Bioacoustics of Nymphargus pijao

Description of the holotype . An adult male in good state of preservation ( Fig. 9 ). Body size moderate (SVL = 22.9 mm ); head wider than the body; HW 34.5% of SLV; head wider than long, HW/HL = 1.0; snout truncate in dorsal profile and lateral profile; END 73.1% of ED; loreal region concave, nostrils elevated producing a slight depression in the internarial area; canthus rostralis round; ED 36.7% of HW; supratympanic fold distinct, tympanic annulus distinguished and round; tympanic membrane differentiated and partially pigmented, IN 48.9% of ED; vomerine teeth absent; tongue round, slightly longer than wide, with posterior distal margin slightly notched, posterior edge not adherent to the floor of the mouth. Humeral spine absent; ulnar tubercles absent; ulnar fold present, not enameled, low; Finger I shorter than Finger II, discs round to subtruncate and wider than tympanum, the diameter of the disc of Finger III equivalent to 53.1% of ED; concealed prepollex. Hand webbing formula I-II (absent), II-III (absent), III 3 - – 21/4 IV; subarticular tubercles round, supernumerary tubercles at base of fingers, palmar tubercle slightly longer than wide, thenar tubercle elliptical, longer than palmar tubercle. Hind limbs slender, almost the same width of forearm, TL 61.8% of SLV; tarsal tubercles absent; translucent tarsal fold present (not enameled), low; inner metatarsal tubercle elliptic, outer metatarsal tubercle absent; subarticular tubercles round; webbing formulae on feet I 2 - – 21/3 II 11/4 – 21/4 III 2 + –2 + IV 21/4 –2 - V; toe discs narrower than finger discs. Smooth dorsal texture with seven low warts associated with pale light spots (in life, white in preservative) and with clusters of melanophores forming small flecks, dorsal skin smooth with abundant microspicules (only visible under stereo microscope), low warts associate with yellow spots in life, and white in preservative; ventral skin on belly and thighs granular; cloacal opening directed posteriorly, concealed by a dermal fold; pair of enlarged flat warts on the posteroventral surface of thighs. Measurements of holotype (in mm) . SVL = 22.9; Head width = 7.9; Head length = 7.6; Interorbital distance = 2.8; Eye-nostril distance = 2.1; Internarial distance = 1.4; Width disc of Finger III = 1.8; Tibia length = 14.1; Foot length = 10.9; Eye diameter = 2.9; Tympanum diameter = 1.1; Hand length = 8.9; Forearm length = 5.7. Coloration of the holotype . Dorsum light green in life, with tiny clusters of melanophores forming small flecks and some yellow or cream-yellowish dots, concentrated on the dorsal surface of the head and body. Parietal peritoneum covered by iridophores, more than 50%. Visceral peritoneum translucent except for the pericardium that is covered by iridophores (M. Rada pers. obs , on freshly collected type series). Slightly pale-yellow toes and hands with few melanophores on dorsal surfaces. The iris is silver with fine reticulations and small punctuations. The bone color is green ( Fig. 8A ). In ethanol, dorsal surfaces of head and dorsum are pale lavender-colored with large light cream-colored dots, with dark lavender clusters of melanophores forming small flecks and cream-colored spots. Melanophores are present on fingers III and IV, less dense on Finger II, and abundant along toes IV and V, less dense in the other fingers. Translucent testis. Dark lavender iris with darker reticulations and small flecks ( Figs. 9 and 10A–B ). Variation. Adult individuals vary from dark green to light