Brachycephalus herculeus, Folly & Condez & Vrcibradic & Rocha & Machado & Lopes & Pombal Jr., 2024

Folly, Manuella, Condez, Thais H., Vrcibradic, Davor, Rocha, Carlos F. D., Machado, Alessandra S., Lopes, Ricardo T. & Pombal Jr., Jose P., 2024, A new species of Brachycephalus (Anura: Brachycephalidae) from the northern portion of the state of Rio de Janeiro, southeastern Brazil, Vertebrate Zoology 74, pp. 1-21 : 1

Brachycephalus herculeus sp. nov.

Figure 1

Chresonymy.

Brachycephalus sp. - Siqueira et al. (2011)

Brachycephalus sp. 2 - Condez et al. (2020); Folly et al. (2022)

Holotype.

MNRJ 42408, adult male, Parque Estadual do Desengano, vicinity of Estalagem Morumbeca, municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil (21°52 ’42.3” S, 41°55 ’11.9” W; 1100 m a.s.l.), collected by D. Vrcibradic on 3 June 2006.

Paratypes.

MNRJ 42407, adult, cleared-and-stained specimen, collected with the holotype. MNRJ 42409, adult male, collected by C.C. Siqueira on 6 June 2006; MNRJ 42410, adult male, collected by H.G. Bergallo on May 2006, all in the same site as the holotype. MNRJ 52718, adult female, and MNRJ 52719-52721, adult males, Parque Estadual do Desengano, Estalagem Morumbeca, municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil (21°52′33″S, 41°55′08″W; 1050 m a.s.l.), collected by A. Chagas, A. Kury, C. Sampaio and T. Moreira, from 13-17 May 2008. CFBH 28049, adult female, CFBH 28124, 28129-30, 28135, adult males, and CFBH 28128, 28132-34, 28136-38, individuals of undetermined sex, including juveniles, Parque Estadual do Desengano, trail to Pico do Desengano, municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil (21°52′49.1″S, 41°55′0.9″W; 1167 m a.s.l.), collected by M.T.C. Thomé, F.A. Brusquetti, and T.H. Condez, from 21-28 February 2011. CFBH 27342-45, Parque Estadual do Desengano, trail to Pico do Desengano, municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil (21°52′45.3″S, 41°55′07.0″W; 1130 m a.s.l.), collected by C. Canedo, T. Brunes, M. Gehara and M.T.C. Thomé, on 20 November 2010.

Diagnosis.

Brachycephalus herculeus sp. nov. is distinguished from all its congeners by the following combination of characters: (1) skin on head and dorsum with dermal ossification; (2) skull with hyperossification of postorbital crests, which can be seen externally; (3) fourth presacral vertebra with transverse processes hyperossified, not ornamented and not visible externally; (4) urostyle crest extending up to 2/3 of urostyle length; (5) long oesophageal process of hyolaryngeal apparatus; (6) general color in life orange with dorsal green irregular patch; (7) presence of osteoderms; (8) presence of black connective tissue scattered over dorsal musculature; (9) medium body size for the genus (SVL of adults: 11.8-14.7 mm for males and 13.9-15.2 mm for females; (10) advertisement call characterized by one note repeated in sequence, commonly comprised of 8-12 pulses.

Comparisons with other species.

The presence of hyperossification in the skeleton and skin with dermal ossification on the head and dorsum distinguish the new species from all members of the B. ephippium species group ( B. actaeus , B. ephippium , B. auroguttatus , B. boticario , B. brunneus , B. coloratus , B. curupira , B. ferruginus , B. fuscolineatus , B. izecksohni , B. leopardus , B. mariaeterezae , B. mirissimus , B. olivaceus , B. ephippium , B. pombali , B. quiririensis , B. tabuleiro , B. ephippium and B. verrucosus ; Pombal et al. 1998, 2018; Ribeiro et al. 2005, 2015, 2017; Alves et al. 2006; Garey et al. 2012; Pie et al. 2015; Bornschein et al. 2016; Monteiro et al. 2018a; Mângia et al. 2023), and from B. clarissae , B. ephippium , B. ephippium , B. pulex , B. puri , and B. ephippium , all of which lack hyperossification ( Izecksohn 1971; Giaretta and Sawaya 1998; Napoli et al. 2011; Condez et al. 2016; Almeida-Silva et al. 2021; Folly et al. 2022). Also, the absence of metacarpal and metatarsal tubercles distinguishes B. ephippium sp. nov. from B. ephippium (metacarpal and metatarsal tubercles present; Giaretta and Sawaya 1998) and from B. auroguttatus , B. boticario , B. brunneus , B. fuscolineatus , B. izecksohni , B. leopardus , B. mariaeterezae , B. olivaceus , B. ephippium , B. pulex , B. quiririensis , B. ephippium and B. verrucosus (outer metatarsal tubercles present, Giaretta and Sawaya 1998; Ribeiro et al. 2005. 2015; Alves et al. 2009; Haddad et al. 2010; Napoli et al. 2011; Pie and Ribeiro 2015).

