rainforest of Espirito Santo, southeastern Brazil. South American Journal of Herpetology 1, 87 - 93. http: // dx. doi. org / 10.2994 / 1808 - 9798 (2006) 1 [87: anbabf] 2.0. co; 2","Kaplan, M. (2002) Histology of the anteroventral part of the breast-shoulder apparatus of Brachycephalus ephippium (Brachycephalidae) with comments on the validity of the genus Psyllophryne (Brachycephalidae). Amphibia-Reptilia, 23, 225 - 227.","Clemente-Carvalho, R. B. G., Klaczko, J., Perez, I. S., Alves, A. C. R., Haddad, C. F. B. & Reis, S. F. (2011) Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae). Molecular Phylogenetics and Evolution, 61, 79 - 89. http: // dx. doi. org / 10.1016 / j. ympev. 2011.05.017","Hedges, S. B., Duellman, W. E. & Heinicke, M. P. (2008 a) New World direct-developing frogs (Anura, Terrarana), molecular phylogeny, classification, biogeography, and conservation. Zootaxa, 1737, 1 - 182.","Pyron, R. A. & Wiens, J. J. (2011) A large-scale phylogeny of Amphibia including over 2,800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution, 61, 543 - 583. http: // dx. doi. org / 10.1016 / j. ympev. 2011.06.012","Pombal, J. P. Jr. & Izecksohn, E. (2011) Uma nova especie de Brachycephalus (Anura, Brachycephalidae). Papeis Avulsos de Zoologia, Sao Paulo, 51, 443 - 451. http: // dx. doi. org / 10.1590 / s 0031 - 10492007001300001","Frost, D. R. (2014) Amphibian Species of the World, an Online Reference. Version 6.0 American Museum of Natural History, New York, USA. Available at http: // research. amnh. org / herpetology / amphibia / index. html (accessed February 04 2014)"]}

A new species of Brachycephalus is described from Serra da Bocaina, State of Rio de Janeiro, Brazil. The new species is characterized by having, in preservative, body uniformly pale cream, dermal ossification of the vertebrae composed of a bulge forming a row, and absence of ossified warts on the body.

Ischnocnema nigriventris (Lutz, 1925) Hylaplesia nigriventris Lutz 1925: original description, type locality ���Itatiaia and Serra do Cubat��o���. Basanitia nigriventris��� Bokermann, 1966: first combination with Basanitia. Modification of one of the type localities, ���Serra do Cubat��o��� corrected to Paranapiacaba, Santo Andr�� municipality. Eleutherodactylus nigriventris ���Lynch 1968: first combination with Eleutherodactylus. Eleutherodactylus nigroventris��� Lynch 1976: Misspelling for Eleutherodactylus nigriventris. Eleutherodactylus nigriventris��� Heyer, 1985: designation of a lectotype and restriction of the type locality to Paranapiacaba, Santo Andr�� municipality. Eleutherodactylus (Eleutherodactylus) nigriventris��� Lynch & Duellman, 1997. Ischnocnema nigriventris��� Heinicke, Duellman & Hedges, 2007: first combination with Ischnocnema. Lectotype: AL-MN 719, a juvenile male from Paranapiacaba, Santo Andr�� municipality, state of S��o Paulo, Brazil, designated by Heyer (1985). Paralectotypes: AL-MN 721, USNM 96846, juveniles, both from Paranapiacaba, Santo Andr�� municipality, state of S��o Paulo, Brazil. AL-MN 720 juvenile of Ischnocnema guentheri (see Heyer, 1985) from Paranapiacaba, Santo Andr�� municipality, state of S��o Paulo, Brazil., Published as part of Berneck, Bianca V. M., Targino, Mariane & Garcia, Paulo Christiano De Anchietta, 2013, Rediscovery and re-description of Ischnocnema nigriventris (Lutz, 1925) (Anura: Terrarana: Brachycephalidae), pp. 131-142 in Zootaxa 3694 (2) on page 132, DOI: 10.11646/zootaxa.3694.2.2, http://zenodo.org/record/222286

Berneck, Bianca V. M., Targino, Mariane, Garcia, Paulo Christiano De Anchietta (2013): Rediscovery and re-description of Ischnocnema nigriventris (Lutz, 1925) (Anura: Terrarana: Brachycephalidae). Zootaxa 3694 (2): 131-142, DOI: http://dx.doi.org/10.11646/zootaxa.3694.2.2

Re-description of Ischnocnema nigriventris Ischnocnema nigriventris is allocated to the Ischnocnema lactea species series (sensu Canedo and Haddad, 2012). The species series includes species with tibia length Ischnocnema, males 18���20 SVL, the two females are 24 SVL (see Table 1 for measurements); head wider than long; snout short and rounded in profile and nearly rounded in dorsal view (Figure 1); large and prominent eyes, diameter smaller than interorbital distance and 40 % of head length; on the upper eyelid several small and medium tubercles, one of them larger than the others; tympanic membrane undifferentiated and tympanic annulus hidden; supratympanic fold barely visible, starting behind the eye, covering the upper half of tympanic annulus and reaching the arm insertion; canthus rostralis distinct and straight, with sparse, small tubercles; usually two postrictal tubercles; nostril laterally oriented; internostril distance smaller than eye diameter, corresponding to 28 % of head width; vomerine teeth small, in two rows of about five teeth each, lying between and posterior to the rounded choanae; tongue posteriorly free, male vocal slits lateral to the insertion of the tongue, single vocal sac not expanded externally; small premaxillary and maxillary teeth. Arms and fingers slender; Finger length III>IV>II>I; disk of first finger smaller than second; disks of the third and fourth wider than second; except for Finger I, which is rounded and not expanded, the others are expanded and are emarginated with ungual flap notched in dorsal view (see figs. 3 and 4 in Savage 1987); Fringes