Bioacoustics of Boana guarinimirim

Diagnosis .— Boana guarinimirim sp. nov. can be distinguished from most of its congeners of the B. pulchella group by the following combination of characters: 1) dorsal pattern composed of longitudinal lines and stripes; 2) a small SVL of 24.1–28.1 mm (26.0 6 1.1; Table 3 ) in males; 3) no spots or vertical bars on hidden surfaces of legs, groin, and flanks; 4) presence of calcar appendage; 5) presence of supracloacal crest (characters 4, 5 in Fig. 3 ); 6) relatively conspicuous discs on fingers and toes; 7) HW 30–34% ( 32 6 1 ) of SVL; 8) vocal repertoire composed of two different types of calls: the ‘‘A'' call (a short and multipulsed note) and the ‘‘B'' call (a trilled note); 9) call dominant frequency (both call types) ranging from 4.5 to 6.6 kHz (5.8 6 0.5). Morphological Comparisons .— Boana guarinimirim sp. nov. can be distinguished from all species in the B. pulchella group, except from those in the B. polytaenia clade, by the combination of the following morphological traits: the presence of dorsal pattern composed of longitudinal lines and stripes (absent in B. aguilari , B. balzani , B. caipora , B. callipleura , B. cambui , B. cordobae , B. curupi B. cymbalum , B. ericae , B. freicanecae , B. gladiator , B. joaquini , B. marginata , B. marianitae , B. melanopleura , B. palaestes , B. poaju , B. prasina , B. pulchella , B. riojana , B. semiguttata , and B. stellae ); the smaller male SVL of 24.1–28.1 mm ( B. aguilari [33.7–43.8 mm], B. balzani [33.3–49.9 mm], B. bischoffi [36.0–47.0 mm], B. callipleura [37.2–43.3 mm], B. cordobae [39.0–50.0 mm], B. cymbalum [44.8– 46.2 mm], B. freicanecae [37.3–42.2 mm], B. gladiator [35.3–49.4 mm], B. goiana [29.0–33.0 mm], B. guentheri [33.0–40.0 mm], B. joaquini [40.3–56.4 mm], B. marginata [37.5–53.6 mm], B. marianitae [36.5–56.8 mm], B. melanopleura [43.6–50.0 mm], B. palaestes [36.2– 50.4 mm], B. poaju [33.5–42.7 mm], B. prasina [41.0–47.0 mm], B. pulchella [37.0–49.0 mm], B. riojana [48.0–56.0 mm], B. semiguttata [36.1–45.2 mm], and B. stellae [40.7–49.9 mm]); and the absence of vertical bars or spots on hidden parts of thighs, flanks, or groin (present in B. aguilari , B. balzani , B. bischoffi , B. caingua , B. caipora , B. callipleura , B. cordobae , B. curupi , B. cymbalum , B. ericae , B. gladiator , B. guentheri , B. joaquini , B. marianitae , B. melanopleura , B. poaju , B. prasina , B. pulchella , B. riojana , B. semiguttata , and B. stellae ; Boulenger, 1912 ; Bokermann, 1963 ; Barrio, 1965 ; Lutz, 1973 ; Heyer et al., 1990 ; Duellman et al., 1997 ; Cruz and Caramaschi, 1998, Garcia et al., 2001 , 2003 , 2007 , 2008 ; Caramaschi and Cruz, 2004; Kwet, 2008 ; Köhler et al., 2010 ; Lehr et al., 2010 ; Pinheiro et al., 2016 ). , SVL HL HW ED END IND EW IOD Boana guarinimirim (15) 24.1–28.1 (26.0 6 1.1) 8.1–9.1 (8.5 6 0.3) 7.7–8.7 (8.2 6 0.3) 2.7–3.4 (3.1 6 0.2) 1.3–2.4 (1.8 6 0.3) 1.6–2.3 (1.8 6 0.2) 2.1–2.7 (2.3 6 0.1) 2.6–3.1 (2.8 6 0.1) Boana sp. 4 (5) 23.0–26.6 (24.5 6 1.4) 7.1–7.6 (7.4 6 0.2) 7.6–8.4 (7.9 6 0.4) 2.7–3.1 (2.9 6 0.2) 1.2–1.6 (1.5 6 0.2) 1.7–2.0 (1.8 6 0.1) 2.3–2.5 (2.4 6 0.0) 2.5–2.9 (2.7 6 0.2) B. polytaenia (53) 23.2–34.7 (27.9 6 2.3) 7.3–11.5 (8.9 6 0.8) 7.4–11.0 (8.8 6 0.7) 2.5–3.6 (3.0 6 0.2) 1.4–2.3 (1.8 6 0.2) 1.3–2.6 (1.9 6 0.3) 1.9–3.2 (2.4 6 0.2) 2.4–3.6 (2.8 6 0.2) B. botumirim (20) 25.9–31.8 (29.2 6 1.4) 8.1–9.9 (9.3 6 0.6) 6.4–9.1 (8.2 6 0.6) 2.9–3.5 (3.1 6 0.2) 1.7–2.3 (1.9 6 0.1) 2.9–3.5 (3.1 6 0.2) 2.2–2.8 (2.6 6 0.2) 2.8–3.3 (3.0 6 0.1) B. buriti (20) 28.2–31.9 (30.0 6 1.9) 8.1–9.0 (8.5 6 0.5) 7.5–8.0 (7.7 6 035) 2.6–3.0 (2.8 6 0.2) 1.9–2.0 (2.0 6 0.1) 2.4–2.6 (2.5 6 0.1) 1.9–2.0 (2.0 6 0.1) 2.7–3.1 (2.9 6 0.2) B. cipoensis 25.6–31.5 — — — — — — — B. leptolineata 27.2–31.6 8.7 8.6 3.2 2.0 2.6 2.0 — B. jaguariaioensis (4) 23.9–28.8 (26.6 6 2.0) 7.2–8.5 (8.1 6 0.6) 7.0–8.2 (7.8 6 0.5) 2.6–3.0 (2.9 6 0.2) 1.8–2.1 (2.0 6 0.1) 2.1–2.4 (2.2 6 0.1) 1.9–2.2 (2.0 6 0.1) 2.4–3.1 (2.8 6 0.3) B. stenocephala (17) 26.0–30.4 (28.3 6 1.1) 7.9–10.0 (8.6 6 0.7) 7.4–8.2 (7.7 6 0.2) 2.5–2.9 (2.7 6 0.1) 1.7–2.0 (1.8 6 0.1) 2.2–2.7 (2.3 6 0.1) 1.6–1.9 (1.7 6 0.1) 2.6–3.2 (2.9 6 0.2) TD TL TAL FL SL THL 3FD 4TD Boana guarinimirim (15) 1.0–1.5 (1.3 6 0.1) 12.7–16.2 (14.3 6 0.9) 7.6–9.8 (8.7 6 0.6) 10.4–12.9 (11.4 6 0.7) 2.5–3.7 (3.1 6 0.4) 10.1–15.3 (13.3 6 1.1) 0.9–1.3 (1.1 6 0.1) 0.9–1.2 (1.0 6 0.1) Boana sp. 4 (5) 1.3–1.5 (1.4 6 0.1) 12.1–13.7 (13.0 6 0.7) 7.6–8.6 (7.9 6 0.6) 9.5–10.8 (10.2 6 0.7) 2.1–2.7 (2.4 6 0.2) 11.8–13.3 (12.6 6 0.7) 0.8–1.2 (1.0 6 0.2) 1.0–1.2 (1.1 6 0.1) B. polytaenia (53) 1.1–2.1 (1.5 6 0.2) 12.3–17.4 (14.8 6 1.1) 7.4–10.5 (8.9 6 0.7) 9.5–15.1 (11.9 6 1.2) 2.5–3.8 (3.0 6 0.3) 11.8–17.8 (14.4 6 1.2) 0.9–1.5 (1.2 6 0.1) 0.9–1.5 (1.1 6 0.1) B. botumirim (20) 1.0–2.0 (1.4 6 0.3) 14.7–17.3 (15.7 6 0.7) — — — 13.6–17.2 (15.3 6 0.9) — — B. buriti (20) — 14.2–16.4 (15.1 6 10.2) — — — 15.0–16.5 (15.6 6 0.8) — — B. cipoensis — — — — — — — — B. leptolineata 1.3 15.2 — — — — — — B. jaguariaioensis (4) 1.2–1.7 (1.5 6 0.2) 11.5–14.3 (13.1 6 1.2) — — — 11.9–14.2 (13.2 6 1.0) 0.7–0.8 (0.8 6 0.0) 0.5–0.9 (0.7 6 0.1) B. stenocephala (17) — 13.0– 15.0 (14.0 6 00.6) — — — 13.5–16.0 (14.7 6 0.6) — — From species of the B. polytaenia clade, B. guarinimirim sp. nov. can be distinguished by the presence of a calcar appendage and a distinct supracloacal crest ( Fig. 3 , absent in B. botumirim , B. buriti , B. cipoensis , B. jaguariaioensis , B. leptolineata , and B. stenocephala ); relatively more conspicuous discs on fingers and toes (less conspicuous in B. botumirim , B. jaguariaioensis , and B. stenocephala ), and the HW of 30–34% of SVL (mean of 27% in B. botumirim , 26% in B. buriti , 29% in B. jaguariaioensis , and 27% in B. stenocephala ). The male SVL of 24.1 to 28.1 mm of B. guarinimirim sp. nov. is smaller than B. buriti (28.2–31.9 mm; Lutz, 1968 ; Braun and Braun, 1977 ; Cruz and Caramaschi, 1998; Caramaschi and Cruz, 1999; Caramaschi et al., 2009 ; Caramaschi et al., 2010 ). B. guarinimirim cannot be distinguished from B. polytaenia and Boana sp. 4 in any of the analyzed morphological traits Vocal Repertoire .