Brachycephalus herculeus sp. nov. lacks the quadratojugal, as in other species of the B. ephippium and B. ephippium groups (except B. ephippium , in which the quadratojugal may be present or not), whereas in species of the B. ephippium group the quadratojugal is present ( Ribeiro et al. 2005; Alves et al. 2006; Campos et al. 2010; Bornschein et al. 2016; Condez et al. 2016; Monteiro et al. 2018a).

The general orange background color in life of Brachycephalus herculeus sp. nov. distinguishes it from that of the flea-toads B. ephippium , B. ephippium , B. pulex , B. puri , and B. ephippium , which exhibit a brown or gray general body color ( Izecksohn 1971; Giaretta and Sawaya 1998; Napoli et al. 2011; Condez et al. 2016; Almeida-Silva et al. 2021), and B. clarissae , which has the dorsal region mainly light brown and the ventral region yellow with scattered red blotches ( Folly et al. 2022). The new species also has a larger body size (adult SVL: 11.8-15.2 mm; Table 1 ) than the aforementioned flea-toads (maximum SVL <11 mm in all six species; Izecksohn 1971; Almeida-Santos et al. 2011; Napoli et al. 2011; Condez et al. 2016; Almeida-Silva et al. 2021; Folly et al. 2022).

Brachycephalus herculeus sp. nov. can be distinguished from the extremely hyperossified species of the B. ephippium species group ( B. ephippium , B. ephippium , B. ephippium , B. ibitinga , B. margaritatus , and B. rotenbergae ) by the absence of a dorsal bony shield (which is present in those species; Pombal 2010; Pombal and Izecksohn 2011; Guimarães et al. 2017; Nunes et al 2021; Condez et al. 2021). Further, Brachycephalus herculeus sp. nov. exhibits black connective tissue scattered over the dorsal musculature (Fig. 2 ). This feature distinguishes this species from B. izecksohni , B. ephippium , and B. ephippium , all of which lack this pigmentation ( Guimarães et al. 2017). Regarding B. ephippium and B. margaritatus , some specimens of these species were reported to present a few scattered areas with dark pigmentation in the dorsolateral region adjacent to the dorsal musculature, while in B. ephippium there is some internal dark pigmentation around the vertebral column ( Guimarães et al. 2017).

The new species is easily separated from other species of the B. ephippium species group, except B. ephippium , by its general color orange with a green irregular patch on the dorsum (see color pictures of other species of the B. ephippium group in Pombal and Gasparini 2006; Alves et al. 2009; Haddad et al. 2013; Condez et al. 2014; Clemente-Carvalho et al. 2016; Folly et al. 2020). Also, B. ephippium sp. nov. can be separated from the other species of the B. ephippium group, except for B. ephippium and B. ephippium , by the presence of wart-like osteoderms on the skin of the dorsum, flanks, venter, arms, and legs (see Folly et al. 2021a). The new species’ dorsal aspect is similar to that of B. ephippium , which also presents greenish coloration on dorsal surfaces, but differs from the latter in that its dorsal color is leaf green (olive green in B. ephippium ) and does not extend to the flanks and to the sides of the head as it does in B. ephippium .

