Bioacoustics of Physalaemus claptoni

Advertisement call . The advertisement call of Physalaemus claptoni sp. nov. ( Fig. 8A, B ; Table 2 ; n=45 calls from five males; Audio S1) consists of two types of multipulsed notes (herein referred as notes A and B) released in sequence. It is formed by 65–93 pulses, has duration ranging from 0.80– 1.28 s (n=45 advertisement calls), interval between notes A and B from 0.10– 0.19 s (n=45 intervals) and between advertisement calls varying from 0.14– 1.38 s (n=41 intervals). Note rate within advertisement call varied from 1.56–2.51 notes/s (n=90 notes). Note A (n=154 notes) is shorter than B, with duration varying from 0.11– 0.16 s , is formed by 12–17 pulses emitted at a rate of 86.7–119.3 pulses/s. Note A was released at a rate of 0.53–0.63 notes/s. Pulses of note A are homogeneously distributed and have ascending amplitude until mid-note then descending towards the end of the note, being fairly symmetric (peak amplitude from 0.44–0.59). Notes A were also observed being released in sequences without note B, and, sometimes, between advertisement calls. In this case, we considered each note A as a single call. The interval between single A note calls emitted in sequences ranged from 0.7– 1.9 s (n=113 intervals). Note B (n=45 notes, from four males) duration varies from 0.67– 1.01 s and is formed by 53–81 pulses emitted at a rate of 67.5–92.1 pulses/s. Note B was released at a rate of 0.32–0.61 notes/s. Pulses of note B are arranged in 5–7 pulses group, with relative amplitude lower than that of note A. Within those groups, pulses are also homogeneously distributed and with ascending amplitude until its midpoint, then descending towards the end of the pulse group. The first and second pulse groups are generally closer and even in contact to each other than are the others (See Fig. 8 A ). The other pulse groups of note B may have a complete amplitude modulation in between groups, or not. In the latter case, there are a few isolated and low-amplitude pulses in between groups. Frequency bandwidth of note A (n=154 notes) is between 1453.1–3140.6 Hz and of note B (n=45 notes) is between 1335.1–3375.0 Hz. Dominant frequency of note A (n=154 notes) ranged from 1734.4–2765.6 Hz and of note B (n=45 notes) from 1507.3–2859.4 Hz.

Distribution and natural history . Physalaemus claptoni sp. nov. is so far only known from its type locality in the highlands of the Serra do Cipó, southern Espinhaço Range, State of Minas Gerais , southeastern Brazil ( Fig. 11 ). The new species reproduces mostly on small still water collections associated with the edge of a hilltop forest fragment surrounded by campo rupestre (or rupestrian grasslands) that becomes slightly flooded during the rainy season. When collected on the outside border of the forest fragment, males were observed calling from the base of dense grass. When captured inside the forest fragment, males were hidden on the leaf litter, generally close to or inside small puddles. Due to the small species size and difficult access to calling sites, we struggled to collect the specimens to make the description, with at least seven field trips from 2010 to 2017 . Most of the type series were collected in 2014 when the open area around the forest fragment was burned right before the rainy season, strongly decreasing the density and size of the grass, making it easier to see and collect the calling males. Moreover, this occasion was the one with the densest fog and was when more calling males could be identified, with approximately 30 individuals. During the other expeditions to the same site, the number of calling males varied from zero to approximately 12 individuals.