—The vocal repertoire of B. guarinimirim sp. nov. ( Fig. 4 A-B; Table 4) is composed of two types of calls, referred to as the ‘‘A'' and ‘‘B'' calls. The ‘‘A'' call is formed by a single and short multipulsed note and is commonly emitted alone or followed by a ‘‘B'' call but can also be emitted in pairs or in a series of consecutive ‘‘A'' calls. It lasts 18–84 ms ( 48 6 14 ) and reaches the amplitude peak at 1% to 76% ( 20 6 17 ) of its total duration. Pulses can be either well defined (with complete amplitude modulation, n = 27 calls) or poorly defined (concatenated pulses, n = 117 calls) along the call. In calls with well-defined pulses, the pulse rate ranges from 178 to 310 pulses per second (243 6 28), with 5–14 pulses per call. The dominant frequency always corresponds to the second harmonic and ranges from 4.6 to 6.6 kHz (6.2 6 0.6). The minimum frequency ranges from 4.4 to 6.2 kHz (5.4 6 0.5) and the maximum frequency from 5.0 to 7.2 kHz (6.3 6 0.6). The ‘‘B'' call is less common, and is usually preceded by an ‘‘A'' call, but can be emitted alone or followed by an ‘‘A'' call. The ‘‘B'' call lasts 381– 915 ms (574 6 136) and is composed of 5–11 pulses, which are emitted at irregular intervals of 2–284 ms (74 6 36). Pulses have durations of 2–16 ms (8 6 3). The ‘‘B'' call reaches its amplitude peak at 1–97% (48 6 35) of the total call duration. The dominant frequency corresponds to the second visible harmonic and ranges from 4.5 to 6.3 kHz (5.4 6 0.5). The minimum frequency ranges from 3.5 to 5.9 kHz (5.0 6 0.6) and the maximum frequency from 4.7 to 6.8 kHz (5.8 6 0.6). . Acoustic Comparisons .—The call of Boana guarinimirim sp. nov. can be distinguished from the call of B. caingua , B. callipleura , B. cambui , B. cordobae , B. freicanecae , B. goiana , B. melanopleura , B. pulchella , and B. riojana by being pulsed (nonpulsed in those species; Baraquet et al., 2007 , 2014 ; Köhler et al., 2010 ; Lehr et al., 2010 ; Pinheiro et al., 2012 ; Batista et al., 2015 ; Furtado et al., 2016 ; Pinheiro et al., 2016 ; Marinho et al., 2020 ). Species of the B. pulchella group with pulsed calls, except those in the B. polytaenia clade, are B. aguilari , B. balzani , B. bischoffi , B. caipora , B. callipleura , B. curupi , B. ericae , B. gladiator , B. guentheri , B. joaquini , B. marginata , B. marianitae , B. palaestes , B. poaju , B. prasina , B. semiguttata , and B. stellae . The advertisement call of B. guarinimirim can be distinguished from those species, except from B. bischoffi and B. ericae , by being composed of two types of calls (a single and pulsed call, or a series of similar calls in those species; Duellman et al., 1997 ; Garcia et al., 2001 , 2003 , 2007 , 2008 ; Antunes et al., 2008 ; Garcia and Haddad, 2008; Kwet, 2008 ; Köhler et al., 2010 ; Lehr et al., 2010 ; Pombal, 2010 ; Delgado and Haddad, 2015; Forti et al., 2019 ). From B. bischoffi and B. ericae , the calls of B. guarinimirim can be distinguished by its higher dominant frequency of 4.5–6.6 kHz (1.4–2.1 kHz in calls of B. bischoffi and 2.0–3.6 kHz in calls of B. ericae ; Garcia and Haddad, 2008; Pombal, 2010 ). Additionally, the vocal repertoire of B. ericae is composed of three types of calls: calls ‘‘A,'' ‘‘B,'' and ‘‘C.'' The ‘‘C'' call consists of a trill of pulses similar to the ‘‘B'' call of B. guarinimirim sp. nov. and can be distinguished