Brachycephalus herculeus sp. nov. is the sister species of B. ephippium according to our molecular analysis (Fig. 3 ). These species exhibit some of the smallest interspecific genetic distances among all pairs of species within the Brachycephalus vertebralis group (Table 2 ). They also share a prominent pelvic region, which is externally marked as a v-shaped bulge (presumably representing the protrusion of the iliac bones underneath) whose vertex lies at the posterior end of the body and whose tips extend to the middle of the trunk on either side; this characteristic distinguishes Brachycephalus herculeus sp. nov. and B. ephippium from the other species of the B. ephippium group. Notwithstanding, Brachycephalus herculeus sp. nov. is easily distinguished from B. ephippium by the presence of osteoderms on the skin (absent in B. ephippium ), by its greenish dorsal color in life (orange in B. ephippium ), and by the advertisement call comprised by notes with 10-12 pulses (notes with 13-17 pulses in B. ephippium ). Furthermore, in terms of osteology and internal anatomy, the new species can be also distinguished from B. ephippium by its lateroposteriorly oriented parotic plate (posteriorly oriented in B. ephippium ), oesophageal process of hyolaryngeal cartilages longer than arytaenoids (shorter than arytenoids in B. ephippium ), absence of an elbow sesamoid (present in B. ephippium ), paravertebral plates not ornamented (ornamented in B. ephippium ), and dorsal crest of the urostyle extending for 2/3 of its length (extending for all its length in B. ephippium ; Folly et al. 2020, 2021a).

Description of the holotype.

Adult male; head wider than long (HL/HW = 0.42%); head length approximately 18% of SVL; snout short, with length equivalent to 77% of eye diameter, rounded in lateral and dorsal views; nostrils protuberant, oriented posterolaterally; canthus rostralis distinct and straight; loreal region weakly concave; mouth nearly sigmoid; eye slightly protruding in dorsal and lateral views, eye diameter 60% of HL; tympanum absent; choanae relatively small and round; vomerine odontophores absent. Upper arm around 78% of forearm length; length of upper arm plus forearm 58% of SVL; hand 58% of upper-arm length; fingers III and IV distinct; fingers II and V vestigial; tip of finger III rounded, tip of finger IV slightly pointed; finger III smaller than finger IV; metacarpal tubercles absent. Tibia slightly shorter than thigh (TL/THL = 0.90); thigh and tibia 86% of SVL; foot longer than thigh (FL/THL = 0.76); toe I externally absent and toe V vestigial; toes II, III, and IV distinct; toe II reduced; tip of toes II and III rounded, tip of Toe-IV pointed; relative length of toes II <III <IV; metatarsal tubercles absent. Skin on dorsum, flanks, venter, and dorsal and ventral surface of legs and forearms with warty appearance, due to the presence of osteoderms; granular skin on ventrolateral surfaces of body and area around the cloacal opening.

Measurements of holotype (in mm).

SVL 13.4; HL 2.5; HW 6.0; ND 0.1; IND 1.7; ED 1.5; EW 1.1; IOD 2.2; END 0.7; NSD 0.4; THL 6.1; SL 5.5; FL 8.1; TL 2.8; AL 4.4; FAL 3.4; HAL 2.5; FIL 1.3.

Coloration of holotype in preservative (Fig. 1).

Forearms and legs gray; hands, feet, arms, lateral and ventral sides of the body beige; two contiguous dorsal paired dark gray marks bordering the vertebral column; scattered whitish warts (osteoderms) on the dorsum, flanks, arms, and legs; a cream line below each eye.

Color in life (Fig. 4).

General background color orange; central area of head and dorsum and dorsal part of thighs and shanks green. Osteoderms, appearing as light yellow “warts” distributed throughout most of the body and limbs. Hands, feet, elbows, knees, and ankles orange. Ventral surface of body bright yellow. Eyes entirely black, with no visible delimitation between pupil and iris.

Variation.