Advertisement call of Physalaemus rupestris . The advertisement call of Physalaemus rupestris ( Fig. 8C, D ; Table 2 ; n=44 calls from three males; Audio S2) consists of two types of multipulsed notes (herein referred as notes A and B) released in sequence. It is formed by 52–85 pulses, has duration ranging from 1.51– 2.2 s (n=44 advertisement calls), interval between notes A and B from 0.22– 0.42 s (n=44 intervals) and between advertisement calls varying from 0.26– 1.75 s (n=24 intervals). Note rate within advertisement call varied from 0.91–1.32 notes/s. Note A (n=93 notes, from three males) is shorter than B, with duration varying from 0.05– 0.10 s , is formed by 8–11 pulses emitted at a rate of 115.4–183.7 pulses/s. Note A was released at a rate of 0.42–0.76 notes/s. Pulses of note A present descending amplitude modulation and are homogeneously distributed, with the exception of the last two or three, which are increasingly separate to each other. Notes A were also observed being released in sequences without note B, and, sometimes, between advertisement calls. In this case, we are considering each note A as a single call. The interval between single A note calls emitted in sequences ranged from 0.22– 5.27 s (n=64 intervals). Note B (n=44 notes, from three males) duration varies from 1.14– 1.78 s and is formed by 41–75 pulses emitted at a rate of 31.4–43.1 pulses/s. Note B was released at a rate of 0.20–0.34 notes/s. Pulses of note B present descending amplitude modulation and are homogeneously distributed, with the exception of the first three to five, which are usually closer to each other, and the last eight to fifteen, which are increasingly close to each other. In the middle of note B, some pulses may be closer to each other than others, but it occurs with just a few pulses and seemingly at random. Frequency bandwidth of notes A (n=93 notes) and B (n=44 notes) are from 1875.0–3750.0 Hz. Dominant frequency of notes A and B ranged from 3046.9–3421.9 Hz.

Comparison with other species . The presence of an arrow-shaped blotch on the dorsum of the body distinguishes Physalaemus claptoni sp. nov. from all the species of the Physalaemus cuvieri Clade ( sensu Lourenço et al. 2015 ). Additionally, the absence of tarsal tubercle distinguishes P. claptoni sp. nov. from the species of the Physalaemus cuvieri Clade, except P. lateristriga , P. olfersii , P . orophilus , and P. maximus of the Physalaemus olfersii species group, from which the new species is promptly distinguishable by the smaller size of adult males at 16.2–18.2 mm ( P. lateristriga , P. olfersii , P . orophilus , and P. maximus combined SVL of adult males= 23.1–44.8 mm ). Within the P. signifer Clade, the presence of a longitudinal light stripe over the urostyle region distinguishes P. claptoni sp. nov. from all the other species, except from P. rupestris ( Fig. 9 , S 1 ), which also has this stripe over the urostyle region ( Fig. 9A ). By having belly and ventral surface of thigh marbled with dark gray irregularly shaped blotches on a bluish background in live specimens, P. claptoni sp. nov. is set apart from P. angrensis , P. atlanticus , P. nanus , and P. spiniger (belly with flashy orange blotches in those species), from P. maculiventris (which has a pale chest and belly, with posterior region of belly and ventral surface of thigh showing bold black blotches), and from P. obtectus (which has belly with flashy red blotches). The absence of reddish coloration over the axillar and inguinal regions in live individuals distinguishes P. claptoni sp. nov. from P. deimaticus and P. erythros (reddish coloration present over the axillary and inguinal regions on those species). The bright orange dots scattered on the dorsum of the head, upper lip, and body in live specimens of P. claptoni sp. nov. distinguishes it from all the other species within the P. signifer Clade. Ventral surfaces of hand and foot are red in live individuals, which distinguishes P. claptoni sp. nov. from P. angrensis , P. atlanticus , P. maculiventris , P. spiniger (ventral surface of hand and foot orange in these species), and from P. bokermanni , P. caete , P. camacan , P. irroratus , P. moreirae , P. nanus , and P. nattereri (which lack aposematic coloration on ventral surfaces of hand and foot). The small size of P. claptoni sp. nov. adult males at 16.2–18.2 mm promptly distinguishes it from P. atlanticus , P. caete , P. camacan , P. crombiei , P. deimaticus , P. erythros , P. irroratus , P. maculiventris , P. moreirae , P. nattereri , and P. obtectus (combined SVL= 18.9–50.6 mm ). The presence of a non-divided nuptial pad in males distinguishes P. claptoni sp. nov. from P. angrensis , P. atlanticus , P. bokermanni , P. caete , P. camacan , P. crombiei , P. deimaticus , P. erythros , P. moreirae , P. nanus , P. obtectus , P. rupestris ( Fig. 7B ), P. signifer , and P. spiniger (nuptial pad divided in those species). Additionally, P. claptoni sp. nov. is distinguished from P. rupestris by having brown nuptial pad (nuptial pad white-cream in P. rupestris ; Fig. 7B ) and by the proportion between END/ED in males ranging from 0.85–0.93 (END/ED of P. rupestris males ranges from 0.75–0.82). The absence of supernumerary tubercles on foot distinguishes P. claptoni sp. nov. from P. angrensis , P. caete , P. camacan , P. crombiei , P. irroratus , P. moreirae , P. signifer , and P. spiniger (supernumerary tubercles present on foot of those species). The absence of a tarsal fold distinguishes P. claptoni sp. nov. from P. angrensis , P. atlanticus , P. bokermanni , P. caete , P. camacan , P. crombiei , P. irroratus , P. moreirae , P. nanus , P. obtectus , P. signifer , and P. spiniger (tarsal fold present in those species). The smooth texture of posterior region of belly and ventral surfaces of thigh in P. claptoni sp. nov. distinguishes it from P. camacan and P. irroratus (posterior region of belly and ventral surface of thigh granulated in those species). The advertisement call of P. claptoni sp. nov. is composed of two note types (notes A and B), setting it apart from P. atlanticus , P. angrensis , P. bokermanni , P. camacan , P. crombiei , P. erythros , P. maculiventris , P. moreirae , P. obtectus , and P. signifer (advertisement call has only one note type in those species). The advertisement call of P. claptoni sp. nov. and P. rupestris are both composed of two note types (A and B), however are promptly distinguishable from each other by its duration, which ranges from 0.80– 1.28 s in P. claptoni sp. nov. and from 1.51– 2.20 s in P. rupestris . Additionally, note A of P. claptoni sp. nov. has pulses with an ascending amplitude until mid-note and then descends towards the end of the note, while note A of P. rupestris has only descending amplitude modulation; note B of P. claptoni sp. nov. has pulses arranged in 5 to 7 pulse groups, whereas note B of P. rupestris has pulse grouping only at the onset and final portion of the note. Dominant frequency of note A in P. claptoni sp. nov. varies from 1734.4–2765.6 Hz and in P. rupestris from 3046.9–3421.9 Hz. Dominant frequency of note B in P. claptoni sp. nov. ranges from 1507.3–2859.4 Hz and in P. rupestris from 3046.9–3421.9 Hz.

