Brachycephalus bufonoides Miranda-Ribeiro, 1920

Folly, Manuella, Amaral, Lucas Coutinho, Carvalho-E-Silva, Sergio Potsch De & Pombal Jr, José P., 2020, Rediscovery of the toadlet Brachycephalus bufonoides Miranda-Ribeiro, 1920 (Anura: Brachycephalidae) with osteological and acoustic descriptions, Zootaxa 4819 (2), pp. 265-294 : 269-283

Brachycephalus bufonoides Miranda-Ribeiro, 1920

Brachycephalus ephippium var. bufonoides Miranda-Ribeiro, 1920 .

Brachycephalus ephippium: Cochran, 1955 (part).

Brachycephalus bufonoides: Pombal, 2010 .

Lectotype. MZUSP 1459 (designated by Pombal 2010). Type locality: Serra de Macaé , Nova Friburgo, State of Rio de Janeiro, Brazil on September 1909 collected by Ernest Garbe. Paralectotype MZUSP 1458 , collected with the lectotype .

Referred specimens. The Serra Queimada trail (22°19’21’’S, 42°16’19’’W: datum WAG84; 1169 m above sea level), Área de Proteção Ambiental ( APA) de Macaé de Cima, municipality of Nova Friburgo, state of Rio de Janeiro, souTHeasTern Brazil: MNRJ 91685 (male) anD ZUFRJ 15424−26 (males), 15428−30 (males) collecTeD on 3 OcTober 2015 by M. Folly anD L. C. Amaral; anD MNRJ 91686 (female), MNRJ 91687−88 (males), ZUFRJ 15525 (male), ZUFRJ 15527 (male), ZUFRJ 15528−29 (females), ZUFRJ 15530−31 (males), ZUFRJ 15534−35 (males), and ZUFRJ 15536 (female) collected on 20 November 2015 by Manuella Folly, Lucas C. Amaral, Sergio P. de Carvalho-e-Silva, and José P. Pombal Jr.

Diagnosis. The specimens show absence of sternum; eight presacral vertebrae; palatal shelf of maxilla lacking pterygoid process; neopalatines and columella absents; arrow-shaped terminal phalanges; one phalange in Finger V, very short phalange in Toe I. These characters have been proposed as diagnostic of Brachycephalus ( Hedges et al. 2008) . Brachycephalus bufonoides belongs to this genus by the aforementioned characters and also by the small size (<18 mm in both males and females). Furthermore, it is assigned to the B. ephippium species group by: (1) bufoniform body shape; and (2) hyperossified skull and presence of hyperossified spinal processes of sacral and presacral vertebrae (intermediate condition sensu Clemente-Carvalho et al. 2012). Adults of B. bufonoides are distinguished from all of its congeners by the combination of the following characters: (1) skin on head and dorsum with dermal hyperossification; (2) skull with hyperossification of postorbital crests; (3) a pair of hyperossified bulges about equidistant between postorbital crests; (4) fourth presacral vertebra with transverse process hyperossified, ornamented and sacral diapophyses hyperossified, which can be seen externally (lineage of B. vertebralis sensu Condez et al. 2020 ); (5) presence of dermal ossification as separated bulges of each vertebrae; (6) general background color orange with different intensities of dark orange blotches on dorsum, including bordering of sacral region; (7) absence of osteoderms and presence of warts on the dorsolateral surface of body; (8) medium body size (SVL of adults: 12.0– 14.5 mm for males and 14.7–16.3 mm for females; Table 1 ); (9) rough dorsum; (10) advertisement calls with 13 to 17 pulses; (11) presence of pulse period modulation; and (12) advertisement calls with notes longer than 0.2 s (0.22 to 0.31 s).

Comparisons with the other species. Brachycephalus bufonoides ( Fig. 2 ) has skin on the head and vertebrae with dermal ossification, and a hyperossified process of the fourth vertebra. These characteristics distinguish the species from B. pernix species group ( B. actaeus , B. albolineatus , 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. pernix , B. pombali , B. quiririensis , B. tridactylus , and B. verrucosus ; Pombal et al. 1998; Ribeiro et al. 2005; Alves et al. 2006; Haddad et al. 2010; Garey et al. 2012; Pie and Ribeiro 2015; Bornschein et al. 2016; Monteiro et al. 2018a; Ribeiro et al. 2017; Pie et al. 2018) and from B. didactylus , B. hermogenesi , B. pulex , and B. sulfuratus that completely lack hyperossification ( Izecksohn, 1971; Giaretta and Sawaya, 1998; Napoli et al. 2011; Clemente-Carvalho et al. 2012; Condez et al. 2016).