Morphometric variation is given in Table 1 . On average, females (SVL = 14.5 ± 0.4 mm; 13.9-15.2 mm, n = 9) of B. ephippium are larger ( Welch’s t-test, t = 4.6443, df = 15.579, p <0.0003) than males (SVL = 12.8 ± 1.1 mm; 11.8-14.7 mm, n = 11). Semicircular or rounded snout in dorsal view and rounded in lateral view. The density of osteoderms in the skin may vary among individuals, but osteoderms are always less numerous on the mid-dorsal and mid-ventral regions and practically absent on the dorsum of head and gular region (Figs 4 - 6 ). Spinal plates weakly to well-delimited externally (Fig. 5 ). In preservative, dorsal and lateral surfaces of body can be cream to gray, varying especially on the head and limbs. A dissected preserved specimen (CFBH 28130), show scattered areas with pigmentation (dark tissue) in the dorsolateral region adjacent to the dorsal musculature, also around the line of the spinal vertebrae (Fig. 2 ). The visibility of black connective tissue scattered over the dorsal musculature is externally represented, in most specimens (70% of the type-series), by paired contiguous dorsal dark gray marks bordering the vertebral column (Fig. 5 ).

Distribution (Fig. 7).

Brachycephalus herculeus sp. nov., is known only from the type-locality, the Parque Estadual do Desengano, in the municipality of Santa Maria Madalena, state of Rio de Janeiro, southeastern Brazil. This site is located within the northern portion of the Serra do Mar Mountain Range.

Natural history.

The new species lives amidst the leaf-litter and can be found active during the day. Most individuals were found hidden amidst the leaf-litter, under fallen trunks, and among roots. The holotype and paratype (MNRJ 42407) were collected on the ground, during nocturnal surveys. The holotype was collected using quadrat sampling (see Siqueira et al. 2011) and thus may have been disturbed by the revolving of the leaf litter during the survey, so it is uncertain if it was active at night. The latter specimen may also have been found inactive under a decomposing leaf at the time. Paratype (MNRJ 42409) was found during the afternoon (at 1500 h) inside a bromeliad ( Vriesea sp.) that was attached to a tree stump, ca. 80 cm from the ground. The specimens collected in 2011, during the rainy season, were on sloping terrain bordering trails in the forest. Males were not observed exposed when calling. An amplectant pair was found hidden under the leaf-litter in February 2011, during the morning after a rainy night. When exposed, the male changed its axillary amplexus to inguinal, as described for B. rotenbergae ( Pombal et al., 1994, where it was called B. ephippium ) and the pair moved away looking for shelter. Minutes after having hidden among the leaf litter, the female left the place and the male remained immobile. No eggs were found.

Advertisement call (Fig. 8).

The advertisement call of the new species is characterized by one pulsed note repeated in sequence, at a rate of 2.5-2.7 notes/s (2.6 ± 0.08 s; n = 60; Fig. 8 ). Call repetition may last for minutes and suggests a long period of calling activity. The call (or note) has a duration of 0.21-0.33 s (0.25 ± 0.05 s; n = 60) and the inter-call (or inter-note) interval is 0.19-0.20 s (0.19 ± 0.00 s; n = 80) long. Notes are comprised of 10-12 pulses (11.0 ± 1.0; n = 60), with each pulse lasting 0.02 ± 0.00 s (n = 60), and the pulse repetition rate is 27-31 pulses/s (29.0 ± 1.70 pulses/s; n = 60). The lower frequency range is 3.7-4.3 kHz (3.9 ± 0.26 kHz; n = 60), that of the upper frequency is 6.0-6.4 kHz (6.2 ± 0.17 kHz; n = 60), and that of the dominant frequency is 4.5-5.1 kHz (4.8 ± 0.24 kHz; n = 60).