or webbing absent; palmar and thenar tubercles weakly developed; Finger I and Finger II with only one subarticular tubercle; Finger III and Finger IV with two subarticular tubercles; supernumerary tubercles absent; translucent nuptial pad on Finger I of most adult males (80 %), divided into two parts: one obscures the medial margin of the thenar tubercle and the other occupies the medial margin of the base of the thumb. Legs slender; thigh and tibia slightly longer than SVL in males, and barely shorter than SVL in females; tibia length 50 % of male SVL, and 48 % of female SVL; feet not webbed, and foot length corresponding to 50 % SVL; Toe length IV>V=III>II>I; Toe I disk is rounded and not expanded, the others are expanded emarginated disks, with ungual flap notched in dorsal view; inner metatarsal tubercle large, protruding, and ovoid; outer metatarsal tubercle small, conical and projecting; supernumerary metatarsal tubercles small and sparse; subarticular tubercles conical, projecting forward; calcar tubercle present and well developed. Dorsal skin shagreen with tubercles or warts, ranging in number, belly weakly areolate to areolate, flanks and posterior surface of thigh coarsely areolate. In life, the dorsum is dark brown with lighter or darker brown blotches. Males have bright yellow mottling on a dark background on the inguinal region and hidden areas of hindlimbs, while in females the mottling is bright orange. At night, specimens showed a light brown belly while during daytime those same specimens showed a deeply dark brown belly. The dorsal and ventral areas of the iris are silver, and the medial transverse band is yellowish copper (Fig. 3). In preservative, dorsal coloration ranges from dark brown to light brown, and some specimens may show many sparse patches (Fig 2). The belly and gular region range from light brown to dark brown, always with a lot of sparse dark spots. The most common venter feature is dark and areolate (76 %). The head is brown with lateral beige bars rising from the ventral margin of maxilla to eye ventral portion; sparse beige blotches cover the maxilla region, and a dark brown stripe outlines the supratympanic fold. Some specimens may show a vertebral white line that can be wide (5 %) or slender (29 %) (Fig. 2 A and D). This line extends from snout to vent. Other specimens show a large dorsolateral band starting behind the eyes and reaching the proximal quarter of the thigh, leg, and feet (17 %) (Fig. 2 B and C). Some specimens show a W-shaped dark brown mark between the eyes (Fig. 2 B). A cream interorbital bar is sometimes present (Fig. 2 C and D), as well as a semilunar cream mark at the tip of the snout (Fig. 2 B and D). Some specimens exhibit transversal dark brown stripes in the dorsal surfaces of hind limbs while, in others, the stripes are randomly disposed. The bright yellow and orange regions become white in preservative. All brown coloration becomes lighter brown in preservative. Diagnosis. Ischnocnema nigriventris can be distinguished from other Ischnocnema by the following combination of character states: (1) snout short and nearly rounded in dorsal view and rounded in profile view; (2) head wider than long; (3) prominent conical tubercles on the upper eyelid; (4) first finger shorter than second; (5) disks on fingers II���IV expanded and emarginated, with ungual flap notched in dorsal view; (6) translucent, double glandular nuptial pad on the thumbs of males; (7) vomerine teeth present; (8) single vocal sac not expanded externally; (9) skin on dorsum shagreen with tubercles or warts, flanks, belly and posterior portion of thighs areolate (10) prominent calcar on the heel; tibia length Comparison with other species of Ischnocnema. Ischnocnema nigriventris is distinguished from all other species of the I. lactea species as follows (character states for other members of the series in parenthesis): upper eyelid tubercles large, conical (absent, or present as low and small rounded granules); expanded disks in fingers II��� IV (Finger IV with no expanded disk in I. concolor and I. vizottoi); nuptial pads present (absent in, I. melanopygia, and I. spanios; not reported in I. paranaensis; I. lactea is only known from its female holotype; the condition is unknown for the poorly preserved male holotype of I. gehrti; not reported in the original description of I. vizottoi but observable as a translucent inconspicuous glandular pad); well-developed calcar tubercles (absent in I. concolor, and I. paranaensis; unknown in I. lactea, and I. gerhti whose type specimens are in bad condition); vomerine teeth present (absent in I. randorum and I. paranaensis); palmar tubercles inconspicuous but present (indistinct in I. spanios and I. paranaensis); dorsum shagreen with tubercles or warts (smooth in I. concolor, I. vizottoi, I. melanopygia, I. gehrti, I. lactea, I. paranaensis, I. spanios, and I. randorum); in life, bright yellow or orange mottling or as blotches in the inguinal region and hidden areas of hind limbs (no bright coloration in these areas occur in I. concolor, I. vizottoi, and I. paranaensis; the coloration in life is unknown for I. lactea and I. gehrti; I holti presents red or orange coloration in the inguinal region but not mottled or as blotches); iris silver with a ventral yellowish copper band (iris green in I. holti, dorsally bluish in