By having a hyperossified dorsal shield or hyperossified spinal process of sacral and presacral vertebrae, Brachycephalus bufonoides belongs to B. ephippium species group. This species can be distinguished from B. darkside , B. ephippium , B. garbeanus , and B. margaritatus by its presence of dermal ossification on the vertebrae (bony shields on dorsum in these species: Pombal 2010; Pombal and Izecksohn 2011; Guimarães et al. 2017). Brachycephalus bufonoides has a transverse process of the fourth presacral vertebra and sacral diapophyses hyperossified and ornamented, which is very distinct of the large bone plate exhibited by B. ephippium , B. darkside , B. garbeanus , and B. margaritatus ; and also distinct from the comparatively less ornamented skeleton (intermediate condition sensu Clemente-Carvalho et al. 2012; B. vertebralis lineage sensu Condez et al. 2020); B. vertebralis lineage sensu Condez et al. 2020 of B. alipioi , B. crispus , B. guarani , B. nodoterga , B. pitanga , B. toby , and B. vertebralis ( Pombal and Gasparini 2006; Haddad et al. 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014). Absence of osteoderms distinguishes B. bufonoides species from B. crispus , B. margaritatus , and B. nodoterga . Brachycephalus bufonoides has skin on its head with dermal ossification and the presence of dermal ossification on the vertebrae while B. atelopoide has no dorsal shields and cephalic ossification ( Miranda-Ribeiro 1920; Pombal 2010).The orange coloration with different intensities of dark orange blotches on the dorsum of B. bufonoides differs from B. alipioi , B. pitanga , B. guarani , and B. toby (orange uniform in B. alipioi: Pombal and Gasparini 2006 ); orange with red irregular blotches in B. pitanga ( Alves et al. 2009) ; orange with a dark brown vertebrate stripe in B. guarani ( Clemente-Carvalho et al. 2012) ; orange with dorsal greenish in B. toby ( Haddad et al. 2010) . Further, B. bufonoides can be distinguished from B. crispus and B. nodoterga by its dorsum without dermal ossification like warts on the skin [with ossification is B. crispus and B. nodoterga ( Condez et al. 2014) ].

Redescription of the species. Measurements for 15 males and four females are given in Table 1 . Vocal slits not observed in males; body robust and bufoniform ( Figs. 1–3 ); head wider than long (HL/HW x = 0.51 in males and females); head length approximately 19% of SVL in males and 20% in females; snout short with length almost half of eye diameter (END/ED x = 0.56 in males and 0.57 in females), rounded in dorsal and lateral views; nostrils, directed anterolaterally, elliptical, not protuberant; canthus rostralis distinct, almost straight; loreal region vertical; eye not protruding dorsally, mean of eye diameter 57% of HL in males and 46% in females; tympanum not visible; supratympanic fold absent; a hyperossified postorbital crest; a pair of hyperossified bulges about equidistant between postorbital crest; tongue long and narrow, without indentation on its free posterior border; choanae elliptical, anterior to the eye; vomerine odontophores absent; premaxillary and maxillary teeth absent. Arm slightly shorter than forearm (AL/FAL x = 0.90 in males and 0.87 in females), slender; total arm length with mean of 42% SVL in males and 39% in females; hands with the same length in males (AL/HAL x = 1.0) and almost of the same length of upper arm in females (AL/HAL x = 0.95); Finger III and IV robust, distinct; IV longer than III; Finger II very short and Finger V vestigial; pointed tip of Finger III and IV; subarticular tubercles, inner, and outer metacarpal tubercles absent. Shank as long as thigh (SL/TL x = 0.96 in males and 0.95 in females); total leg length with mean of 84% of SVL in both males and females; thigh length larger than foot length (TL/FL x = 0.78 in males and 0.80 in females); Toe II, III, and IV distinct, robust; Toe I and V reduced; tip of Toe II rounded, tip of Toe III and IV pointed; relative lengTH of Toes I ≈ V <II <III <IV; subarTicular, inner anD ouTer meTaTarsal Tubercles absenT. Skin on HeaD, verTebral column and fourth vertebra with dermal hyperossifications; skin on dorsolateral surface of body, flanks, and dorsal surface of thighs granular; presence of warts on the dorsolateral surface of body; skin on venter and ventral surfaces of the legs smooth; granular skin on ventrolateral surfaces of body and area around the cloacal opening.

Coloration in life. General background coloration orange; dorsum with a dark orange blotch bordering the spine; protruding dorsal hyperossifications lighter orange than the dorsum color ( Fig. 1 ); finger IV and toe V with black tips; a light-yellow line below the eye, eye black in color. Dorsum varies from orange to light orange blotches bordering to the spine varying in different intensities.

Coloration in preservative and variation. Upper surfaces gray ( Figs. 3 A–D); protruding dorsal hyperossifications beige ( Fig. 3E ), flanks light brown; tips of Finger IV and toe V gray; a cream line below the eyes. Dorsal surfaces of body can be gray to brown, varying specially from below the head to the cloaca. In robust specimens, the ossified structures on the head, dorsum and diapophyses are less distinct ( Fig. 3D ) than thinner specimens that have such ossified structures much evident ( Figs. 3A, C ). The hyperossification in the spinal processes of presacral vertebrae is completely ossified forming an irregular (MNRJ 91688 and MNRJ 91687) or regular oval shape (ZU-FRJ 15428 and ZUFRJ 15529).

Natural history. Specimens of Brachycephalus bufonoides were active during the day were found on forest floor or amidst leaf litter. Some individuals were found under fallen tree trunks where the moisture is higher. Specimens were observed walking slowly on the leaf-litter. Generally, males were observed and heard calling under the leaves of the leaf-litter or branches, but they can be found calling exposed on the surface of leaf-litter or timber (males observed on cloudy and rainy days). Individuals were seen calling from both above and under the leaf-litter, except for one individual, which was calling on a fallen tree trunk.