The main structure of the advertisement call of the new species, characterized by one note repeated in sequence, commonly comprised of 10-12 pulses, distinguishes it from the advertisement call of B. actaeus , B. ephippium , B. ephippium , B. mirissimus , B. olivaceus , B. ephippium , B. quiririensis , B. ephippium (in which the notes are comprised of 1-4 pulses; Monteiro et al. 2018a, b; Bornschein et al. 2018; Pie et al. 2018; Bornschein et al. 2019), B. ephippium (notes comprised of 4-7 pulses; Condez et al. 2016), and B. ephippium (notes comprised of 6-8 pulses; Guimarães et al. 2017). Additionally, the dominant frequency of the advertisement call in the new species, ranging from 4.5-5.1 kHz (average of 4.8 ± 0.24 kHz) is also distinct from the ones described for B. ephippium (average of 6.8 ± 0.24 kHz; Verdade et al. 2008), B. ephippium (6.2-7.2 kHz, average of 6.7 ± 0.3 kHz; Condez et al. 2016), B. olivaceus (6.4-7.0 kHz, average of 6.2 ± 0.2 kHz; Monteiro et al. 2018b), B. quiririensis (6.2-6.5 kHz, average of 6.3 ± 0.2 kHz; Monteiro et al. 2018b), B. actaeus (6.6-7.3 kHz, average of 6.9 ± 0.3 kHz; Monteiro et al. 2018a), B. rotenbergae (2.8-4.5 kHz, average of 3.8 ± 0.35 kHz; Nunes et al. 2021), and B. ephippium (2.9-3.8 kHz, average of 3.4 ± 1.8; Guimarães et al. 2017). The call duration in the new species (0.21-0.33 s, average of 0.25 ± 0.05 s) differs from that of B. ephippium (0.08-0.16 s, average of 0.11 ± 0.13 s; Guimarães et al. 2017). Some temporal and spectral parameters of the advertisement call of Brachycephalus herculeus sp. nov. overlap with those described for B. ephippium , B. ephippium , B. ephippium , and B. ibitinga ( Condez et al. 2014; Oliveira and Haddad 2017; Folly et al. 2020; Condez et al. 2021). Among these species, the number of pulses per call (10-12 pulses) distinguishes the new species from B. ephippium (notes comprised of 13-17 pulses; Folly et al. 2020). The inter-call (or inter-note) interval for the new species (0.19-0.20 s) differs from the ones described for B. ephippium (average of 0.35 s; Condez et al. 2014), B. ephippium (average of 0.27 s; Oliveira and Haddad 2017), and B. ibitinga (0.28-0.37 s, average of 0.32 s; Condez et al. 2021). The note repetition rate (2.5-2.7 notes/s, average of 2.6 notes/s) reported for the new species differs from those of B. ephippium (average of 1.7 notes/s; Condez et al. 2014), B. ephippium (2.0-2.4 notes/s; Folly et al. 2020), and B. ibitinga (average of 1.8 notes/s; Condez et al. 2021).

Etymology.

Herculeus is a Latin adjective meaning very great, difficult, or dangerous; requiring the strength or courage of Hercules to challenge or accomplish a mission, as herculean labor or task (derived from Greek Mythology). The specific epithet was chosen to represent the “herculean” task that is, for such a small species, to survive in one of the most threatened forest environments in the world, the Brazilian Atlantic Forest.

Molecular analysis.

We obtained a final aligned matrix of 5,456 base pairs considering the selected mitochondrial and nuclear gene fragments. The optimal partition scheme and nucleotide substitution models selected for each data block were GTR+I+G (12S rRNA, 16S rRNA), K80+G (fist codon position of COI), GTR+I (second codon positions of COI and cyt b), GTR+G (three codon positions of Tyr; first codon positions of RAG1 and RAG2; third positions of COI and cyt b), SYM+I+G (first codon position of cyt b), HKY (second codon positions of RAG1 and RAG2), HKY+G (third codon position of RAG1), K80+I (third codon position of RAG2). Our main results corroborate many of the previously published phylogenetic hypotheses for the genus Brachycephalus but differs from those with respect to the position of the flea-toads B. clarissae , B. ephippium , B. puri , B. ephippium , and the candidate species B. sp. 7 and B. sp. 8 (Fig. 3 ). Nevertheless, our hypothesis supports the position of B. pulex as sister to all other Brachycephalus species, as suggested in previous studies ( Almeida-Silva et al. 2021; Dos Reis et al. 2021; Lyra et al. 2021). It also corroborates the position of B. ephippium as the sister species of the clade comprised of the B. ephippium and B. ephippium species groups ( Condez et al. 2020; Dos Reis et al. 2021; Almeida-Silva et al. 2021; Folly et al. 2022). However, our analysis diverges from previous studies in the phylogenetic positions of B. ephippium , which is known to be fairly controversial in the literature (see Lyra et al. 2021), and of B. clarissae (sister to the B. ephippium group instead of the B. ephippium group (as B. ephippium lineage) as in Condez et al. 2020 or the B. ephippium + B. ephippium groups as in Folly et al. 2022). Brachycephalus sulfuratus was recovered as the sister species to a clade containing B. puri and two undescribed taxa ( Brachycephalus sp. 7 and Brachycephalus sp. 8), but its position relative to the pumpkin-toadlets remains unstable (e.g., Condez et al. 2020; Folly et al. 2020, 2022; Lyra et al. 2021). The position of B. puri in our tree was distinct from that presented at the original description of the species ( Almeida-Silva et al. 2021), probably due to the inclusion of samples of the two flea-toad candidate species in our dataset. Brachycephalus herculeus , new species, was recovered as monophyletic in our phylogenetic reconstruction, and was nested within the B. ephippium species group, as the sister species of B. ephippium (Fig. 3 ). Based on the genetic distances for the 16S rRNA gene (1548 bp), the sequences of different individuals of the new species did not differ from each other (p -distance = 0; n = 11). Within the B. ephippium species group, the smallest genetic divergences between the new species and other congeners were 0.5-0.8% (from its sister species B. ephippium ) and the greatest was 4.9% (from B. ephippium ; Table 2 ).