I. spanios, golden to lemon yellow in I. randorum, I. melanopygia, I. concolor, and I vizottoi, and dark violet in I. lactea; not reported for I. gehrti and I. paranaensis); snout nearly rounded in dorsal view (truncate in I. gehrti, the others are also nearly rounded, rounded or sub-acuminate with no substantial difference; head wider than long (head longer than wide in I. melanopygia, I. randorum, and I. spanios). Ischnocnema nigriventris differs from species in the I. guentheri species series by having a tibia length shorter than 55 % of SVL; snout nearly rounded in dorsal view; disks expanded and emarginated; translucent glandular nuptial pads. (I. guentheri species series: legs with tibia length> 60 % SVL; acuminate snout in dorsal view; disks usually small or slightly expanded, large only in I. hoehnei and I. vinhai; and conspicuous white glandular nuptial pads, unknown in I. vinhai; see Heyer, 1984; Hedges et al. 2008; Canedo et al. 2010). Ischnocnema nigriventris differs from species in the I. parva species series by its shagreen dorsum with tubercles. Finger I smaller than Finger II; disks expanded and emarginated; and translucent and glandular nuptial pads. (I. parva species series: smooth dorsum; Finger I as long as Finger II; small and pointed disks; and conspicuous white glandular nuptial pads [contra Hedges et al. 2008]). Ischnocnema nigriventris differs from the I. verrucosa species series by its rounded snout in dorsal view; Finger I shorter than II; and expanded disks. (I. verrucosa species series: snout sub acuminate in dorsal view; Finger I as long as II; and small disks, Hedges et al. 2008; Canedo et al. 2010). Ischnocnema nigriventris differs from I. manezinho and I. sambaqui, recently removed from the I. lactea species series and not allocated to any series (Canedo and Haddad, 2012), by its smaller size, males 18���20 mm; presence of nuptial pads; and bright coloration in hidden areas in life (bigger size, I. sambaqui males 32���40 mm, and I. manezinho males 22���28 mm; absence of nuptial pads and bright colors in hidden areas in life). Vocalization. Two calls were recorded for two specimens of Ischnocnema nigriventris. The first (considered a territorial call due to continuous and high emission rate) consists of a single, non-pulsed note emitted at regular intervals of 2.8���3.3 seconds (Figure 4). Note duration is 30���41 ms (36.1 �� 3.8 ms, n = 8 calls of two individuals). The frequency spectrum is 2004���3685 Hz and the peak frequency (= dominant frequency) is 2928���3014 Hz (2965 �� 46.0, n = 8 calls of two individuals). The second call (Figure 5), considered here as the advertisement call, shows two to four non-pulsed notes, emitted after long irregular intervals. The duration of the call is 194���565 ms, the frequency spectrum is 1955���3932 Hz, and the peak frequency is 2756���2928 Hz (n = 19 calls of three individuals). The first note differs from the others by its lower intensity (the amplitude of the first note is about 11���33 % lower than the others) and lower frequency range (the first note ranged from 2151���2774 Hz). Note duration is similar, the first note is 30���89 ms (54.2 �� 2.0 ms) and the others are 28���88 ms (41.7 �� 2.0 ms). The interval between the notes is 113���157 ms (132 �� 12 ms). The major differences in call with respect to other Ischnocnema from I. lactea series (sensu Canedo and Haddad, 2012) and I. manezinho and I. sambaqui (former I. lactea series and unassigned to species series in Canedo and Haddad, 2012) are shown in Table 2. I. nigriventris I. nigriventris I. randorum Call 1 Call 2 (advertissment call) (Heyer et al. 1990) Natural history. Ischnocnema nigriventris starts its vocalization activity just before dusk, with its territorial call. The males are usually found perching on trees or shrubs. On some occasions we found males calling from about three meters above the ground. On others, a couple in amplexus was observed moving on the leaf litter. Individuals were found active all year round, even during the dry season (June), although they were more abundant in September during the early stages of the rainy season when the forest was very humid. Two dissected females contained large, unpigmented ovarian ovules. One (CFBH 23478) with 20 ovules, nine in the left ovary and eleven in the right, and the second (MZUSP 136724) with 18 ovules in total. Ovule diameter: 2.5���3.2 (x = 2.8mm). Distribution. Ischnocnema nigriventris is only known from the Parque das Neblinas (Bertioga municipality), Paranapiacaba (Santo Andr�� municipality), and Borac��ia (Sales��polis municipality), all localities in the Serra do Mar, eastern S��o Paulo State, Brazil (Figure 6). Remarks. Lutz (1925) did not designate type specimens in the original description of Hylaplesia nigriventris, only provided a single measurement (SVL 21mm), and mentioned two localities, Serra de Cubat��o and Itatiaia, for the distribution of the species. Cochran (1961) lists one cotype, measuring 6.5 mm total length, from Serra de Cubat��o and deposited in the United States Natural History Museum (USNM 96846). Bokermann (1966) cited two specimens labeled as cotypes from Adolpho Lutz Collection (AL-MN 720 and 721) and corrected the type locality to Paranapiacaba in Santo Andr��, state of S��o Paulo, Brazil. Heyer (1985) found three specimens, labeled as cotypes, in the Adolpho Lutz