from it by the longer duration of 381–915 ms in the ‘‘B'' call of B. guarinimirim sp. nov. (‘‘C'' call duration of 104–560 ms in B. ericae ). Among species within the Boana polytaenia clade, the calls of B. buriti and B. cipoensis remains undescribed. The calls of B. guarinimirim sp. nov. can be distinguished from the calls of B. botumirim and B. stenocephala by the higher dominant frequency of 4.5–6.6 kHz (dominant frequency of 3.3–4.1 kHz in ‘‘A'' calls of B. botumirim and B. stenocephala ; Caramaschi et al., 2009 ; Martins et al., 2016 ) and by being composed of two distinct call types (‘‘B'' call unknown in B. botumirim ). Also, the call of B. guarinimirim can be distinguished from the calls of B. jaguariaioensis and B. stenenocephala by the ‘‘A'' call being composed of only one note (‘‘A'' call composed by a sequence of short notes in B. jaguariaioensis and B. stenocephala ; Martins et al., 2016 ; Guerra et al., 2017 ). The described calls of B. leptolineata ( Forti et al., 2019 ) cannot be distinguished from the calls of B. guarinimirim based on any of the analyzed traits; however, these species are easily distinguished by morphological features and are not closely related ( Faivovich et al., 2021 ). Although the DAPC analysis pointed to a discrimination between B. guarinimirim and its distant taxon B. polytaenia ( Fig. 1B ), we could not find any acoustic trait that did not overlap in our analysis (Table 4). Additionally, B. guarinimirim could not be distinguished from Boana sp. 4 based on acoustic traits.

Description of the Holotype .— Adult male ( Figs. 2 , 6 ); SVL 24.1; body slender; head wider than body, slightly longer than wider (HL/HW = 1.11); snout rounded in dorsal view (SL/HL = 0.37; SL/HW = 0.41), and in profile; END shorter than ED (END/ED = 0.55); canthus rostralis distinct, slightly curved in dorsal view; loreal region oblique, slightly concave; internarial region not depressed; nostrils not protuberant, dorsolaterally directed; interorbital area flat, shorter than ED (IOD/ED = 0.94), shorter than HW (IOD/HW = 0.36). Eyes large and protuberant (ED/HL = 0.35; ED/HW = 0.39); palpebral membrane translucent, without reticulations, pigmented on its lower portion and margin. Supratympanic fold absent. Tympanum small (TD/ED = 0.42), inconspicuous, directed laterally. Arm slender, lacking an axillary membrane; forearm slightly hypertrophied in comparison with the arm, with a dermal ridge along the ventral side extending from the proximal limit of hand to the elbow. Fingers bearing round discs; disc diameter on finger III narrower than tympanum (3FD/TD 0.74); relative finger length: I, II, IV, III; webbing formula I–II 2 - – 3 III–IV ; lateral fringes on fingers absent; subarticular tubercles distinct, circular, nonbifid, and rounded in ventral view; supernumerary tubercles present on fingers and palm; outer metacarpal tubercle rounded, almost indiscernible from supernumerary tubercles ( Fig. 2C ). Nuptial pad absent; prepollex enlarged and pointed as a curved bony spine. Hind limbs long and slender (THL/SVL = 0.42; TL/SVL = 0.53); tarsal dermal ridge extending from the inner metatarsal tubercle to