green with or without clusters of melanophores forming small flecks. Orange-yellowish or light-yellow dots are present on the dorsal surfaces of the head and body. Yellowish to pale green toes and hands with melanophores on dorsal surfaces. The iris ranges in color from silverwhite to faint gold with fine reticulations and minute rounded flecks. Dorsal color of specimens in ethanol varies from lavender to pale lavender-colored with different concentrations of minute dark lavender flecks and light cream-colored dots. Melanophores present on fingers III and IV, are less dense on finger II, and abundant along toes IV and V, less dense in the other fingers ( Fig. 8A, E–G ). Measurement and body proportion are shown in Table 1 . Variation in webbing of hands and feet are shown in Table 2 . Vomerine odontophores vary from absent to present. Measurements Males (n = 27) Females (n = 7) Body size (SVL) 24.3 ± 1.17 (22.1–27.3) 26.1 ± 1.5 (24.6–28.6) Head width (HW) 7.8 ± 0.4 (6.6–8.8) 8.4 ± 0.6 (7.7–9.1) Head length (HL) 6.4 ± 0.4 (5.7–7.7) 6.8 ± 0.4 (6.3–7.5) Horizontal eye diameter (ED) 3.0 ± 0.5 (2.2–4.2) 3.5 ± 0.6 (2.8–4.7) Inter-orbital distance (IOD) 3.2 ± 0.3 (2.7–3.8) 3.6 ± 0.3 (3.1–4.0) Eye-nostril distance (EN) 2.4 ± 0.2 (2.0–2.6) 2.6 ± 0.3 (2.13–3.0) Internarial distance (IN) 2.18 ± 0.2 (1.9–2.6) 2.3 ± 0.2 (1.9–2.6) Width of the disc on the third finger (3DW) 1.4 ± 0.2 (1.1–1.7) 1.5 ± 0.1 (1.4–1.8) Tibia length (TL) 14.3 ± 0.6 (12.9–15.6) 15.3 ± 0.9 (14.2–16.6) Foot length (FL) 11.1 ± 0.6 (10.1–12.7) 11.9 ± 0.3 (10.8–13.1) Voucher Webbing formula of hands Webbing formula of feet ARUQ 491 III 2-–2+ IV I 2+–2+ II 21/2–21/3 III 11/2–3- IV 3-–2- V ARUQ 647 III 3–21/4 IV I 2+–21/4 II 1+–2 III 1+–31/2 IV 31/2–2 V ARUQ 648 III 3–21/4 IV I 2-–21/2 II 1-–21/4 III 11/2–2 IV 3+–1+ V ARUQ 743 II 2-–31/4 III 3+–21/2 IV I 2–2- II 1+–2+ III 1-–2+ IV 21/2–2- V ARUQ 1377 III 3 - – 21 /4 IV I 2 - – 21 /3 II 11 /4 – 21 /4 III 2 + –2 + IV 21 /4 –2 - V ICN 16007 III 3–21/2 IV I 2-–2+ II 1+–2+ III 1+–21/2 IV 21/2–1+ V ICN 16057 II 2-–31/3 III 3-–21/2 IV I 1+–2+ II 11/2–2+ III 11/2–11/3 IV 21/2–1+ V ICN 16075 III 3-–2+ IV I 2+–2+ II 11/2–21/2 III 1+–11/2 IV 2-–1+ V ICN 16080 III 3-–3- IV I 2-–2+ II 1+–2- III 1+–2+ IV 2+–1+ V ICN 16081 III 3-–2+ IV I 2+–2+ II 1+–2+ III 1+–1+ IV 2-–1+ V ICN 19635 III 2-–1+ IV I 2-–21/2 II 11/3–21/4 III 1+–21/4 IV 21/4–11/4 V ICN 19727 II 2+–31/3 III 21/4–21/3 IV I 11/4–2+ II 1+–21/2 III 1+–21/2 IV 2+–1+ V ICN 19728 II 2+–3+ III 21/2–21/2 IV I 2-–21/2 II 11/2–21/4 III 2-–21/3 IV 21/4–11/2 V ICN 24933 II 3+–31/4 III 3-–21/4 IV I 2–21/3 II 1+–21/3 III 11/2–21/3 IV 21/4–2- V ICN 24938 II 2+–31/4 III 3+–21/3 IV I 2-–21/2 II 1+–21/2 III 1+–2-–21/3 IV 11/4–2- V ICN 30139 II 2+–31/2 III 3-–2+ IV I 2–21/2 II 1+–21/4 III 2-–21/4 IV 21/3–1+ V ICN 30818 II 2+–31/4 III 3-–21/4 IV I 2–21/3 II 11/2–21/4 III 11/4–21/4 IV 21/4–11/4 V ICN 31323 II 21/2–31/4 III 3–21/2 IV I 2-–21/2 II 11/3–21/3 III 2-–21/2 IV 21/4–11/2 V ICN 41394 II 2+–31/3 III 3-–21/2 IV I 2-–21/3 II 11/2–1+ III 1+–2- IV 11/2–11/4 V Bioacoustics. According to Arcila-Pérez et al. (2017) , the advertisement call of N. pijao is a pulsed note with one or more amplitude peaks, the call duration is on average 0.023 ± 0.007s (range = 0.013 –0.037 s) with a dominant frequency of 3.68 ± 0.09 kHz (3.62–3.79 kHz). In-situ tadpole rearing. We collected a clutch with 49 embryos at the developmental Stage 9–11 sensu Gosner (1960) and Salazar-Nicholls & del Pino (2015). The in-situ management and care provided to tadpoles of N. pijao was successful