Vocalizations. The individuals found calling in the field were recorded at air temperature of 20.8 oC, and relative humidity of the air of 78%. Calls were emitted as a regular series of low-intensity buzzes (around 120 dB at about 1-meter recording distance). The advertisement call duration is of up to 3 minutes (specimens usually stopped calling when disturbed), and consists of a series of regularly emitted notes, with constant general amplitude ( Fig. 4 ). Notes consisted of a number of pulses, ranging from 13 to 17 pulses/note (x = 15.05 ± 0.88; = 15; Mo = 16; n = 352), with most of them consisting of 14 to 16 pulses (96.7%). Amplitude modulation increased until the second quarter, with a mild descending modulation from end of second quarter until the end of the note. Note duration ranges from 222.09 to 308.53 ms (x = 271.33 ± 18.70; = 276.00; Mo = 290.07; n = 352). Inter-note interval duration ranges from 147.58 to 265.00 ms (x = 209.05 ± 24.68; = 219.00; Mo = 232.00; n = 298). Note period ranges from 401.82 to 545.00 ms (x = 482.10 ± 35.61; = 495.00; Mo = 503.00; n = 298). Note repetition rate ranges from 1.98 to 2.43 note/s (x = 2.15 ± 0.20; = 2.04; n = 3). Pulse period ranges from 9.89 to 25.13 ms (x = 18.18 ± 3.39; n = 729). There is a pulse period modulation to shorter duration of the periods starting through the second third of the note, and continuing modulation to longer duration of the periods through the final third of the note. Pulse repetition rate ranges from 47.55 to 71.28 pulses/s (x = 56.20 ± 4.81; = 55.01; Mo = 51.27; n = 352). Pulse envelope with decay steeper than attack. Dominant frequency of the call ranges from 4.13 to 4.88 kHz (x = 4.55 ± 0.14; = 4.5; Mo = 4.5; n = 344); minimum frequency ranges from 2.49 to 4.24 kHz (x = 3.38 ± 0.37; = 3.43; Mo = 3.37; n = 98), and maximum frequency ranges from 5.58 to 7.05 kHz (x = 6.268 ± 0.37; = 6.25; Mo = 5.94; n = 98).

Comparison with advertisement calls of species of Brachycephalus . Brachycephalus bufonoides differs from the other species of Brachycephalus by a number of distinct advertisement call parameters. Brachycephalus bufonoides has greater number of pulses (range = 13–17) than the species belonging to the B. pernix group: B. actaeus 2–3 pulses ( Monteiro et al. 2018a); B. albolineatus 1–3 pulses ( Bornschein et al. 2017); B. mirissimus 1–3 pulses ( Pie et al. 2018); B. olivaceus 2–3 ( Monteiro et al. 2018b); B. pernix : 3 pulses ( Wistuba 1998); B. quiririensis 3–4 pulses ( Monteiro et al. 2018b); B. tridactylus 1 pulse ( Garey et al. 2012), and also presents longer note duration in B. bufonoides : x = 271.33 ms, while in B. actaeus x = 4 ms ( Monteiro et al. 2018a); B. albolineatus x = 20 ms ( Bornschein et al. 2017); B. olivaceus x = 38 ms ( Monteiro et al. 2018b); B. pernix x = 60 ms ( Wistuba 1998); B. quiririensis x = 50 ms ( Monteiro et al. 2018b); B. tridactylus x = 110 ms ( Garey et al. 2012). It also differs acoustically from most of them by presenting shorter inter-note interval in B. bufonoides x = 0.27 s, while in B. actaeus x = 5.42 s ( Monteiro et al. 2018a); B. albolineatus x = 6.66 s (Bornschein et al. 2018); B. mirissimus x = 5.83 s ( Pie et al. 2018); B. olivaceus : x = 10.69 s ( Monteiro et al. 2018b); B. quiririensis : x = 12.58 s ( Monteiro et al. 2018b), and lower dominant frequency in B. bufonoides x = 4.6 kHz, while in B. actaeus x = 6.9 kHz ( Monteiro et al. 2018a); B. albolineatus x = 6.4 kHz (Bornschein et al. 2018); B. mirissimus x = 6.7 kHz ( Pie et al. 2018); B. olivaceus : x = 6.8 kHz ( Monteiro et al. 2018b); B. quiririensis x = 6.3 kHz ( Monteiro et al. 2018b). The dominant frequency of B. tridactylus x = 4.8 kHz ( Garey et al. 2012) overlaps with the range of B. bufonoides (4.1–4.9 kHz), and so does B. pernix 4.5–6.7, x = 5.6 kHz ( Wistuba 1998). Values for pulse repetition rate and note repetition rate also vary greatly.