heel; calcar appendage present on both heels, small. Toes long, with round discs, the 4th disc diameter slightly smaller than that on finger (4TD/3FD = 0.90); relative toe length I <II <III = V <IV; webbing formula I2 + –2 + II 1 + –2 + III 2–3 + IV 2 + –1 + V; presence of lateral fringes on toes. Presence of a dermal ridge extending from the base of the disc to the heel. Subarticular tubercles circular, slightly conical in profile; numerous supernumerary tubercles present on toes and sole; outer metatarsal tubercle rounded in ventral view, inner metatarsal elliptical in ventral view ( Fig. 2D ). Dorsal skin smooth. Pectoral, abdominal areas, and ventral surfaces of thighs granular. Pectoral fold absent. Cloacal opening directed posteroventrally at upper level of thighs; cloacal sheath (veil) absent; a white supracloacal dermal ridge present; cloacal tubercles present around vent, scattered, extending to midlevel of thighs. Tongue ovoid, barely free behind; dentigerous processes of vomers prominent, in two separate series between and slightly behind choanae, bearing four (right) and three (left) visible teeth. Choanae elliptical. Vocal slits extending from midlateral base of tongue, almost reaching the angle of jaws. Vocal sac single, median, subgular. Call duration (ms) Pulses per call Pulses per second Call rise time (%) Dominant frequency (kHz) Minimum frequency (kHz) Maximum frequency (kHz) Interval between pulses (ms) Pulses duration (ms) Species ‘‘ A' ' call ‘‘ B' ' call ‘‘ A' ' call ‘‘ B' ' call ‘‘ A' ' call ‘‘ A' ' call ‘‘ B' ' call ‘‘ A' ' call ‘‘ B' ' call ‘‘ A' ' call ‘‘ B' ' call ‘‘ A' ' call ‘‘ B' ' call ‘‘ B' ' call ‘‘ B' ' call Reference Boana guarinimirim 18 – 84 381 – 915 5 –14 5 – 11 178 –310 1 – 76 1 – 97 4.6 –6.6 4.5 – 6.3 4.4 –6.2 3.5 – 5.9 5.0 – 7.2 4.7 – 6.8 2 – 284 2 – 16 Present work (16 / 11) (48 6 14) (574 6 136) (243 6 28) (20 6 17) (48 6 35) (6.2 6 0.6) (5.4 6 0.5) (5.4 6 0.5) (5.0 6 0.6) (6.3 6 0.6) (5.8 6 0.6) (74 6 36) (8 6 3) Boana sp. 4 (8 / 5) 44 – 68 (61 6 11) 55 – 124 (74 6 14) 346 – 1,018 (724 6 195) 570 – 1,501 (1,050 6 314) — 4 – 10 – 1 – 24 (29 6 16) 0 – 86 (36 6 17) 0 – 81 (17 – 47) 12– 93 (55 6 24) 6.0 – 6.5 (6.5 6 0.2) 3.4 –7.5 (5.8 6 1.3) 5.8 – 6.7 (6.3 6 0.1) 3.4 – 7.0 (5.6 6 1.3) 5.6 – 6.1 (6.1 6 0.2) 3.0 – 6.9 (5.4 6 1.3) 5.6 – 6.3 (6.0 6 0.2) 2.8 – 6.6 (5.2 6 1.3) 6.6 – 7.0 (7.0 6 0.3) 3.7 – 7.9 (6.5 6 1.5) 6.4 – 6.9 (6.7 6 0.1) 3.8 – 7.4 (5.9 6 1.3) 6 – 210 (105 6 8) 4 – 236 (78 6 37) 2 – 23 (11 6 3) 2 – 23 (11 6 5) Present work B. polytaenia (6 / 8) l – 14 7– 23 182 – 240 (223 6 1) Present work B. botumirim (3 / —) 37 – 64 — 5 – 11 — — — — 3.3 – 4.0 — — — — — — — Caramaschi B. leptolineata (4 /4) (50 6 5) 20 – 160 190 – 640 4 – 10 — — — — (3.7 6 1.4) 3.6 – 4.8 3.8 – 4.9 3.4 – 4.7 3.0 – 4.4 3.9 – 5.1 3.9 – 6.5 — — et al. 2009 Forti et al. (60 6 20) (391 6 63) (4.2 6 0.4) (4.3 6 0.4) (4.0 6 0.4) (3.9 6 0.4) (4.5 6 0.4) (4.6 6 0.5) 2019 B . jaguariaioensis 21 – 508 170 –426 14 – 18 7 –12 — — — 4.5 – 4.9 4.5 – 4.9 4.1 – 4.7 4.3 –4.5 4.7 – 5.2 4.9 – 5.1 3 –68 3 – 12 Guerra et al. (17 / 17) (271 6 106) (278 6 72) (4.7 6 0.1) (4.7 6 0.1) (4.5 6 0.1) (4.4 6 1.0) (5.0 6 0.1) (5.0 6 0.1) (23 6 16 ) (8 6 1) 2017 B. stenocephala (5 / 1 Color Variation .