Bioacoustics of Ranitomeya aquamarina

Advertisement call. The advertisement call of Ranitomeya aquamarina sp. nov. ( n = 7 males ) consist of a long-lasting trill of 21–45 notes ( n = 44 calls) — most commonly of 32–38 notes ( n = 24 calls) — a call duration of 984 ± 197 ms (647–1,424 ms) — and silence between calls of 5.8– 115.3s (most commonly between 7 and 18 s) (n = 24 silence between calls). Notes are distinct, separated by silence intervals, with note duration of 11.7 ± 0.14 ms (9.6–14.8 ms), a silence between notes of 19.4 ± 0.2 ms (15.6–22.7 ms), and a note rate of 32.8 ± 2.2s (28–36). Calls are emitted with a minimum frequency ( LF ) of 5,139 ± 283 Hz ( 4,699 –5,860 Hz), a maximum frequency ( HF ) of 6,054 ± 255 Hz ( 5,545 –6,600 Hz) and a dominant frequency ( DF ) of 5,633 ± 289 Hz ( 4,996 –6,288 Hz) (Fig. 6 ). However, the first note is emitted at approximately 300 Hz – a lower frequency compared to the subsequent notes ( LF of 4,921 ± 187 Hz, HF of 5,683 ± 211 Hz and a DF of 5,340 ± 202 Hz). Temporal and spectral traits, summarized according to individual call arrangement, are presented in Table 3 . Variables Mean SD Minimum Maximum CD – Call duration (ms) 984 197 647 1,424 SBC – Silence between calls (s) 19.3 20.6 5.8 115.3 NN – Number of notes per call 32.4 6.7 21 45 ND – Note duration (ms) 11.8 0.13 9.6 14.8 SBN – Silence between notes (ms) 19.4 0.19 15.6 22.7 NR – Note rate (notes per second) 32.4 2.4 28 36 LF – Minimum frequency (Hz) 5,132 272 4,699 5,860 HF – Maximum frequency (Hz) 6,059 244 5,545 6,600 DF – Dominant frequency (Hz) 5,640 277 4,996 6,288

Differential diagnosis. External morphology. The new species differs from all currently recognized Ranitomeya species ( R. amazonica , R. benedicta , R. cyanovittata , R. defleri , R. fantastica , R. flavovittata , R. imitator , R. reticulata , R. sirensis , R. summersi , R. toraro , R. uakarii , R. vanzolinii , R. variabilis , R. ventrimaculata , and R. yavaricola ) by its unique coloration (light yellowish green to light metallic turquoise-green dorsal stripe pattern, medium metallic chrome orange limbs with dark carmine spotting, and conspicuous sulfur yellow ocellus-like spot on the dorsal surface of the thighs). Ranitomeya aquamarina sp. nov. is generally most similar to R. cyanovittata and R. yavaricola but it can be easily distinguished from R. cyanovittata by light yellowish green to light metallic turquoise green dorsal stripes and medium metallic chrome orange limbs with dark carmine spotting (dorsal stripes turquoise blue, limbs with bluish reticulation and black spots; Pérez-Peña et al. 2010 ) and from R. yavaricola by light yellowish green to light metallic turquoise green dorsal stripes with no or only minor breaks and by dark carmine spotting on the limbs (dorsal stripes sage color, formed by points, that can become dashes, limbs solid bronze; Pérez-Peña et al. 2010 ). In addition, Ranitomeya aquamarina sp. nov. is distinguished by its smaller male SVL ( 15.4–17.7 mm ) from R. fantastica (~ 20 mm ; Boulenger 1884 ), R. imitator (~ 19 mm ; Schulte 1986 ) and R. summersi ( 17.5–19.5 mm ; Brown et al. 2008 ); and larger than R. cyanovittata ( 13.8 mm ; Pérez-Peña et al. 2010 ), R. sirensis ( 14.7–15.4 mm ; Aichinger 1991 ), R. toraro ( 14.8–15.6 mm ; Brown et al. 2011 ) and R. uakari ( 14.8–15.5 mm ; Brown et al. 2006 ); by its larger female SVL ( 17.3–18.5 mm ) from R. sirensis ( 16.8 mm ; Aichinger 1991 ), R. toraro ( 16.2–16.7 mm ; Brown et al. 2011 ), R. uakari ( 15.7–16.2 mm ; Brown et al. 2006 ); R. yavaricola ( 16.7–16.8 mm ; Pérez-Peña et al. 2010 ); by its greater female head width ( 5.7–5.9 mm ) from R. cyanovittata ( 5.6 mm ; Pérez-Peña et al. 2010 ), R. sirensis ( 5.4 mm ; Aichinger 1991 ), R. toraro (5.0– 5.3 mm ; Brown et al. 2011 ), R. uakarii (5.0– 5.2 mm ; Brown et al. 2006 ), R. yavaricola ( 5.1–5.7 mm ; Pérez-Peña et al. 2010 ); by its greater male head length ( 4.8–5.4 mm ) from R. cyanovittata ( 3.6 mm ; Pérez-Peña et al. 2010 ) and R. sirensis (3.0– 3.8 mm ; Aichinger 1991 ), but smaller than R. imitator (~ 6 mm ) and R. yavaricola ( 5.5–6.6 mm ; Pérez-Peña et al. 2010 ); by its smaller female head length ( 5.1–5.2 mm ) from R. toraro ( 5.5 mm ; Brown et al. 2011 ) and R. yavaricola ( 5.9–6.3 mm ; Pérez-Peña et al. 2010 ) and larger than R. sirensis (4.0 mm; Aichinger 1991 ). Bioacoustics. The advertisement call of R. aquamarina sp. nov. is distinguished by its longer call duration (647–1,424 ms) from the call of R. amazonica (160–360 ms; Brown et al. 2011 ), R. benedicta (100–170 ms; Brown et al. 2008 ), R. defleri (410–620 ms; Twomey and Brown 2009 ), R. fantastica (180–320 ms; Brown et al. 2011 ), R. reticulata (180–290 ms; Brown et al. 2011 ), R. summersi (380–500 ms; Brown et al. 2008 ), R. uakarii (260–290 ms; Brown et al. 2006 , Brown et al. 2011 ), R. vanzolinii (570–640 ms; Brown et al. 2011 ), R. variabilis (140–440 ms; Brown et al. 2011 ) and R. ventrimaculata (320–380 ms; Brown et al. 2011 ). Furthermore, it differs by its higher dominant frequency ( 4,996 –6,288 Hz) from the calls of R. benedicta ( 3,190 –4,240 Hz; Brown et al. 2008 ), R. fantastica ( 2,950 –3,790 Hz; Brown et al. 2011 ), R. reticulata ( 4,140 –4,480 Hz; Brown et al. 2011 ), R. summersi (2760–3220 Hz; Brown et al. 2008 ), R. uakarii ( 3,790 –4,130 Hz; Brown et al. 2006 , Brown et al. 2011 ), and R. ventrimaculata (4190–4400 Hz; Brown et al. 2011 ); by its lower number of notes (21–45) from the calls of R. reticulata (48–94; Brown et al. 2011 ) and R. ventrimaculata (58–63; Brown et al. 2011 ); by greater number of notes from the calls of R. fantastica (10–13; Brown et al. 2011 ), R. summersi (14–16; Brown et al. 2008 ), R. uakarii (14–16; Brown et al. 2006 , Brown et al. 2011 ), and R. vanzolinii (16–17; Brown et al. 2011 ); by its smaller note rate (28–36 notes / s) from the calls of R. amazonica (85–138 notes / s; Brown et al. 2011 ), R. defleri (94–104 notes / s; Twomey and Brown 2009 ), R. fantastica (41–57 notes / s; Brown et al. 2011 ), R. reticulata (270–382 notes / s; Brown et al. 2011 ), R. summersi (39–40 notes / s; Brown et al. 2008 ), R. uakarii (50–58 notes / s; Brown et al. 2006 ; Brown et al. 2011 ), R. variabilis (106–297 notes / s; Brown et al. 2011 ), and R. ventrimaculata (166–181 notes / s; Brown et al. 2011 ). The advertisement call of R. aquamarina sp. nov. is highly similar to the calls of all other species of the R. vanzolinii group, which have long-lasting trills, but it can still be distinguished from the call of R. vanzolinii , which has slightly lower note rate of 26–28 notes / s ( Brown et al. 2011 ). On the other hand, based on the literature, the call of R. aquamarina sp. nov. is indistinguishable from the calls of R. flavovittata , R. imitator , R. sirensis , and R. yavaricola ( Perez-Peña et al. 2010 ; Brown et al. 2011 ). There is no available information about minimum and maximum frequencies, note duration and inter-notes interval in these species. In addition, the calls of R. toraro and R. cyanovittata remain completely unknown. Tadpole morphology. There