Osteological description.

Skull (Figs 9 , 10 ). Shape and proportions: Isolated bony structures (osteoderms) are predominantly distributed on the lateral surfaces of the body. The skull is wider than long (length/width range 85-95%). The diameter of the orbit is 39-47% of the total length of the skull. The skull is widest at the prootics, and the jaw articulation lies anterior to the posterior end of the skull at the occipital condyles. The upper jaws are relatively short, with the posterior apex lying at the level of the optic fenestra. Large additional elements associated with the skull (parotic plate) and vertebrae (vertebral and paravertebral plates) are dermal bones. - Skull plate. Exostosis and co-ossification are present in the skull forming an ornamented ridge-like pattern. Thus, nasals, dermal sphenethmoid, frontoparietals, squamosals, and the parotic plate are synostosed and exostosed, forming a dorsal skull plate. A marginal extension of the frontoparietals is fused to the parotic plate, dorsally covering the exoccipitals and prootics. There is a space between the parotic plate and a post-orbital crest forming a small concavity that reaches half of the width of the orbit. The parotic plate is connected and fused medially to the frontoparietal and has a lateroposterior orientation related to the main axis of the skull. The anterior extension of the parotic plate is small, not covering the articulation between squamosal and prootic and the posterior extension does not reach the posterior border of the occipital condyle. The post-orbital crest is latero-posteriorly projected and well developed, reaching the height of occipital condyle. - Dermal investing bones. Nasals: Exostosed. These bones are completely synostosed with the sphenethmoid but are not in anteromedial contact. An attenuate maxillary process contacts the preorbital process of the pars facialis of the maxilla. Frontoparietals: Expanded and exostosed. - Neurocranium. Sphenethmoid: Expanded. This bone is dorsally covered by frontoparietals and nasals. The cartilaginous optic fenestrae are absent. Fused Exoccipitals and Prootics: Not expanded and nor exostosed. The epiotic eminence (anterior and posterior) is undetectable. These bones are dorsally covered by the skull plate (frontoparietal fused with parotic plate). - Ventral investing bones. Parasphenoid: The cultriform process is synostosed with the sphenethmoid (Fig. 9 ). The parasphenoid alae are broad and laterally oriented beneath the otic capsule; the distal margins of the alae are wider than the proximal margins, terminating between the first third and the midpoint of the otic capsule. The posterior margin (posteromedial process) of the parasphenoid terminates in a triangular apex that lye the foramen magnum. Neopalatine: Absent. Vomers: Reduced. The anterior process is triangular with a rounded tip and is around the same length of the prechoanal process. The reduced prechoanal process forms the anterior and anteromedial margins of the choana and terminates in a rounded tip. The postchoanal process forms the posteromedial margin of the choana. The posteromedial margins of the vomers are moderately separated from each other, diverging abruptly anteriorly. The pre and postchoanal processes are around the same length. The dentigerous process is absent. - Maxillary arcade. Premaxillae: Each premaxilla is broad and separated from each other by a short gap in anterior view. The pars dentalis of each premaxilla lack teeth. In anterior view, the height of the alary process corresponds to around the length of premaxillae, and the distal tip of each alary process is pointed. The basal parts of the alary processes converge medially. The distal tips diverge from one another. Laterally, the alary processes are curved. The distal end of each pars palatina is sharp and converges to each other almost touching one another. Maxillae: The anterior end of the maxilla overlaps the posterolateral end of the premaxilla. Maxilla toothless; its posterior end is rounded and does not reach the ventral ramus of the squamosal. The pars facialis is high and thin, extending for less than half the length of the maxilla. Quadratojugals: Absent. - Suspensory apparatus. Pterygoids: The anterior ramus of each pterygoid is long, cylindrical, terminating in a truncated apex and extending to the final third of the maxilla. The medial ramus is short, slightly laminar, has a truncated end, and invests the prootic. The posterior ramus is long, triangular, and has a truncated point. Squamosals: Expanded and exostosed. They are composed of three rami that render each squamosal as a “T” shaped element composed by the