and it was possible to record individuals on distinct stages. The time between the beginning of rearing tadpoles ( i.e. , egg hatching) and metamorphosis ( Fig. 13C ) was approximately 10 months (from March 2019 to January 2020 ). Comparative diagnosis. Nymphargus pijao can be distinguished from other centrolenids by its truncated snout (both in dorsal and lateral profile; Fig. 10 ); medium body size of adults (SVL in males = 22.1–27.3 mm , n = 27, in females = 24.6–28.6 mm , n = 7; Table 1 ); humeral spine absent ( crista ventralis small in reproductive males); vomerine teeth absent or present (1–2) on very reduced dentigerous processes of the vomer; dorsum green with tiny clusters of melanophores and yellow or green-yellowish spots; skin texture smooth with minute spicules (only visible under magnification); webbing between the outer fingers absent, webbing of foot reduced ( Fig. 12 ), and parietal peritoneum white in more than 1/2, visceral peritoneum translucent (not covered by iridophores). Among the species of Nymphargus ( Fig. 16 ), N. pijao is most similar to N. buenaventura , N. cristinae ( Ruiz-Carranza & Lynch, 1995b ) , N. griffithsi , and N. lasgralarias . However, N. pijao is distinguishable from these species because the snout shape in N. cristinae is subacuminated in dorsal profile and adult males are slightly larger (although their overlap in size; SVL in adult males 26.0– 31.1 mm ; n = 12); N. buenaventura has iridophores covering renal capsules (white kidneys = iridophores present) and is uniformly green with small scattered pale-yellow spots (see Fig. 16 dorsum green with tiny clusters of melanophores and yellow or green-yellowish spots in N. pijao ); N. lasgralarias has a dorsum uniformly green lacking spots ( Guayasamin et al. 2020 ). Finally, reproductive males of N. griffithsi have moderate to enlarged crista ventralis of the humerus (here considered as humeral spine present, absent in N. pijao see Figs. 11 and 16A–B , but see Guayasamin et al. 2020 ). Furthermore, none of the most similar species compared above is sister of the new species. The genus Nymphargus includes 38 additional species: N. anomalus , N. armatus , N. balionotus , N. bejaranoi , N. cariticommatus , N. caucanus ( Rada et al. , 2017 ) , N. chami , N. chancas , N. cochranae ( Goin, 1961 ) , N. colomai ( Guayasamin & Hutter, 2020 ) , N. garciae , N. grandisonae , N. humboldti , N. ignotus ( Lynch, 1990 ) , N. laurae , N. lindae , N. luminosus ( Ruiz-Carranza & Lynch, 1995b ) , N. luteopunctatus ( Ruiz-Carranza & Lynch, 1996 ) , N. manduriacu , N. mariae , N. megacheirus . N. mixomaculatus , N. nephelophila ( Ruiz-Carranza & Lynch, 1991d ) , N. ocellatus ( Boulenger, 1918 ) , N. oreonympha , N. phenax ( Cannatella & Duellman, 1982 ) , N. pluvialis , N. posadae N. prasinus ( Duellman, 1981 ) , N. rosada , N. ruizi ( Lynch, 1993 ) , N. siren , N. spilotus , N. sucre , N. truebae ( Duellman, 1976 ) and N. vicenteruedai , of which N. pijao differs (character/state in parenthesis). From N. anomalus , N. caucanus , N. colomai , N. ignotus , N. rosada and N. sucre differs because the dorsal coloration pattern in these species is pale brown, brownish-yellow, yellowish green, grey olive, or pink (green). The skin of sexually active males of N. armatus , N. bejaranoi , N. chami , N. chancas , N. garciae , N. mariae , N. megacheirus , N. megista , N. mixomaculatus , N. nephelophila , N. oreonympha , N. phenax , N. pluvialis , N. ruizi , N. truebae , and N. vicenteruedai is covered by medium to large, enameled