Collection, AL-MN 719 (SVL 17.8 mm), AL-MN 720 (SVL 8.2 mm), and AL-MN 721 (SVL 7.0 mm), all from Paranapiacaba. The specimen AL-MN 720 was identified as a juvenile of Eleutherodactylus guentheri (now Ischnocnema guentheri) and, since none of these specimens reached the size informed in the original description, Heyer (1985) considered that this may be the length of the lost specimen from Itatiaia. From the remaining syntypes, he designated AL-MN 719 as lectotype (Figure 7). In the same work, he associated the lectotype with the specimen MZUSP 37787 from Boraceia, clearly cospecific with specimens studied herein. However, we found an unpublished illustration at the Museu Nacional, Rio de Janeiro, having the following handwritten information ��� Hylaplesia nigriventris, 21 mm. n. esp. Campo Bello, Alto da Serra de Cubat��o Alto Itatiaia��� (Figure 8). The illustrated specimen seems to exactly match the original description, on the basis of its color pattern and the recorded length (��� 21mm ���). As none of the syntypes from Paranapiacaba reach 21mm SVL, this illustrated specimen could be the lost syntype from Itatiaia. The illustration does not show any bright coloration on the inguinal region and hidden areas of the hind limbs, nor the presence of a calcar on the heels, or tubercles on the upper eyelid, as found on the extant syntypes of I. nigriventris and our freshly collected specimens. For those reasons, the specimens studied herein and the syntypes from Paranapiacaba may not be conspecific with the figured specimen and, consequently, with the original description, probably from the Itatiaia specimen, as already pointed by Heyer (1985). Since the specimens from Paranapiacaba are the only ones available, and considering that a lectotype has already been designated by Heyer (1985), in accordance with the Article 74.1. 3 of the I.C.Z.N., the name I. nigriventris is attributed to the syntypes, all from Paranapiacaba. The remaining lectotype and paralectotypes are juveniles, their color pattern is completely dark, and most of their fingers and toes are missing (Fig. 7). The specimens we collected share the following characters with them: series of warty tubercles on the upper eyelid and dorsum; presence of prominent calcar on the heels; and the expanded emarginated disks, with ungual flap notched in dorsal view. There are three species of the Ischnocnema lactea species series in Itatiaia, I. melanopygia, I. concolor, and I. holti (Targino & Carvalho-e-Silva, 2008; Targino et al. 2009). The illustrated specimen (Fig. 8) does not seem to correspond to any of them, although the expanded disks and W-shaped marking between the eyes may resemble I. holti. However, the shape of the head is different (rounded in I. holti, as shown in the illustration). The illustrated specimen has expanded disks on the first finger and toe: we consider this a mistake since this character does not occur in any known species of Ischnocnema. There are three species of the I. lactea series that are still known only from their type specimens, generally poorly preserved. These are I. gehrti, I. lactea, and I. paranaensis. The type locality of I. gehrti is also Paranapiacaba, but the type has smooth skin, truncate snout in dorsal view (as already observed by Pombal & Cruz, 1999), and has no palpebral tubercles. These character states are also listed in the original description of I. gehrti (Miranda���Ribeiro, 1926). The type of Ischnocnema lactea is from Iguape, state of S��o Paulo, SVL 32 mm, and the description makes no reference to bright coloration in life or palpebral tubercles (Miranda���Ribeiro, 1923). The type of Ischnocnema paranaensis is from Pico Paran��, state of Paran��, and it has smooth skin, vomerine teeth absent, palpebral and calcar tubercles absent (Langone & Segalla, 1996)., Published as part of Berneck, Bianca V. M., Targino, Mariane & Garcia, Paulo Christiano De Anchietta, 2013, Rediscovery and re-description of Ischnocnema nigriventris (Lutz, 1925) (Anura: Terrarana: Brachycephalidae), pp. 131-142 in Zootaxa 3694 (2) on pages 133-141, DOI: 10.11646/zootaxa.3694.2.2, http://zenodo.org/record/222286

The Pumpkin toadlet, Brachycephalus ephippium (Spix, 1824), is an orangish daily anuran, which inhabits the litter of the Atlantic Forest of Southeastem Brazil. During a survey of this species' natural history in the vicinities of Campinas (22o52'S, 46o49'W), Sao Paulo State, field observations were made on the oviposition behavior, in which after the male left the oviposition site, the female covered the surface of ali eggs with soil particles. She pressed and rolled each egg against the ground using her hindfeet, until the eggs were indistinguishable from the litter background. A clutch was obtained in laboratory and two others were found in the field. After 25 days of development the embryo possess a big yolk sac, differentiated mouth and a small tail with caudal membranes poorly developed. After 41 days, fingers and toes are fully formed, the tail is relatively smaller than the previous state, the abdomen is large due to the yolk within, and the mouth possess two egg tooth. After 54 days, the tail remains as a small appendix, there is only one egg teeth, and body is densely pigmented. The hatehling oceurred after 64 days of development, the young possess reddish brown coloration, vestigial tail, and no egg teeth.