The species from Brachycephalus ephippium group have an overall similar structure of the call, consisting in constant repetition of pulsed notes. Brachycephalus bufonoides can be distinguished from the other species of the B. ephippium group by a combination of different call structures. If differs most from B. darkside for presenting greater number of pulses ( B. bufonoides 13–17 pulses, x = 15) in B. darkside 5–8 pulses, x = 6 ( Guimarães et al. 2017), while is similar or overlaps parts of the range with the other species: B. crispus 7–12 pulses, x = 10 ( Condez et al. 2014); B. ephippium 5–15 pulses, x = 12 ( Pombal et al. 1994); B. pitanga : 7–14 pulses, x = 11 ( Araújo et al. 2012; Tandel et al. 2014). The note duration also is close to or overlaps some of its range with two of the species ( B. bufonoides : 221–309, x = 271 ms): B. crispus x = 280 ms ( Condez et al. 2014); B. pitanga : 150–250, x = 190 ms ( Tandel et al. 2014), x = 170 ms ( Araújo et al. 2012), but the value is lower in B. darkside x = 111 ms: Guimarães et al. 2017) and B. ephippium (x = 112 ms: Pombal et al. 1994). The interval between notes of B. bufonoides (148–265, x = 209 ms) differs mostly from that of B. crispus (x = 350 ms: Condez et al. 2014), but overlaps slightly with the other species ( B. darkside 122–215, x = 159.5 ms: Guimarães et al. 2017); B. ephippium (123–149, x = 134 ms: Pombal et al. 1994); B. pitanga (200–430, x = 280 ms: Tandel et al. 2014). The pulse repetition rate (x = 56.20 pulses/s in B. bufonoides ) is distinct from that of B. crispus x = 17.4 pulses/s: Condez et al. 2014) but is similar to the rate of B. darkside x = 56.9 pulses/s: Guimarães et al. 2017) and B. pitanga x = 62.4 pulses/s: Araújo et al. 2012). Furthermore, note repetition rate of B. bufonoides (x = 2.15 notes/s, or 129 notes/min) is similar to the rate of B. crispus x = 1.67 notes/s: Condez et al. 2014) and B. pitanga x = 159 notes/min: Araújo et al. 2012) but differs from that of B. darkside x = 211 notes/min: Guimarães et al. 2017). Brachycephalus bufonoides can be further distinguished from B. darkside for its higher dominant frequency ( B. bufonoides : 4.1–4.9 kHz, x = 4.6 kHz) while in B. darkside : 2.8–3.8 kHz ( Guimarães et al. 2017), but differ less from the other species ( B. ephippium : x = 3.9 kHz ( Goutte et al. 2017), lowest frequency = 3.4 kHz, highest frequency = 5.3 kHz ( Pombal et al. 1994), and overlaps with the range of B. crispus : x = 4.6 kHz: Condez et al. 2014 and B. pitanga : x = 4.9 kHz ( Araújo et al. 2012) and x = 4.8 kHz ( Tandel et al. 2014).

The advertisement call of B. hermogenesi (x = 6.8 kHz: Verdade et al. 2008) and B. sulfuratus (x = 6.7 kHz: Condez et al. 2016) differ from B. bufonoides by presenting higher dominant frequency. Some overlap between the note duration of B. sulfuratus (131–233, x = 195 ms: Condez et al. 2016) and B. bufonoides (221–309 ms) was noticed, however a greater number of pulses per note in B. bufonoides (x = 15 pulses) than in B. sulfuratus 7–11 pulses, x = 9 ( Condez et al. 2016) was observed, which results in difference in the pulse rate between B. bufonoides (x = 56.2 pulses/s) and B. sulfuratus (6.1–12.3, x = 9.3 pulses/s: Condez et al. 2016). The note repetition rate of B. bufonoides (x = 2.15 notes/s) is greater than B. hermogenesi (x = 1.09 notes/s: Verdade et al. 2008). Furthermore, even though the values were not given by Verdade et al. (2008), the analyzing the graphs provided, B. hermogenesi seems to also differ in number of pulses per note, its repetition rate, and other parameters.

Type locality and distribution. Brachycephalus bufonoides was originally collected from Serra de Macaé region ( Miranda-Ribeiro 1920) which was attributed to the municipality of Nova Friburgo and mountain range of municipality of Macaé, State of Rio de Janeiro (IBGE 1959). Bokermann (1966) attributed the type-locality of B. bufonoides to the municipality of Nova Friburgo, State of Rio de Janeiro, Brazil. A report presented by Ihering and Ihering (1911) on expeditions made by Museu Paulista (where E. Garbe worked) between 1906 and 1909, simply states the year and general locality names. Labels of bird specimens collected by E. Garbe in this same expedi-tion also failed to provide further information (L.F. Silveira, pers. comm.). Perhaps, nowadays it is impossible to determine the precise locality (or localities) where B. bufonoides was collected by E. Garbe. The Área de Proteção Ambiental (APA) de Macaé de Cima, municipality of Nova Friburgo, state of Rio de Janeiro ( Fig. 5 ), where the specimens of B. bufonoides were now collected (present study), is recognized as inserted in Serra de Macaé, the same mountain range of the type-locality of B. bufonoides .

Molecular analysis. Considering DNA sequences of the 16S gene (999 aligned basepairs, 78 terminals), the topology generated, on the basis of both phylogenetic analyses (BI and MP), supported the recognition of Brachycephalus bufonoides and of B. ephippium species group (specimens appear clustered into eight exclusive lineages, which are supported by high posterior probability and bootstrap values ( Fig. 6 ), that corroborates the morphological identification.

Furthermore, the population of Brachycephalus bufonoides was recovered as a monophyletic group. The genetic distances between this species and the sister clade composed by B. alipioi , B. pitanga , B. vertebralis , B. toby , and B. nodoterga range from 3.2–5.8%, corroborating the distinction between these species. We found low genetic distances among four of five analyzed specimens (ZUFRJ 15424, 15426–28) of B. bufonoides , with maximum values corresponding to 0.001% between the specimen from Serra Queimada trail, APA de Macaé de Cima, Lumiar, municipality of Nova Friburgo, RJ. However, the specimen ZUFRJ 15429 had the value between 0.021 and 0.022% of distance from the other four specimens.