is little information available about the tadpoles of the Ranitomeya species, but we did find information for ten species ( R. amazonica , R. benedicta , R. defleri , R. flavovittata , R. imitator , R. reticulata , R. toraro , R. uakarii , R. vanzolinii and R. variabilis ). The tadpoles of R. aquamarina sp. nov. differ from the tadpoles of all these species by absence of emarginate marginal papillae. Because there is great variation between the initial (25–27), intermediate (28–32), and final (37–40) stages, we compared them using the ratios between measurements (the characteristics of compared species are given in parentheses). The labial tooth row formula in R. aquamarina sp. nov. is 2 (2) / 3 (1) in all stages and differs from the formula of R. toraro 2 (2) / 2 (1) ( Brown et al. 2011 ). The ratios (in percentages) tail length / total length are in R. aquamarina sp. nov. (63 to 64 % in all stages) greater than in R. amazonica (45 % st. 29, Brown et al. 2011 ), R. flavovittata (57 % st. 26; Brown et al. 2011 ), R. imitator (62 % st. 26; Brown et al. 2011 ), R. reticulata (41 % st. 30; Brown et al. 2011 ), R. uakarii (62 %, st. 29; Brown et al. 2011 ) and R. variabilis (37 % st. 28; Brown et al. 2011 ), and smaller than in R. toraro (64.2 % st. 25; Brown et al. 2011 ), R. vanzolinii (67.9 % st. 38; Brown et al. 2011 ) and R. yavaricola (64.4 % st. 25; Pérez-Peña et al. 2010 ). The ratios oral disc width / body width are in R. aquamarina sp. nov. (42 % at stage 26, 36 % at stage 29, and 35 % at stage 39) greater than in R. amazonica (29 % st. 26, 22 % st. 29, 33 % st. 38; Brown et al. 2011 , Klein et al. 2020 ), R. flavovittata (28 % st. 26; Brown et al. 2011 ), R. imitator (38 % st. 26; Brown et al. 2011 ), R. toraro (36 % st. 25; Brown et al. 2011 ), R. reticulata (14 % st. 30; Brown et al. 2011 ), R. uakarii (35 %, st. 29; Brown et al. 2011 ), and smaller than in R. vanzolinii (38.9 % st. 38; Brown et al. 2011 ). The ratios tail muscle width / tail muscle height are in R. aquamarina sp. nov. (91 % at stage 26 and 115 % at stage 29) greater than in R. flavovittata (63 % st. 26; Brown et al. 2011 ), R. imitator (52 % st. 26; Brown et al. 2011 ), R. amazonica (76 % st. 29; Brown et al. 2011 ), R. reticulata (92 % st. 30; Brown et al. 2011 ), R. uakari (88 %, st. 29; Brown et al. 2011 ), and R. variabilis (72 % st. 30; Brown et al. 2011 ), and smaller than in R. toraro (100 % st. 25; Brown et al. 2011 ). Posterior tooth row formula of R. aquamarina sp. nov. ( P- 1 > P- 2 > P- 3 in all stages) differs from the formulas of all other described tadpoles: R. amazonica ( P- 1 = P- 2 > P- 3 , P- 3 = 80 % of P- 1 ; Brown et al. 2011 ), R. benedicta ( P- 1 = P- 2 = P- 3 ; Klein et al. 2020 ), R. flavovittata ( P- 1 = P- 2 > P- 3 , P- 3 = 80 % of P- 1 ; Brown et al. 2011 ), R. imitator ( P- 1 = P- 2 > P- 3 , P- 3 = 55 % of P- 1 ; Brown et al. 2011 ; Klein et al. 2020 ), R. reticulata ( P- 1 = P- 2 > P- 3 , P- 3 = 80 % of P- 1 ; Brown et al. 2011 ; Klein et al. 2020 ), R. toraro ( P- 1 > P- 2 ; Brown et al. 2011 ), R. uakarii ( P- 1 = P- 2 > P- 3 , P- 3 = 75 % of P- 1 and P- 2 ; Brown et al. 2011 ), R. vanzolinii ( P- 1 < P- 2 = P- 3 , P- 1 = 44.6 % of P- 2 ; Brown et al. 2011 ), R. variabilis ( P- 1 = P- 2 > P- 3 , P- 3 = 75 % of P- 1 ; Brown et al. 2011 ) and R. yavaricola ( P- 1 = P- 2 > P- 3 ; Pérez-Peña et al. 2010 ). In life, tadpoles of R. aquamarina sp. nov. have a translucent brownish head in all stages, which differs from all the other tadpoles described: R. amazonica (head and body black