spicules (abundant minute spicules, only visible under magnification in N. pijao ). Moreover, dorsal coloration of these lineages (except N. chami and N. phenax and some individuals of N. garciae ) includes dark spots or a combination of dark and yellow spots (dark spots absent). Other green colored species are N. cochranae , N. laurae , N. lindae , N. luteopunctatus , N. manduriacu and N. ocellatus ; however, those species have dark ocelli surrounding yellow or orange spots (yellow or green-yellowish spots present, but ocelli absent N. pijao ). Other pale green, with or without yellow dorsal spots glassfrogs are Nymphargus cariticommatus , N. posadae , N. prasinus , N. siren and N. wileyi . The dorsum of these species is smooth or shagreen, similar to N. pijao , but with the following differences: N. posadae has large-sized reproductive males (SVL= 32.7–34.1 mm , n = 3) with skin covered with numerous small warts and some scattered larger warts without spicules visible, and N. prasinus has large-sized reproductive males (SVL= 33.0– 34.5 mm , n = 3) with uniformly smooth skin without spicules visible, while N. pijao has smaller reproductive males (SVL= 22.1–27.3 mm , n = 27) with smooth skin but with abundant minute spicules (only visible under magnification). Moreover, the peritoneum around the esophagus and kidneys in N. cariticommatus and N. wileyi is white, while in N. pijao it is invariably translucent (reviewed in old and freshly preserved individuals). Differences between N. siren and N. pijao include lateral snout shape (slightly sloping in N. siren ; truncated in N. pijao ) and reproductive male size, N. siren is conspicuously smaller (although there is a small overlap; SVL = 19.8–22.6 mm ; n = 24) than N . pijao . Additionally, both species have allopatric distribution, N. siren occurring in the Amazonian slope of the northern Ecuador and southern Colombia (eastern Cordillera) and N. pijao distributed in both, Western and Central Cordillera (western slope) of Colombia . We also compared Nymphargus pijao with species from Ecuador and Colombia ( N. balionotus , N. grandisonae , N.humboldti , N. luminosus and N. spilotus ) . These species are lime green with large red dorsal spots ( N. grandisonae ), lime green or green with yellow spots (more abundant in N. luminosus and N. spilotus , less abundant in N. humboldti ) or lime green with a combination of dark and yellow dorsal dots and spots ( N. balionotus ). Additionally, the skin dorsum of reproductive males is shagreened with minute spicules (skin texture smooth with minute spicules only visible under magnification in N. pijao ). The dorsum is uniformly green with tiny clusters of melanophores and yellow or green-yellowish spots in N. pijao . In addition, adult males of N. grandisonae and N. balionotus have a moderate to enlarged crista ventralis of the humerus (see Guayasamin et al. 2020 ), coded as humeral spine absent (very low crista ventralis ) in N. pijao ( Fig. 3E ). Reproductive males of N. spilotus differ from N. pijao , in having the dorsal skin texture granular without spicules (smooth with abundant minute spicules only visible under magnification) and because the yellow dorsal spots are bigger and more abundant in N. spilotus (smaller and less conspicuous in N. pijao , Fig. 8E–G ). In addition, the two species, although closely related, are not sister ( i.e. , N. spilotus is sister to N. rosada , whereas N. pijao is sister to N. laurae + N. siren + N. humboldti ; Fig. 5 ). The advertisement call has been described only for ten (23%) species of the genus Nymphargus : N. balionotus ( Maynard et al. 2020 ) , N. bejaranoi ( Márquez et al. 1996 ) , N. grandisonae ( Hutter et al. 2013 ) , N. griffithsi , N. lasgralarias ( Hutter & Guayasamin 2012 ) , N. manduriacu ( Guayasamin et al. 2019 ) , N. oreonympha ( Mendoza-Henao et al. 2021 ) , N. pijao (reported as N. griffhithsi in Arcila-Pérez et al. 2017 ), N. pluvialis , and N. truebae ( Catenazzi et al. 2009 ) . Of the advertisement calls described for species of the genus Nymphargus , the most similar to N. pijao in terms of dominant frequency (3.68 ± 0.09 kHz), is that emitted by N. lasgralarias (3.70 ± 0.13 kHz) ( Arcila-Pérez et al. 2017 ). The call dominant frequency in N. griffithsi is 2.4 ± 0.5 kHz ( Hutter & Guayasamin 2012 ). The remaining species have a call with higher dominant frequency, except for N. mariae and N. grandisonae which have a call with lower dominant frequency. Regarding temporal features of the call, they differ partially among N. pijao , N. lasgralarias , and N. griffithsi . Call duration in N. pijao (0.023 ± 0.007 s ) is similar to that in N. lasgralarias (0.026 ± 0.006 s ), but shorter than in N. griffithsi (0.122 ± 0.009 s ); the number of pulses per call was similar between N. pijao and N. lasgralarias (2 ± 0.8 and 1.5 ± 0.6, respectively), but was higher in N. griffithsi (2.4 ± 0.5) ( Arcila-Pérez et al. 2017 ). Tadpole external morphology. The following general tadpole description was based on a Stage 35 individual (ARUQ 1243; Fig. 14 ) obtained from the study area (creek El Impes, municipality of Pijao, department of Quindío , Colombia ) (see Table 3 for measurements in several stages). From a lateral view, the body is elliptical, depressed (BH/BL = 53.1%), and the snout is rounded ( Fig. 14A ). From a dorsal view, the body is elongated, elliptical, wider anteromedially, and with a rounded snout. The eyes are well-developed, positioned dorsally, and directed anterolaterally ( Fig. 14B ). The nares are reniform, located dorsally, and directed anterolaterally; nares have a marginal rim and small triangular fleshy projection on a sagittal margin. The spiracle is sinistral in position, tubular, short, and located posterolaterally at 72.3% of the body length (BL). It is directed posterolaterally in dorsal view, dorsal at angle of ca 45° in lateral view; inner wall present, distally free from the body; opening elliptical ( Fig. 14A ). Vent tube dextral, tubular, positioned at the level of ventral fin, fused to ventral fin medially ( Fig. 14A ). Tail long (TAL/TL = 69.2%) and low (BH/MTH = 83.7%); caudal muscles not reaching rounded tip; dorsal fin arched, originating on tail, ventral fin arched; dorsal fin slightly higher than ventral fin. Myotomes V-shaped, arranged in serial blocks; maximum tail height reached at 2/3 of tail. Lateral line stitches are conspicuous, including X, Y and Z lines. Stage n Total length (TL) Body length (BL) Body Width (BW) Body height (BH) Internarial distance (IND) Interorbital distance (IOD) 25 1 18.1 5.0 2.8 2.5 - 0.8 26 1 20.8 6.7 2.9 2.6 1.6 1.1 27 2 (24.9–30.8) (7.9–8.1) (3.3–4.1) (3.0–4.0) (1.4–1.9) (1.0–1.4) 28 2 (33.0–34.6) (10.1–10.7) (4.5–4.7) (3.6–5.3) (1.9–2.1) (1.5–1.6) 29 1 35.2 8.0 5.3 5.1 2.6 1.4 33 1 37.6 9.2 7.5 5.3 2.4 1.5 35 1 38.9 11.3 6.6 6.0 2.3 1.7 36 1 39.2 11.3 6.5 5.8 1.9 1.8 37 1 42.6 12.2 5.3 5.9 2.3 3.7 39 1 40.6 11.2 7.4 5.9 2.0 2.0 Continued. Stage n Spiracle-snout distance (SSD) Eye-snout distance (ESD) Maximum tail height (MTH) Tail length (TAL) Tail muscle height (TMH) Oral disc