The advertisement call of Eleutherodactylus fraudator is described from Laguna Verde, Departamento Santa Cruz, Bolivia. The call consists of a short, single, unpulsed note repeated in regular succession. Call duration is the shortest and call repetition rate the highest known within the E. fraudator species group. The bioacoustic structures found in the advertisement call of E. fraudator are in accordance with the hypothesis of apomorphic characters found within this species group.

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Made available in DSpace on 2018-11-26T17:10:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-10-27 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundacao Grupo Boticario de Protecao a Natureza A new species of Brachycephalus (Anura: Brachycephalidae) is described from the Atlantic Forest of northeastern state of Santa Catarina, southern Brazil. Nine specimens (eight adults and a juvenile) were collected from the leaf litter of montane forests 79 835 m above sea level (a.s.l.). The new species is a member of the pernix group by its bufoniform shape and the absence of dermal co-ossification and is distinguished from all its congeners by a combination of its general coloration (dorsal region of head, dorsum, legs, arms, and flanks light, brownish green to dark, olive green, with darker region in the middle of the dorsum and a white line along the vertebral column in most specimens) and by its smooth dorsum. The geographical distribution of the new species is highly reduced (extent of occurrence estimated as 25.04 ha, or possibly 34.37 ha). In addition, its habitat has experienced some level of degradation, raising concerns about the future conservation of the species. Preliminary density estimates suggest one calling individual every 3-4 m(2) at 815-835 m a.s.l. and every 100 m2 at 790 m a.s.l. Together with the recently described B. boticario and B. fuscolineatus, the new species is among the southernmost species, of Brachycephalus known to date. Univ Estadual Paulista, Campus Litoral Paulista, Sao Paulo, Brazil Mater Nat Inst Estudos Ambientais, Curitiba, Parana, Brazil Pontificia Univ Catolica Parana, Escola Ciencias Vida, Curitiba, Parana, Brazil Univ Florida, Florida Museum Nat Hist, Gainesville, FL 32611 USA Univ Fed Parana, Dept Zool, BR-80060000 Curitiba, Parana, Brazil Univ Estadual Paulista, Campus Litoral Paulista, Sao Paulo, Brazil CNPq: 571334/2008-3 Fundacao Grupo Boticario de Protecao a Natureza: 0895_20111 Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014

We describe a new species of Brachycephalus that is morphologically similar to the flea-toads B. didactylus, B. hermogenesi, and B. pulex . The new species occurs from the sea level up to 1000 m and it is widely distributed throughout southern Atlantic Forest. Brachycephalus sulfuratus sp. nov. is distinguished from all of its congeners by the combination of the following 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.

The genus Brachycephalus Fitzinger, 1826 comprises 17 tiny species, eight of which were described only in the past 10 years (Frost 2012). Vocal communication is still poorly studied in this group, and despite its importance for taxonomic studies (e.g. Padial & De La Riva 2009) only two species of the group have had their calls described (Pombal et al. 1994; Verdade et al. 2008).