Osteology of Brachycephalus bufonoides

Skull ( Figs. 7–8 )

Shape and proportions. The skull is wider than long (length/width range 74–84%, N = 3). The length of the orbit is about half the total length of the skull (orbit/length 47–59%). The skull is widest at the prootics, and the jaw articulation lies well anterior of the posterior end of the skull at the occipital condyles. The jaws are relatively short, with the posterior apex lying to the level of the optic fenestra. Dermal roofing bones of the skull ornamented with co-ossification of skin to bones. Nasal, sphenethmoid, frontoparietals, prootics, and exoccipitals are synostosed, hyperossified and sculptured forming a dorsal cranial plate.

Dermal investing bones. Nasals – sculptured. These bones are completely synostosis with sphenethmoid. An attenuate maxillary process is not in contact with the preorbital process of the pars facialis of the maxilla. ZUFRJ 15536 has no sculpture on the nasal bone. Frontoparietals – hyperossified and sculptured. The paired frontoparietals overlie the taenia tecti marginalis of the braincase. The bones are synostosed medially and completely obscure the frontoparietal fontanelle. Frontoparietals are also synostosed with nasals, sphenethmoid and fused exoccipitals and prootics.

Neurocranium. Sphenethmoid – hyperossified and sculptured. This bone is fused with frontoparietals and nasals. The optic fenestra cartilage is absent. The sphenethmoid of two specimens (ZUFRJ 15536 and ZUFRJ 15535) is not sculptured. Fused Exoccipitals and Prootics – hyperossified and sculptured. The exoccipital portions of this composite element form the posterior end of the braincase. The prootic portion of the bone forms the posterolateral walls of the braincase and, together with its cartilaginous crista parotica, the middle ear; the head of the squamosal articulates with the lateral margin of the crista parotica. The epiotic eminence (anterior and posterior) is indistinguishable.

Ventral investing bones. Parasphenoid –The cultriform process is synostosed with sphenethmoid. The parasphenoid alae are broad and posterolaterally oriented beneath the otic capsule; the distal margins of the alae are truncate and terminate after the midpoint of the otic capsule where almost contact the medial process of the pterygoid. The parasphenoid terminates in a broad truncate or triangular (MZUSP 1459) posteromedial process lying or not (MZUSP 1459) the foramen magnum. Neopalatine – absent. Vomers – reduced. The anterior process is curved, rounded and extends toward the maxilla. The reduced prechoanal process forms the anterior and anteromedial mar-gin of the choana and terminates in a rounded point. 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 specimen (ZUFRJ 15535) has broad separation between vomers. ZUFRJ 15536 has greater length of all three processes than ZUFRJ 15430 and 15535. The dentigerous process is absent.

Maxillary arcade. Premaxillae –Each premaxilla is broad and are separated from each other by a short space in front view. The pars dentalis of each premaxilla is lacking. In front view, the height of the alary process corresponds to half the length of premaxillae, and the distal tip of each alary process is bifurcate (“U”-shape). 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. The posterior end of the maxilla is sharp; and does not have teeth. The pars facialis is high, thin and reduced, extending for about half the length of the maxilla. Quadratojugals – absent.

Suspensory apparatus. Pterygoids –The anterior ramus of each pterygoid is long; cylindrical; terminated in a truncate point; and extends to the median margin of the maxilla. The medial ramus is short; slightly laminar; has a truncated end; and invests the prootic. The posterior ramus is short; triangular; has a sharp point. Squamosals –They are composed of three rami that give to the squamosal a “L” shape: the ventral ramus, the otic ramus (posterior ramus) and the zigomatic ramus (anterior ramus). The ventral ramus is the longest of the three, and has a truncated distal end, which is wider than the proximal portion. In lateral view, the zigomatic ramus is shorter than the ventral ramus and the distal end is sharp or rounded (MZUSP 1459), toward to the maxilla, but does not contact it. In lateral view the otic ramus is shorter than the zigomatic ramus, and the distal end is rounded. In dorsal view, the distal end of the otic ramus is truncated, toward the parotic plate, but does not overlap it. Mandible –The mentomeckelian bones are located in the anterior part of the mandible and are separated from each other by a short space. In frontal view, the mentomeckelians have a sharp point anteriorly. They 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. Angulosplenial bones situated in the lateroposterior part of the mandible in ZUFRJ 15430 and 15535, corresponding to about 80% of the lower jaw’s length. ZUFRJ 15536 have longer angulosplenial corresponding to around 90% of lower jaw’s length. The anterior end of each angulosplenial is roundly pointed, and the posterior end is robust and rounded. The posterior end of the angulosplenial bears well-developed pars articularis process.

Hyolaryngeal Skeleton ( Fig. 9 )

Hyoid plate is rectangular i.e., longer than wide. Length of hyoid plate around four times its smallest width. Anterior processes long and straight, forming a deep hyoglossal sinus, which deepens to nearly the height of the alary processes. Alary and posterolateral processes much reduced. Posteromedial processes diverging widely to embrace a broad larynx. Arytaenoids of cricoid are short, semicircular and narrowly separated from each other. Cricoid with short oesophageal process. Laterally contiguous with the posteromedial process, the lateral process of the cricoid fitting over the end of the posteromedial process.