to gray; Brown et al. 2011 , Klein et al. 2020 ), R. benedicta (head and body dark gray, with a reddish area anterior and posterior to the eye; Klein et al. 2020 ), R. imitator (head beige strongly dotted with yellowish green spots; Klein et al. 2020 ), R. reticulata (head and body gray; Brown et al. 2011 ), and R. toraro (head and body gray; Brown et al. 2011 ), R. uakari (head gray; Brown et al. 2011 ), R. vanzolinii (head and body dark gray to black; Klein et al. 2020 ), R. variabilis (head and body gray; Brown et al. 2011 ) and R. yavaricola (light grey; Pérez-Peña et al. 2010 ). When preserved, the tadpoles of R. aquamarina sp. nov. are cream with brown reticules on the lateral, dorsal, anterior half belly, spiracles, tail muscle and fins and differ from R. amazonica (dorsum dark gray and hindlimbs bluish gray, spotted with dark dots; Klein et al. 2020 ), R. benedicta (dorsum and hindlimbs dark gray; Klein et al. 2020 ), R. imitator (beige, densely spotted with gray dots; Klein et al. 2020 ) and R. vanzolinii (dorsum of body grayish brown, tail musculature light yellowish brown and fins translucent; Brown et al. 2011 ).

Characterization. This new species of Ranitomeya is characterized by the following combination of characters: (1) dorsal color jet black with three parallel stripes metallic light yellowish green to metallic light turquoise-green, mid-dorsal stripe extending from between eyes to slightly before the vent, dorsolateral stripes extending from the snout to the groin, where they become medium sulfur yellow; (2) venter jet black with metallic olive-yellow to metallic light yellowish green reticulations on belly, and gular region metallic light yellowish green to olive-yellow; ventrolateral stripes light yellowish green; extending from through the loreal region, to the thighs integrating into the ventral reticulate pattern, becoming medium sulfur yellow on the arms; (3) limbs medium metallic chrome orange with dark carmine spotting, presence of a conspicuous sulfur yellow spot on the dorsal surface of the thighs, forming an ‘ ocellus’ like pattern; (4) dorsal skin shagreen to granular, and smooth on head; (5) gular and ventral skin shagreen to granular; (6) limbs smooth to shagreen; (7) SVL in adult males of 15.4–17.7 mm ( n = 8) and in females of 17.3–18.5 mm ( n = 5); (8) sexual dimorphism, females with greater SVL , BW and KK ; presence of vocal slits in males, located near jaw articulation; (9) head width 0.8–1.0 × body width; (10) head width 1.1–1.2 × larger than head length; (11) head width 31–34 % of SVL ; (12) snout moderately long ( SL 36–42 % of HL ), rounded in dorsal view and rounded to protruding in lateral view; (13) canthus rostralis rounded, loreal region flat; (14) nostril directed frontolaterally at the angle of the snout, internarial distance 33–39 % of head width; (15) tympanum visible, tympanic membrane poorly differentiated, tympanum diameter 38–48 % of eye diameter; (16) tongue ovoid, attached anteriorly; (17) dentigerous processes of vomers absent; (18) choanae ovoid and small, located marginally in the maxilla; (19) hand 24–28 % of SVL , arm 25–30 % of SVL ; (20) fingers III> IV> II> I, Finger I 58–68 % of Finger II, finger discs rounded on Finger I, and expanded and truncate on fingers III and IV; (21) thenar tubercle elliptical, palmar tubercle large and ovoid; (22) proximal subarticular tubercles ovoid, present in each finger; distal subarticular tubercle present only on Finger III; (23) knee-knee distance 80–84 % of SVL , femur 94–98 % of