Ischnocnema izecksohni inhabits the gallery forests from the Quadrilátero Ferrífero, Southern Espinhaço range, state of Minas Gerais, southeastern Brazil, and it is considered endemic to this region. Its closest related species is I. nasuta according to the original description. We describe the advertisement call of I. izecksohni based on specimens recorded and collected at the municipality of Nova Lima, state of Minas Gerais, distant about 10 km straight line from its type locality. The advertisement call consists of a group of notes emitted sporadically without a regular interval between the calls. Call duration (n = 36 calls in four individuals) ranged from 1.03 to 1.85 s (= 1.52 ± 0.21 s) and the call rise time from 0.66 to 1.52 s (= 1.16 ± 0.25 s), with 34-57 notes per call (= 47.42 ± 6.03). Peak frequency ranged from 2250 to 2625 Hz, the dominant frequency from 1317.8 to 3128.0 Hz and interval between notes from 22.00 to 41.00 ms (= 28.63 ± 0.03 ms). From the examination of herpetological collections, morphological and bioacoustical data we extended the species known distribution ca. 200 km eastward, to ten new localities, all of them outside the Quadrilátero Ferrífero region, at the Mantiqueira mountain range. We analyzed color patterns and we find some dorsal patterns not described at the original description of I. izecksohni. We also make some comments concerning the taxonomic status of I. izecksohni and I. nasuta. Ischnocnema izecksohni habita as matas de galeria do Quadrilátero Ferrífero, sul da Cadeia do Espinhaço, estado de Minas Gerais, sudeste do Brasil e é considerada endêmica desta região. Sua espécie mais próxima, de acordo com a descrição original, é I. nasuta. Descreve-se o canto de I. izecksohni baseado em espécimes gravados e coletados em Nova Lima, MG, que dista 10 km em linha reta da localidade tipo da espécie. O canto de anúncio consiste em um grupo de notas emitidas esporadicamente sem um intervalo regular entre os cantos. A duração do canto (n = 36 cantos em quatro indivíduos) variou de 1,03 até 1,85 s (= 1,52 ± 0,21 s) e o tempo até a amplitude máxima do canto de 0,66 to 1,52 s (= 1,16 ± 0,25 s), com 34-57 notas por canto (= 47.42 ± 6.03). A freqüência de pico variou de 2250 a 2625 Hz, a freqüência dominante de 1317,8 a 3128,0 Hz e o intervalo entre as notas de 22,00 to 41,00 ms (= 28,63 ± 0,03 ms). A partir do exame de coleções herpetológicas, dados morfológicos e bioacústicos estendeu-se a distribuição da espécie para cerca de 200 km a leste, para mais dez localidades, todas elas fora do Quadrilátero Ferrífero, no complexo da Mantiqueira. Foram analisados padrões de coloração e alguns padrões dorsais além daquele presente na descrição original foram encontrados. São feitos comentários acerca do status taxonômico de I. izecksohni e I. nasuta.

Brachycephalus pulex sp. nov. Figures 1���3 Holotype. MNRJ 69646 (ex-UESC 8357), adult specimen, collected at the Serra Bonita mountain (15 o 23 'S, 39 o 33 'W, 830 m above sea level), Municipality of Camacan, State of Bahia, Brazil, on 0 4 July 2010, by I.R. Dias. Paratopotypes. MZUESC 8352, adult specimen, collected on 25 March 2010, by I.R. Dias. MZUESC 8604��� 8605, adult specimens, collected on 26 July 2010, by I.R. Dias. UFBA 10415���10416, juveniles, collected on 16 April 2009, by M.F Napoli and A. Y.C. Vargas. UFBA 10455, sub-adult, collected on 11 September 2010, by I.R. Dias. Diagnosis. The new species is promptly included in the genus Brachycephalus by the small body size (snout��� vent length less than 18 mm), reduced number of toes, terminal digits not expanded, toes without circumferential grooves, and digits apically pointed. Brachycephalus pulex is diagnosed from all its congeners by the combination of the following character states: (1) toe II externally absent; (2) toe V vestigial; (3) fingers I and IV externally absent; (4) body leptodactyliform; (5) small body size (adult specimens SVL 8.0��� 8.4 mm); (6) dorsal ground color maculated with variegated browns without yellow or orange vivid colors; (7) dorsum with a x-shaped mark from posterior corners of eyes to sacral region; (8) an inverted depigmented v-shaped mark on chest, bordered above by a dark brown stripe on each side; (9) smooth skin texture with no dermal co-ossification on top of the head and central part of the back body. Comparisons with other species. Brachycephalus pulex is closely allied to B. didactylus and B. hermogenesi by the small body size (combined SVL of B. didactylus and B. hermogenesi 8.6���10.2 mm; B. pulex 8.0��� 8.4 mm), body leptodactyliform, dorsal ground color maculated with variegated browns without yellow or orange vivid colors, a dark brown x-shaped mark on dorsum, and short and pointed snout with a rounded tip in dorsal view. These features promptly diagnose B. pulex from B. alipioi, B. atelopoide, B. brunneus, B. bufonoides, B. ephippium, B. ferruginus, B. garbeana, B. izecksohni, B. nodoterga, B. pernix, B. pitanga, B. pombali, and B. vertebralis, which have body size larger (combined SVL 9.3���19.7 mm), body bufoniform, general body color vivid yellow to orange in life and pale cream in preservative (dorsal background color in B. brunneus brown, but not variegated), and rounded or mucronate snout shape in dorsal view. Brachycephalus pulex has smooth skin texture with no dermal co-ossification on top of the head and central part of the back body, whereas B. ephippium, B. nodoterga, and B. vertebralis have such characters. The