ventral ramus, the otic ramus (posterior ramus), and the zygomatic ramus (anterior ramus). The ventral ramus is the longest of the three and has a rounded distal end, which is wider than its proximal portion. In lateral view, the zygomatic ramus is reduced, shorter than the ventral ramus and the distal end is rounded towards the maxilla, but without contacting it. In lateral view, the otic ramus is longer than the zygomatic ramus, and its distal end is rounded. In dorsal view, the distal end of the otic ramus is truncated, towards the parotic plate, overlapping it. Mandible: The mentomeckelian bones are in the anterior part of the mandible and are separated from each other by a short space. In anterior view, the mentomeckelians are sharp anteriorly and are fused to the dentaries laterally. Each dentary invests laterally less than half of the angulosplenial bone and has a pointed posterior end. They do not overlap the Meckel’s cartilage anterolaterally. The anterior end of each angulosplenial is roundly pointed, and the posterior end is robust and rounded. The posterior end of the angulosplenial bears a well-developed pars articularis process. - Auditory Apparatus. Columellae absent. Fenestra ovalis posteriorly oriented. An enlarged and completely ossified operculum nearly fills the fenestra ovalis. - Hyolaryngeal skeleton (Fig. 10 ). The hyoid plate is longer than wide, with its length around four times its smallest width. The anterior processes are long and form a deep hyoglossal sinus, which deepens to nearly the height of the alary processes. The alary and posterolateral processes are much reduced, and the former is longer than the posterolateral processes. The posteromedial processes diverge widely to embrace the broad larynx. The arytenoids are short, semicircular and narrowly separated from each other, while the oesophageal process is longer than the arytenoids. - Postcranium (Fig. 9 ). Pectoral girdle: The clavicle, procoracoid, epicoracoid, coracoid, and scapula are completely ossified and fused, with a large fenestra separating the clavicle from coracoid; suprascapula not expanded, with anterior half ossified as cleithrum; omosternum and sternum absent. The two sides of the coracoid are well-developed with a concave-shaped coracoid and around the same height giving a quadrangular appearance to the scapular apparatus. The distal edge of the clavicle bears a dorsal projection. Vertebral column: The vertebral column is composed of eight presacral, non-imbricate vertebrae. Transverse processes of Presacrals III, VI and VII are perpendicular to the notochordal axis, those of Presacrals IV and V are inclined posteriorly, and those of Presacrals II and VIII are inclined anteriorly. Relative lengths of the transverse process of presacrals IV> SD> III> V-VIII> II> I. Sacral diapophyses (SD) are moderately expanded and slightly posteriorly oriented; distal end of diapophyses with a flat, slightly calcified cartilage that articulates with the ilial shaft of the pelvic girdle. Sesamoids are present on both sides of sacral diapophyses. There are two types of bony elements associated with the vertebral column: (1) spinal plates, which lie dorsal to the vertebra and are generally ornamented (except for the posteriormost ones, which might not be ornamented); and, (2) the paravertebral plates, which are associated with the transverse processes of vertebrae IV; they are poorly developed, not covering the distal part of the transverse processes, and are not ornamented. The dorsal crest of the urostyle extends for 2/3 of its length. The ilium crest extends for the whole length of this bone. Manus: Phalangeal formula of hand 1-2-3-1. The carpus is composed of a radiale, ulnare, element y fused with Carpal 2, and a large postaxial assumed to represent a fusion of centrale with Carpals 3-5. Prepollex with two elements ossified and very reduced. Tips of the terminal phalangeal elements of fingers arrow shaped. A palmar sesamoid is present. Pes: Phalangeal formula of foot 1-2-3-4-1. Tarsus composed of tibiale, fibulare, three individual elements, including distal tarsal 2-3, distal tarsal 1, element y. Distal tarsal 1 is the smallest and articulates with element y, distal tarsal 2-3, and metatarsal I and II. Distal tarsal 2-3 articulates mainly with metatarsal III, but also with metatarsal II and IV, and with distal tarsal I. Prehallux has a single element. Tips of the terminal phalangeal elements of toes II-V arrow-shaped, toes I and V reduced, digit IV elongated. Plantar and cartilage sesamoids are present in all specimens. Three sesamoids are present at the knee region, the larger one might be the graciella sesamoid.