width (ODW) 25 1 - 2.0 2.7 12.2 - 1.4 26 1 3.9 2.3 2.4 14.4 1.9 1.5 27 2 (4.6–5.2) (2.3–2.4) (4.5–5.1) (17.7–21.6) (2.3–2.8) (0.5–2.1) 28 2 (6.6–7.0) (3.3–3.4) (5.7–6.0) (22.6–24.7) (2.8–4.5) (2.5–2.6) 29 1 - 3.3 7.1 24.8 3.3 2.7 33 1 7.2 2.8 6.4 26.9 4.4 2.2 35 1 8.2 2.9 7.2 26.9 5.8 3.1 36 1 8.0 3.4 7.4 29.7 6.0 2.5 37 1 8.5 3.4 7.5 31.5 3.1 3.3 39 1 9.0 3.7 8.8 28.3 4.2 3.5 The mouth is positioned anteroventrally, and the oral disc is 52.7% of body width. It is not emarginated laterally and is bordered by a single row of ca., 63 conical, alternated, marginal papillae; upper lip with large diastema; marginal papillae longest on the lateral lip; submarginal papillae absent. The labial tooth row exhibits a formula 2(2)/3(1); A-1 = A-2, P-1 = P-2> P3. There is a large gap in A2, extending through the descending border of the upper jaw; P1 and P2 are well developed, P2 with a small medial gap. Jaw sheaths present, serrate, keratinized; upper jaw sheath arch-shaped; lower jaw sheath U-shaped; upper jaw sheath wider than lower jaw sheath ( Fig. 14C ). Tadpole color in life and preservative. In life, the tadpole of Nymphargus pijao is almost translucent, but with a very slight green coloration on the skin, giving the tadpole a green-whitish color in appearance. The dorsum is slightly pigmented with some pale brown scattered punctuations, especially concentrated between the nares and eyes ( Fig. 15B ), in the posterior section of the body, and along the tail ventral skin of the body is translucent ( Fig. 15A ). Coiled intestines visible through the skin; liver orange pink; heart sinus hyobranchialis and other parts of the circulatory system are pink ( Fig. 15C ). The tail musculature is translucent, and the tail fins are also translucent or slightly greenish tinted. In preservative, the dorsum, tail musculature, and venter of the tadpole are pale white or cream. Melanophores on dorsal, tail fins and tail musculature turn very pale brown. Tadpole variation and ontogenetic development . The following description of variability for the tadpole of Nymphargus pijao is based on an individual at Stage 35 (see previous description) and other eleven tadpoles (ARUQ 1235–1245, MPUJ ANFB 10967) at Stages 25 to 39 ( Table 3 ). The labial tooth row formula (LTRF) is variable: three different LTFR were observed varying from 2/2 to 2/3. Apparently, there is no relation between LTRF and development stages; however, the most common LTRF was 2(2)/3(1) (n = 10; Stages 25–28, 35–39), whereas the least frequently observed LTRF was found 2(2)/2(1)(2) (n = 1; Stage 38). Additionally, two individuals at Stages 29 and 33, exhibited 2(2)/3(1)(2), an unusual LTRF among centrolenids (see comparisons section ahead). Major differences in pigment pattern were observed in the sample: the body, tail musculature and fins are white in early stages and greenish in the late ones (Stages 36–39; Fig. 15 ). In Stages 25–29, the eyes of N. pijao are small and Cshaped in dorsal view, whereas they are larger and round from Stage 33 to 39 ( Fig. 14B ). Natural history . Males of Nymphargus pijao were usually found on the upper surfaces of leaves between 50–200 cm height nearby streams. The amplexus of N. pijao is axillary ( sensu Duellman & Trueb 1986 ; Carvajal-Castro et al. 2020 ). Egg clutches were observed on leaves of plants of the families Araceae , Arecaceae , Orchidaceae , Piperaceae , and Thelypteridaceae at ~ 1.3–1.9 m above water ( Fig. 13A–B ). More details about the natural history of N. pijao are found in Arcila-Pérez et al. (2017) .