toe II externally absent diagnoses B. pulex from all congeners, in which toe II is developed. Also, an only vestigial toe V diagnoses B. pulex from B. hermogenesi, which presents a small, but developed, toe V. The external absence of fingers I and IV diagnoses B. pulex from B. didactylus and B. hermogenesi, in which such fingers are vestigial. An inverted depigmented v-shaped mark on chest bordered above by a dark brown stripe on each side diagnoses B. pulex from B. didactylus, which has a dark brown trapezoidal mark on chest, and from B. hermogenesi (without dark brown marks on chest). Description of holotype. Body slender, leptodactyliform (Fig. 1). Head wider than long, its length 38 % of snout���vent length; snout long with length almost equal to eye diameter, rounded in lateral and dorsal views (Fig. 2 A���B); nostrils directed anterolateraly, elliptical, elongated, almost perpendicular to the canthus rostralis; canthus rostralis rounded; loreal region weakly concave; lips nearly sigmoid; eye slightly protruding in dorsal and lateral views, eye diameter 42 % of head length; tympanum indistinct; vocal sac indistinct and vocal slits absent; tongue longer than wide, free behind; choanae relatively small, rounded; vomerine odontophores absent. Upper arm and forearm relatively slender, upper arm approximately as long as forearm; fingers I and IV absent; finger II distinct, short; finger III long, robust; tip of finger II nearly rounded, tip of finger III pointed; relative lengths of fingers II Color of holotype. In preservative (70 % ethyl alcohol), dorsal surfaces light brown, darkened by numerous brown pin dots; eyeball blackish brown; dorsum maculated by dark brown marks: a thin and medially interrupted interorbital stripe, a x-shaped mark from posterior corners of eyes to sacral region, somewhat dimmed in its ends, and an inverted v-shaped mark from sacrum to vent, continuous with the x-shaped mark. Forearms with scattered dark brown blotches somewhat anastomosed, absent on upper arms; outer edges of fingers II and III dark brown. Groin with a dark brown blotch, continuous with a dark brown diagonal transverse large stripe on thigh; knee dark brown; posterior surfaces of thighs with a continuous and curved dark brown stripe just below the vent; tibia with two dark brown stripes cranially, and a third one over the heel; foot with five small dark brown blotches from tarsus to fourth toe. Background color of ventral surfaces pale cream, darkened by numerous brown pin dots; chest with an inverted depigmented v-shaped mark bordered above by two dark brown stripes on each side; edges of throat and belly delimited by distinct depigmented circular areas, also scattered on the inferior surfaces of thighs and tarsus. Palm of hand and sole of foot widely depigmented. Color in life. Description based in the paratype MZUESC 8604. Dorsal ground color maculated with variegated browns looking like dead leaves (Fig. 3). A dark brown x-shaped mark is discernible over the dorsum, anastomosed caudally with a dark brown v-shaped mark over the sacrum, which in turn is continuous with dark brown stripes over the thighs, tibia and tarsus. Dorsal ground color surfaces anterior and posterior to the x-shaped mark, reddish brown. Loreal region, flanks, concealed surfaces of thighs, and anterior surface of tibia, faded reddish brown. A light brown v-shaped interorbital area is delimited on the head cranially and caudally by two dark brown stripes, the former narrower than second. A dark brown mid-dorsal stripe extends perpendicularly from the cranial v-shaped stripe to near the tip of snout, which in turn presents the same background color of the v-shaped interorbital area. Tarsus and posterior surface of tibia light brown. Ventral surfaces dark brown (Fig. 3), with an inverted blackish brown v-shaped mark on chest, and white blotches bordering the throat, belly, thighs, and tibia. An xshaped mark is outlined from the groin to the posterior undersurface of thighs by a row of white botches bordered externally by a blackish stripe. From the groin, these white blotches extend cranially to the posterior margins of chest, bordering the belly. Pupil black, iris orange. Variation. Specimens are congruent with respect to the external morphologic characters and color pattern. Snout-vent lengths of juveniles are 5.5 mm (UFBA 10415) and 6.8 mm (UFBA 10416); sub-adult UFBA 10455 is 7.4 mm. Measurements of adult paratopotypes are in Table 1. Etymology. The specific name (pulex), a noun in apposition, refers to the flea, Pulex irritans, and is allusive to the small size and to the striking capacity for large leaps presented by the new species. Geographic distribution and Natural History. Brachycephalus pulex is only known from the type locality, a forest remnant located in a mountain range regionally named Serra Bonita, with elevations ranging from 200 to 950 m above sea level, which leads to altitudinal changes in the vegetation, from evergreen forest with elements of moist lowland semi-deciduous forest, to moist submontane forest near the summits. The Serra Bonita mountain range lies in the municipalities of Camacan and Pau Brasil, in the State of Bahia, Brazil, and is one of the last remnants of moist submontane forest in the region. It covers an area of approximately 7,500 hectares, 130 km from the city of Ilh��us on the Atlantic coast and 526 km from the State capital of Salvador. Approximately 50 % of the land cover is primary