Postcranium

Pectoral girdle ( Fig. 10A ). Clavicle, coracoid, and scapula fused and completely ossified; procoracoid and epicoracoid fused with coracoid but separated from the clavicle by large fenestrae; suprascapula not expanded, anterior half ossified as cleithrum; omosternum not visible and sternum absent.

Vertebral column ( Figs. 10B–D ). Vertebral column composed by eight presacral, non-imbricate vertebrae. First presacral vertebra (atlas) lacks transverse process; transverse of Presacrals II–IV bears moderately stout processes, and Presacrals V–VIII are shorter, less robust processes. Transverse processes of Presacrals III–VI perpendicular to the notochordal axis, those of the Presacrals II, VII–VIII directed anteriorly and of the Presacrals V–VI directed posteriorly. Lengths of the transverse process of presacrals along with that of the sacral diapophyses: IV> SD> III> V> VII>VI> II-VIII. Fourth presacral vertebra with transverse process hyperossified and ornamented which can be seen externally. Sacral diapophyses moderately expanded and directed posteriorly, distal end of diapophyses with a flat, slightly calcified cartilage that articulates with the ilial shaft of the pelvic girdle. Sesamoids on both sides of sacral diapophyses can be seen externally. There are two types of bony elements associated with the vertebral column: (1) spinal plates - lies dorsal to the vertebra. All vertebrae have distinct spinal plates, except for Presacrals I and II that have fused spinal plates. ( 2) ZUFRJ 15535 and 15430 also have spinal plates above the sacrum (absent in ZUFRJ 15536 ); and, (2) the paravertebral plates - associated with the transverse processes of vertebrae IV.

Manus ( Fig. 11A ). Phalangeal formula of the 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 ( Fig. 10A ). One sesamoid occurring at level of the basis of metatarsal V in ZUFRJ 15536.

Pes ( Fig. 11B ). Phalangeal formula of foot 1–2–3–4–1. Tarsus ( Fig. 10B ) 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, also with metatarsal II and IV and with distal tarsal I. There are 2 small sesamoids under the tarsals. Prehallux has one very reduced element. Tips of the terminal phalangeal elements of toes II–IV arrow-shaped, toes I and V reduced with tips of terminal phalangeal elements pointed, elongate on digit IV.

Osteological comparisons of species of Brachycephalus . Trends of reduction and loss of skull bones were observed among groups of species in Brachycephalus . Among these reductions and losses, columellae are always absent in all analysed species. Species of B. pernix group have: (1) reduced neopalatines in B. albolineatus ( Bornschein et al. 2016) , B. brunneus ( Ribeiro et al. 2005) , B. coloratus and B. curupira ( Ribeiro et al. 2017) and B. izecksohni ( Ribeiro et al. 2005) or absence of neopalatines in B. actaeus ( Monteiro et al. 2018a) , B. ferruginus and B. pombali ( Alves et al. 2006) ; (2) quadratojugals present in B. actaeus , B. albolineatus , B. coloratus , B. curupira , B. ferruginus , B. pombali , B. brunneus , B. izecksohni , and B. pernix ( Ribeiro et al. 2005; Alves et al. 2006; Bornschein et al. 2016; Ribeiro et al. 2017; Monteiro et al. 2018a); (3) maxillary odontoids present, few in number (6-8) in B. actaeus , B. brunneus , B. curupira , B. ferruginus , B. izecksohni , B. pernix and B. pombali ( Ribeiro et al. 2005; Alves et al. 2006; Ribeiro et al. 2017; Monteiro et al. 2018a) or absent in B. coloratus ( Ribeiro et al. 2017) ; (4) vomers reduced in B. actaeus , B. brunneus , B. curupira , B. ferruginus , B. izecksohni , B. pernix and B. pombali ( Ribeiro et al. 2005; Alves et al. 2006; Ribeiro et al. 2017; Monteiro et al. 2018a), co-ossified with sphenethmoid in B. albolineatus and B. coloratus ( Bornschein et al. 2016; Ribeiro et al. 2017); (5) squamosals robust with anterior zygomatic ramus long in B. actaeus , B. albolineatus , B. brunneus , B. coloratus , B. curupira , B. ferruginus , B. izecksohni , B. pernix and B. pombali ( Ribeiro et al. 2005; Alves et al. 2006; Bornschein et al. 2016; Ribeiro et al. 2017; Monteiro et al. 2018a); (6) pterygoids relatively robust in B. actaeus , B. albolineatus , B. brunneus , B. coloratus , B. curupira , B. ferruginus , B. izecksohni , B. pernix and B. pombali ( Ribeiro et al. 2005; Alves et al. 2006; Bornschein et al. 2016; Ribeiro et al. 2017; Monteiro et al. 2018a); (7) cultriform process of parasphenoid not fused to sphenethmoid in B. actaeus , B. brunneus , B. coloratus , B. curupira , B. ferruginus , B. izecksohni , B. pernix and B. pombali ( Ribeiro et al. 2005; Alves et al. 2006; Ribeiro et al. 2017; Monteiro et al. 2018a), fused in B. albolineatus ( Bornschein et al. 2016) . Species of B. didactylus group have ( Ribeiro et al. 2005; Alves et al. 2006; Condez et al. 2016): (1) absent neopalatines in B. hermogenesi and B. sulfuratus ; (2) quadratojugals present in B. hermogenesi and B. sulfuratus ; (3) maxillary odontoids present, numerous in B. hermogenesi ; (4) vomers reduced in B. hermogenesi ; (5) squamosals with anterior zygomatic ramus reduced and ramus ventral laterally expanded in B. hermogenesi ; (6) pterygoids relatively robust B. hermogenesi ; (7) cultriform process of parasphenoid not fused to sphenethmoid in B. hermogenesi or fused in B. sulfuratus . Species of B. ephippium group have: (1) absence of neopalatine in B. bufonoides , B. crispus , B. darkside , B. guarani , B. toby ( Haddad et al. 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014; Guimarães et al. 2017); (2) absence of quadratojugals in B. bufonoides , B. darkside , B. ephippium , B. guarani , B. toby ( Campos et al. 2010; Haddad et al. 2010; Clemente-Carvalho et al. 2012; Guimarães et al. 2017), present in B. crispus ( Condez et al. 2014) ; (3) maxillary odontoids absent in B. bufonoides , B. crispus , B. darkside , B. guarani , B. toby ( Haddad et al. 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014; Guimarães et al. 2017); (4) vomers reduced in B. bufonoides , B. darkside ( Guimarães et al. 2017) and B. ephippium ( Campos et al. 2010) , fused in B. crispus and B. guarani ( Clemente-Carvalho et al. 2012; Condez et al. 2014); (5) squamosals with zygomatic ramus short and ornamented in B. bufonoides , B. crispus , B. darkside , B. ephippium , B. guarani ( Campos et al. 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014; Guimarães et al. 2017); (6) fusion of the cultriform process of parasphenoid to sphenethmoid in B. crispus ( Condez et al. 2014) and B. bufonoides and not fusion on B. darkside , and B. ephippium ( Campos et al. 2010; Guimarães et al. 2017).