—In preservative, dorsum covered with three dark brown, grey, or dark grey thick lines (referred to next as stripes) and multiple interposed light brown or grey thinner longitudinal lines (referred to next as lines) extending from snout to cloacal region. Stripes fragmented on the sacral region in the individuals AAG-UFU 6772, AAG-UFU 6774–75, AAG-UFU 6798–99, CFBH 46037–40, and CFBH 46042–43. Dorsum background light brown, brown, or light grey. A dark brown dorsolateral stripe extends from the snout to the groin encompassing the nostrils, canthus rostralis, and tympanum, being interrupted at the eye level. Loreal region with lines also extending to the groin encompassing the flank region. Flank background white, sometimes with a dark brown stripe margin in its ventrolateral region. Stripes and lines extend from the mid portion of fingers III and IV to the elbow dorsally. Additionally, a stripe extending from the elbow to the mid portion of finger IV is present at the distal margin of the forearm (fragmented in individuals AAG-UFU 6772–5, AAG-UFU 6798, CFBH 46037–40, and CFBH 46042). Dorsal region of arms have multiple lines. At hind limbs, stripes encompass the dorsal surface of thighs, shank, and the distal border of the tarsal and metatarsal region, reaching toes IV and V (absent in individual AAG-UFU 6800; individuals AAG-UFU 6773, AAG-UFU 6776, CFBH 46027, and CFBH 46039–42 have circular to elliptical dots in between the lines). Usually, a dark brown stripe bordered with white stripes is present at the distal border of the shank (absent in individual AAG-UFU 6775). The hidden surfaces of the thighs are beige or pale orange, without any marks or stripes. The iris is light brown or grey without stripes and the palpebral membrane is translucent, with its lower portion and upper margin partially pigmented. The upper lip is brown with a white line margin in its border. The ventral coloration of limbs is pale yellow, and the ventral skin ridge of the forearm is white (bordered by a dark brown line in individuals AAG-UFU 6772–74, AAG-UFU 6776, CFBH46038–40, and CFBH 46041–42). Mentonian region is white. White granules on chest and belly. No transparency on the ventral surfaces (individuals CFBH 46037 and CFBH 46039 share a depigmented area on the ventral surface and the ventral surface of CFBH 46040 is irregularly pigmented). Calcar appendage white in dorsal view, brown in ventral view. Supracloacal crest white or pale yellow, bordered by a brown line. In life ( Figs. 6–7 ), the lines covering the dorsum and dorsal surfaces of limbs are brownish-red, brownish-orange, or pale brown. The stripes on the dorsum, canthus rostralis, dorsolateral, flanks, and distal edge of the shank are pale to dark brown. The hidden surfaces of the thighs and groin; the proximal borders of the shank, palm, and sole; and the ventral surface of discs of the fingers and toes are orange. The irises are beige to copper. ) 165 – 588 (312.8 6 74.0) 287 7– 23 10 7– 135 (109 6 7) — — 3.6 – 4.1 (3.9 6 0.1) 3.6 – 4.1 (3.9 6 0.2) — — — — — 1 – 16 (14 6 1) Martins et al. 2016