forest, and the remaining is a mosaic of forests in advanced stages of recuperation mixed with ���cabruca��� (cocoa trees planted under natural forest canopy) and small areas of pasture. In the Serra Bonita there are several springs that provide fresh water for the inhabitants of Camacan and Pau Brasil. Brachycephalus pulex was captured within the Serra Bonita Reserve Complex (SBR), a Private Natural Heritage Reserve (RPPN), which comprises ca. 1,800 hectares of a ���consortium��� of privately owned properties (Instituto Uira��u 2009). This is the first record of a Brachycephalus for the State of Bahia and the northernmost record for the coastal Atlantic Forest (but see below). The new species was captured in a primary forest remnant provided with streams near the top of the mountain (800���930 m a.s.l.), within an area of dense leaf litter and several epiphytic bromeliads. At the time of specimens capture (December 2009 to July 2010), the minimum mean air temperature was 16.9 ��C (12���20 ��C), the maximum 23.9 ��C (20���28 ��C), and the average monthly precipitation was 188.6 mm (22.5���315 mm). The specimens were captured beneath the leaf litter, except by one individual that was on a tree trunk 30 cm above the ground. One specimen when disturbed during the survey of anurans jumped away many times quickly and stopped over a dry leaf, remaining immovable (deaf feigning) even after we raise the leaf. This behavior was also observed for both juvenile paratypes. No vocalization was heard, although the advertisement call of the closely allied B. hermogenesi has been recorded and described by Verdade et al. (2008), and also for B. ephippium (Pombal et al. 1994). Remarks. The northern geographic distribution limit of the genus Brachycephalus deserves some considerations. Hedges et al. (2008) stated that the genus Brachycephalus is restricted to the Atlantic Coastal Forest in the states of Rio de Janeiro, S��o Paulo, and Paran�� in southeastern Brazil. In the same article, the authors provided a map with the geographic distribution of the genera Brachycephalus and Ischnocnema, in which the range distribution of the former extends to near the Brazilian State of Alagoas (ca. 10 o S). This mapped geographic distribution is contradictory to the information presented in the article itself and is not congruent with the literature, as the northernmost geographic distribution limit known for the genus Brachycephalus is that of B. alipioi in the municipalities of Vargem Alta, Forno Grande and Santa Teresa (ca. 20 o S), all in the State of Esp��rito Santo, Brazil (Pombal & Gasparini 2006), and that of B. ephippium in the Serra do Brigadeiro (ca. 20 o S) (Pombal 2001; Dayrell et al. 2006), a regional designation of the Serra da Mantiqueira mountain range in the State of Minas Gerais, Brazil. Dayrell et al. (2006) and Frost (2010) included in the geographic distribution range of B. ephippium the State of Bahia, Brazil. Indeed, the type locality of B. ephippium lies in the Municipality of Ilh��us, State of Bahia (Bokermann 1966), but Pombal et al. (1998) and Pombal and Gasparini (2006) already contested this information pointing out that possibly Spix���s material was incorrectly labeled and the type locality is in error, as B. ephippium is common in the mountains of Rio de Janeiro city, where Spix also collected. In assuming this error in the label of Spix���s material, Pombal and Gasparini (2006) considered B. alipioi as the northernmost species of the genus. Therefore, the presence of B. pulex in the Municipality of Camacan (ca. 15 o S), southern Bahia State, currently corresponds to the northernmost distribution limit of the genus Brachycephalus., Published as part of Napoli, Marcelo Felgueiras, Caramaschi, Ulisses, Cruz, Carlos Alberto Gon��alves & Dias, Iuri Ribeiro, 2011, A new species of flea-toad, genus Brachycephalus Fitzinger (Amphibia: Anura: Brachycephalidae), from the Atlantic rainforest of southern Bahia, Brazil, pp. 33-40 in Zootaxa 2739 on pages 34-39, DOI: 10.5281/zenodo.276578, {"references":["Verdade, V. K., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. C. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta & Sawaya, 1998) (Anura, Brachycephalidae). Journal of Herpetology, 42, 542 - 549.","Pombal, Jr., J. P., Sazima, I. & Haddad, C. F. B. (1994) Breeding behavior of the pumpkin toadlet, Brachycephalus ephippium (Brachycephalidae). Journal of Herpetology, 28, 516 - 519.","Hedges, B., Duellman, W. E. & Heinicke, M. P. (2008) New World direct-developing frogs (Anura: Terrarana): molecular phylogeny, classification, biogeography, and conservation. Zootaxa, 1737, 1 - 182.","Pombal, Jr., J. P. & Gasparini, J. L. (2006) A new Brachycephalus (Anura: Brachycephalidae) from the Atlantic Rainforest of Espirito Santo, southeastern Brazil. South American Journal Herpetology, 1, 87 - 93.","Pombal, Jr., J. P. (2001) A new species of Brachycephalus (Anura: Brachycephalidae) from Atlantic Rain Forest of southeastern Brazil. Amphibia-Reptilia, 22, 179 - 185.","Frost, D. R. (2010) Amphibian Species of the World: an Online Reference. Version 5.4 (08 April, 2010). Electronic Database available from http: // research. amnh. org / vz / herpetology / amphibia / American Museum of Natural History, New York, USA (accessed 24 August 2010).","Pombal, Jr., J. P., Wistuba, E. M. & Bornschein, M. R. (1998) A new species of Brachycephalid (Anura) from the Atlantic Rain Forest of Brazil. Journal of Herpetology, 32, 70 - 74."]}