Figures

Figure 1. Holotype of Brachycephalus herculeus, new species (MNRJ 42408). A Dorsal, B ventral, and C lateral views of the body; D ventral view of foot, and E ventral view of hand. Scale bar = 1 mm.

Figure 2. Ventral and dorsal view of dissected preserved specimens of Brachycephalus herculeus, new species (CFBH 28130), showing scattered areas with pigmentation (dark tissue) in the dorsolateral region adjacent to the dorsal musculature, also around the line of the spinal vertebrae.

Figure 3. The majority rule consensus tree resulting from the Bayesian Inference analysis based on the concatenated alignment of 5456 bp (four mitochondrial and three nuclear genes) showing the relationships within Brachycephalus. Sequences of the new species are highlighted in blue. Numbers associated to nodes represent Bayesian Posterior Probabilities <0.90; all other nodes were fully supported (BPP = 1.0). Voucher numbers of specimens are provided at each terminal, when available. See Table S 2 for the GenBank accession numbers.

Figure 4. Four specimens of Brachycephalus herculeus, new species, in life. Photographs by C. F. D. Rocha (A), C. F. B. Haddad (B) and T. H. Condez (C, D).

Figure 5. Dorsal variation in preserved specimens of Brachycephalus herculeus, new species. A CFBH 28124 (male; SVL 12.0 mm), B CFBH 28129 (male; SVL 12.2 mm), C CFBH 28135 (male; SVL 12.3 mm), D MNRJ 52721 (male; SVL 11.8 mm), E MNRJ 42409 (male; SVL 12.0 mm), F MNRJ 52718 (female; SVL 15.2 mm), G CFBH 28132 (female; SVL 14.1 mm), H CFBH 28136 (female; SVL 15.1 mm), and I CFBH 28138 (female; SVL 14.4 mm). Scale bar = 1 mm.

Figure 6. Ventral variation in preserved specimens of Brachycephalus herculeus, new species. A CFBH 28124 (male; SVL 12.0 mm), B CFBH 28132 (female; SVL 14.1 mm), C MNRJ 52718 (female; SVL 15.2 mm), and D CFBH 28136 (female; SVL 15.1 mm). Scale bar = 1 mm.

Figure 8. Advertisement call of Brachycephalus herculeus, new species (FNJV 58774), recorded at Parque Estadual do Desengano, municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil, 23 February 2011, at 16: 00 p. m., air temperature 20.1 ° C, relative air humidity 88 %. A Spectrogram (above) and oscillogram (low quality; below) of four consecutive calls (or notes), B Spectrogram (above) and oscillogram (low quality; below) of one pulsed call (or note).

Figure 9. High-resolution computed tomography (Micro-CT) scans of paratypes of Brachycephalus herculeus, new species, showing osteological features. A Dorsal, B ventral views of the skeleton (CFBH 28131); C left hand in palmar view (CFBH 28133); D right foot in plantar view (CFBH 28048 and 28137, respectively); E dorsal, F ventral, and G lateral views of the skull (without the lower jaw; CFBH 28133); H dorsal view of vertebral column (CFBH 28137 and 28133, respectively). The dots surround the skeleton in (A) and (B) are osteoderms. Scale bars = 1 mm.

Figure 10. Views of (A) hyolaryngeal skeleton and (B) pelvic girdle of one of the paratypes of Brachycephalus herculeus, new species (MNRJ 42407). Scale bar = 1 mm.