Regarding the appendicular osteology, pes of B. actaeus , B. albolineatus , B. crispus , B. darkside , B. didactylus , B. ephippium , B. ferruginus , B. guarani , B. pombali , and B. toby ( Alberch and Gale 1985; Fabrezi 2001; Alves et al. 2006; Haddad et al. 2010; Clemente-Carvalho et al. 2012; Bornschein et al. 2016; Guimarães et al. 2017; Monteiro et al. 2018a) have one prehallical element as well as in the B. bufonoides described in the present study. On the other hand, manus has been described with two minutes elements for B. actaeus ( Monteiro et al. 2018a) , B. ephippium ( Fabrezi 2001) as well as in B. bufonoides , however only one element seems to be present in prepollex of B. albolineatus , B. crispus , B. darkside , B. ferruginus , B. guarani , B. pombali and B. toby (see Alves et al. 2006; Haddad et al. 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014; Bornschein et al. 2016; Guimarães et al. 2017). Moreover, the element y present on manus of B. bufonoides , as well as in B. ephippium (see Andersen 1978) and B. albolineatus ( Bornschein et al. 2016) , seems not to be present in B. ferruginus and B. pombali (see Alves et al. 2006).

Figures

FIGURE 1. Brachycephalus bufonoides (ZUFRJ 15525) in life.

FIGURE 2. Brachycephalus bufonoides (ZUFRJ 15525). Dorsal view of the body (A), ventral views of the right hand (B), the right foot (C), and lateral view of the head (D). Scale is 5 mm.

FIGURE 3. Variation in preserved specimens of Brachycephalus bufonoides. (A) ZUFRJ 15428 (male; SVL 13.6 mm), (B) ZUFRJ 15529 (female; SVL 16.3 mm), (C) MNRJ 91688 (male; SVL 12.0 mm), (D) MNRJ 91687 (male; SVL 13.0 mm), (D–E) ZUFRJ 15525 (male; SVL 14.1 mm) in dorsal and ventral views, (G–H) B. bufonoides lectotype (MZUSP 1459; SVL 13.5 mm) in dorsal and ventral views and, (I–J) B. bufonoides paralectotype (MZUSP 1458; SVL 16.4 mm) in dorsal and ventral views. Scale bar = 3 mm.

FIGURE 4. Advertisement call of Brachycephalus bufonoides (adult male, ZUFRJ 15425) from Área de Proteção Ambiental (APA) de Macaé de Cima, municipality of Nova Friburgo, state of Rio de Janeiro, Brazil. Oscillogram (above) and spectrogram (middle) of sequence of seven notes. Band filter of 1000 Hz applied. Oscillogram of a note (left below). Notice the slight change in pulse period and amplitude throughout the note. Scatterplot (right below) depicting the pulse period modulation in 21 notes, with line representing a moving average tendency line (rolling window = 45). X-axis: Time of Note not in seconds but normalized.

FIGURE 5. Geographic location of recent collected specimens of Brachycephalus bufonoides. Área de Proteção Ambiental (APA) de Macaé de Cima, municipality of Nova Friburgo, center of the state of Rio de Janeiro, Brazil.