tibia; (24) toes IV> III> V> II> I, Toe I 48–64 % of Toe II, finger discs not expanded and rounded on Finger I to elliptical on toes III and IV and truncate on Finger V; (25) outer metatarsal tubercle ovoid, poorly visible; inner metatarsal tubercle elliptical; (26) proximal subarticular tubercles ovoid on all toes, distal subarticular tubercles on toes III – V; (27) advertisement call with 21–45 notes and average call duration of 647–1,424 ms, note rate (28–36 notes / s) and dominant frequency of 4,996 –6,288 Hz; and (28) tadpole head translucent in life, and posterior tooth rows P- 1 > P- 2 > P- 3 .

Distribution, habitat, natural history, and conservation. Ranitomeya aquamarina sp. nov. is only known from its type locality, in preserved forests on the Eiru River, a tributary of the Juruá River, near the Comunidade de Santo Antônio, municipality of Eirunepé, state of Amazonas, Brazil (Fig. 9 ). We sampled four RAPELD modules in the region, and the new species has only been recorded at one site. We did not find the new species living in sympatry with any other species of the genus. However, other Dendrobatoidea occur at the site: Allobates femoralis , Allobates sp. undescribed (A. P. Lima, unpublished data), Ameerega hahneli and A. trivittata . Ranitomeya aquamarina sp. nov. is diurnal, showing greater activity in the early morning and late afternoon. On rainy days, activity lasts throughout the day. Most individuals were observed in clusters of ‘ banananeira brava ’ ( Phenakospermum guyannense , Strelitziacaea ; Fig. 10 A ), but the species was also found in a phytotelma in the forest understory, ~ 3 m above the ground (Fig. 10 B ). Individuals climb vertically through vegetation (Fig. 10 C ) and are very agile. Eggs are deposited in water accumulated in cavities in the vegetation. We found eggs (Fig. 10 D ) and tadpoles in the ‘ banananeira brava ’ axils and in small holes in trees (Fig. 10 B ). The eggs are small and brown, wrapped in a thick transparent gelatinous layer. Furthermore, we found tadpoles at different stages of development and metamorphizing in the same area, which suggests that reproduction is prolonged, probably occurring during the entire rain season. Juveniles ( n = 5; not collected) and adults ( n = 8) were seen foraging among the dry leaves of the same plants. In juveniles, dorsolateral stripes are uniformly yellow and are not fully formed (Fig. 10 E ). Males perform calling perched on vegetation (Fig. 10 F ). They start calling at dawn (~ 6 am) and remain active until ~ 9 am, with a peak between 7 am and 8 am. After that, their activity remains sporadic until ~ 11 am. They call again in the late afternoon, but with a lower intensity. Most of the time, we observed adults as couples ( n = 8; Fig. 10 G, H ), which strongly suggests that the species is monogamous. Males appear to be territorial, responding to and approaching the playback. Additionally, when we captured the females, the respective males called incessantly. The new species was found in only one of the four sampling sites ( 5 km RAPELD trails) and appears to be strongly associated with ‘ bananeira brava ’ plants. Therefore, this species is not expected to be abundant nor homogeneously distributed throughout its range. Its known extent of occurrence and area of occupancy are restricted, suggesting that its conservation status deserves attention. Nevertheless, we currently do not have enough information to assign Ranitomeya aquamarina sp. nov. to any IUCN category, and here we classify it as Data Deficient (DD).