Diagnosis . The new species is distinguished from all the congener species by the following combination of character states: (1) presence of an arrow-shaped blotch on the dorsum of the body ( Figs. 1A , 3 , 4 , 6A ); (2) presence of a median longitudinal light stripe over the urostyle region ( Figs. 1A , 3 , 4 , 6A ); (3) belly and ventral surface of thighs marbled with dark gray irregularly shaped blotches, on a bluish background ( Fig. 5 ); (4) absence of reddish coloration over the axillar and inguinal regions in live individuals; (5) bright orange dots scattered on the dorsum of the head, upper lip, and body, in live individuals ( Figs. 3 , 4 ); (6) ventral surface of hand and foot red in live individuals; (7) small size (adult male SVL= 16.2–18.2 mm , Table 1 ); (8) presence of a brown, non-divided, nuptial pad in males ( Fig. 7A ); (9) END/ED in males ranging from 0.85–0.93 ( Table 1 ); (10) supernumerary tubercles on foot absent ( Fig. 2D ); (11) tarsal fold absent ( Fig. 1B ); (12) tarsal tubercle absent; (13); texture of posterior region of belly and ventral surface of thigh smooth ( Fig. 1B ); (14) advertisement call composed of two note types (note A + B); (15) advertisement call duration of 0.80– 1.28 s ( Fig. 8A ; Table 2 ); (16) note A with ascending amplitude until mid-note then descending towards the end of the note; (17) note B with pulses arranged into 5–7 groups; (18) dominant frequency of note A from 1734.4–2765.6 Hz; and (19) dominant frequency of note B from 1507.3–2859.4 Hz ( Fig. 8 ).