FIGURE 6. Bayesian Inference tree (lnL = -4368.48) for 16S sequences (999 aligned basepairs, 78 terminals; 2 partitions = for the two fragments: each GTR+G) of species of Brachycephalus. Thicker clades refer to those also found by maximum parsimony. Clade support values include posterior probabilities above branches and parsimony bootstrap below branches. Branches were colored according to their classification into B. pernix (blue), and B. ephippium (green), according to Ribeiro et al. (2015). Photos provided by C. de Luna-Dias (B. ephippium), R. Sawaya (B. nodoterga and B. hermogenesi), and C.F.B. Haddad (B. vertebralis). Photos modified of Alves et al. (2009) (B. pitanga), Haddad et al. (2010) (B. toby), Condez et al. (2016) (B. sulfuratus), Ribeiro et al. (2015) (B. auroguttatus, B. fuscolineatus, B. leopardus, B. mariaeterezae, and B. verrucosus), Bornschein et al. (2016) (B. albolineatus), Ribeiro et al. (2017) (B. coloratus), and Monteiro et al. (2018a) (B. actaeus).

FIGURE 7. Skull of Brachycephalus bufonoides. (A) dorsal and (B) ventral views (ZUFRJ 15536), (C) variation of dorsal view (ZUFRJ 15535), and (D) mandible (ZUFRJ 15535). Scale bar: 1 mm.

FIGURE 8. High-resolution computed tomography (Micro-CT) scans of the lectotype of Brachycephalus bufonoides (MZUSP 1459) showing osteological features. (A) Dorsal view of the skeleton and (B) vertebral column; (C) right foot in plantar view; (D) left hand in palmar view; (E) pectoral girdle in ventral view; (F) dorsal, (G) ventral, and (H) lateral views of the skull (with the lower jaw). Scale bars equal 5 mm.

FIGURE 9. Hyolaryngeal skeleton of Brachycephalus bufonoides in ventral view (ZUFRJ 15535). Scale bar: 1 mm.

FIGURE 10. Postcranium of Brachycephalus bufonoides. (A) Pectoral girdle in dorsal view (ZUFRJ 15536); (B–D) vertebral column in dorsal view (ZUFRJ 15430, ZUFRJ 15535 and ZUFRJ 15536, respectively). Scale bar: 1 mm.

FIGURE 11. Manus and pes of Brachycephalus bufonoides (ZUFRJ 15535). (A) Dorsal views of manus and (B) pes. Scale bar: 1 mm.

Tables

TABLE 1. Measurements in millimeters of collected specimens of Brachycephalus bufonoides. Abbreviations are mean (x); standard deviation (SD); snout–vent length (SVL); axilla–groin length (AGL); head length (HL); head width (HW); nostril diameter (ND); internostril distance (IND); eye diameter (ED); eyelid width (EW); interorbital distance (IOD); eye–nostril distance (END); nostril–tip of snout distance (NSD); thigh length (THL); shank length (SL); foot length (FL); Toe-III length (TL); arm length (AL); forearm length (FAL); hand length (HAL); and Finger-IV length (FIL).

		Males (n=14)			Females (n=4)
	x	SD	Range	x	SD	Range
SVL	13	0.7	12.0–14.5	15.5	0.5	14.7–16.3
AGL	4.7	0.9	2.7–6.1	6.3	0.9	5.5–7.8
HL	2.6	0.3	2.2–3.3	3.1	0.2	2.9–3.4
HW	5.5	0.1	5.3–5.7	6	0.1	5.8–6.2
ND	0.4	0.1	0.2–0.5	0.4	0.04	0.4–0.5
IND	1.8	0.1	1.6–2.1	1.9	0.02	1.89–1.95
ED	1.5	0.1	1.2–1.7	1.4	0.05	1.3–1.5
EW	1	0.1	0.8–1.2	1.07	0.05	1.0–1.1
IOD	2.4	0.1	2.1–2.6	2.6	0.05	2.5–2.7
END	0.8	0.1	0.6–1.0	0.8	0.05	0.7–0.9
NSD	0.6	0.1	0.4–0.8	0.6	0.09	0.5–0.7
THL	5.8	0.4	5.0–6.5	6.6	0.2	6.3–6.9
SL	5.5	0.3	5.0–6.2	6.3	0.02	6.3–6.36
FL	7.4	0.3	6.8–7.9	8.3	0.3	7.8–8.8
TL	2.9	0.2	2.3–3.1	3.2	0.1	3.0–3.3
AL	2.6	0.3	1.8–3.2	2.8	0.2	2.4–3.1
FAL	2.9	0.2	2.5–3.3	3.2	0.1	3.1–3.4
HAL	2.6	0.1	2.4–2.9	3	0.06	2.9–3.0
FIL	0.8	0.1	0.7–1.0	0.9	0.08	0.8–1.0

TABLE 1. Measurements in millimeters of collected specimens of Brachycephalus bufonoides . Abbreviations are mean (x); standard deviation (SD); snout–vent length (SVL); axilla–groin length (AGL); head length (HL); head width (HW); nostril diameter (ND); internostril distance (IND); eye diameter (ED); eyelid width (EW); interorbital distance (IOD); eye–nostril distance (END); nostril–tip of snout distance (NSD); thigh length (THL); shank length (SL); foot length (FL); Toe-III length (TL); arm length (AL); forearm length (FAL); hand length (HAL); and Finger-IV length (FIL).