Bioacoustics of Brachycephalus bufonoides

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.

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 ).

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.