Nymphargus pijao, Montilla & Arcila-Pérez & Toro-Gómez & Vargas-Salinas & Rada, 2023

Montilla, Sebastián O., Arcila-Pérez, Luisa F., Toro-Gómez, María Paula, Vargas-Salinas, Fernando & Rada, Marco, 2023, A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus), Zootaxa 5271 (1), pp. 1-48 : 9-23

Nymphargus pijao sp. nov.

( Figs. 8 , 9 , 10A–B , 11B , 12A–B , 13 , 14 , and 15)

Centrolenella griffithsi Lynch & Duellman, 1973: 33 ; Duellman & Burrowes, 1989: 10

Cochranella griffithsi Ruiz-Carranza & Lynch, 1991a: 97

Nymphargus griffithsi Cisneros-Heredia & McDiarmid, 2007: 34

Nymphargus sp. Guayasamin, Cisneros-Heredia, McDiarmid, Peña & Hutter, 2019: 6

Common name. English: Pijaos’ glassfrog, Spanish: Rana de cristal de los Pijaos.

Generic allocation. Based on our phylogenetic results, we include the new species in the genus Nymphargus . The following morphological characters further support its placement within the genus: absence of iridophores in the hepatic and intestinal peritonea, absence or reduced webbing between fingers III and IV, and absence of humeral spines in adult males ( Cisneros-Heredia & McDiarmid 2007; Guayasamin et al. 2009).

Holotype: Adult male, ARUQ 1377 ( Figs. 8A and 9 ), collected in the stream Sopladero, at 40 km northwest of Corregimiento Uribe , cabaña La Romelia, Parque Nacional Natural Munchique, municipality of El Tambo, department of Cauca, Colombia (2°56′2″N, 76°56′19″W, 2267 m asl), on 17 September 2017 by Gustavo González-Durán.

Paratypes: Adult males ( ICN 26013–26015 , 26018–23 , 26025–30 , 26067 ), and adult female ( ICN 26017 ), collected in the stream Sopladero, at 40 km northwest of Corregimiento Uribe , Parque Nacional Natural Munchique, municipality of El Tambo, department of Cauca, Colombia (2°56′2″N, 76°56′19″W, 1890 m asl), on 01 October 1990 by Pedro M. Ruiz-Carranza. Adult males ( ARUQ 1378–1379 ) collected at Sector La Romelia , Parque Nacional Natural Munchique, municipality of El Tambo, department of Cauca, Colombia (2°42′26″N, 76°52′58″W, 1721–2267 m asl) on 17 September 2017 by Gustavo González-Durán. Adult males ( ICN 16080 , 16081 ) and adult female ( ICN 16007 ) collected at road from Corregimiento Nutibara to Corregimiento La Blanquita , municipality of Frontino, department of Antioquia, Colombia (6°47′55″N, 76°15′3″W, 2000 m asl) by Pedro M. Ruiz-Carranza. Adult male ( ICN 16022 ) collected at road from Corregimiento Nutibara to Corregimiento La Blanquita, municipality of Frontino, department of Antioquia, Colombia (6°45′35″N, 76°18′22″W, 1960 m asl), on 12 July 1987 by Pedro M. Ruiz-Carranza. Adult male ( MPUJ ANFB 10968 ) collected at municipality of Ciudad Bolivar, department of Antioquia, Colombia (1859 m asl), in February 2008 by Taran Grant, Juan Camilo Arredondo and Marco Rada .

Referred material. Adult male ( ICN 59946 ) and adult female ( ICN 59949 ) collected at stream La Vinagra, Alto Tominejo (on trial to Corregimiento Carauta ), vereda Curadiente-Chachafrutal , Corregimiento Nutibara , municipality of Frontino , department of Antioquia , Colombia (6°43′48.2″N, 76°16′2.2″W, 2050 m asl), on 26 and 27 August 2014 by Marco Rada. Adult male ( ICN 16057 ) collected at road from Corregimiento Nutibara to Corregimiento La Blanquita , municipality of Frontino , department of Antioquia, Colombia (6°46′54″N, 76°15′57″W, 1430 m asl), on 18 July 1987 by Pedro M. Ruiz-Carranza. Adult males ( ICN 16075 , 16085 ) collected at road from Corregimiento Nutibara to Corregimiento La Blanquita , municipality of Frontino , department of Antioquia, Colombia (6°47′55″N, 76°15′3″W, 2000 m asl) by Pedro M. Ruiz-Carranza. Adult male ( ICN 19730 ) collected at stream Las Canoas , Parque Nacional Natural Las Orquideas , vereda Calles , municipality of Urrao , department of Antioquia, Colombia (6°30′44″N, 76°14′7″W, 1770–1800 m asl) on 25 May 1988 by Pedro M. Ruiz-Carranza. Adult males ( ICN 19728– 29 ) collected at stream Las Canoas , Parque Nacional Natural Las Orquídeas , vereda Calles , municipality of Urrao , department of Antioquia, Colombia (6°30′44″N, 76°14′7″W, 1770–1800 m asl), on 25 May 1988 by Pedro M. Ruiz-Carranza. Adult male ( ICN 19727 ), and adult female ( ICN 19635 ) collected at stream El Silencio , Parque Nacional Natural Las Orquideas , vereda Calles , municipality of Urrao , department of Antioquia, Colombia (6°30′46″N, 76°16′24″W, 1480–1540 m asl), on 23 May 1988 by Pedro M. Ruiz-Carranza. Adult males ( ICN 24933 , 24937– 38 ) collected at Bremen Reserve , vereda El Roble , municipality of Filandia , department of Quindío, Colombia (4°40′2″N, 75°37′42″W, 1930 m asl), on 07 November 1990 by Pedro M. Ruiz-Carranza. Adult male ( ICN 9922 ) and adult female ( ICN 9921 ), collected near at hacienda San Julian and hacienda El Brillante , vereda San Julián , municipality of Calarcá , department of Quindío, Colombia (2030–2100 m asl), on 31 May 1981 by John D. Lynch. Adul male ( ARUQ 491 ), collected at farm El Jardin , vereda San Rafael municipality of Calarcá , department of Quindío, Colombia (4º30′23.81′′N, 75º36′30.081′′W 1915 m asl), on 23 July 2015 by Carlos Londoño-Guarnizo. Adult females ( ARUQ 568–569 ), collected at farm El Jardin , vereda San Rafael municipality of Calarcá , department of Quindío, Colombia (4º30′23.81′′N, 75º36′30.081′′W 1915 m asl), on 26 September 2015 by Carlos Londoño-Guarnizo. Adult male ( ARUQ 743 ), collected at reserve Las Cascadas de Río Verde , municipality of Cordoba, department of Quindío, Colombia (4º24′048′N, 75º39.271′W, 2096 m asl), on 05 February 2017 by Fernando Vargas-Salinas. Adult males ( ARUQ 647 , 648 ) collected at creek El Impes , Bellavista reserve , municipality of Pijao , department of Quindío, Colombia (4º19′31.8′′N, 75º42′0.36′′W, 1926 m asl), on 06 August 2016 by Fernando Vargas-Salinas. Adult males ( ARUQ 888 ) collected at creek El Impes , Bellavista reserve , municipality of Pijao , department of Quindío, Colombia (4º19′31.8′′N, 75º42′0.36′′W, 1926 m asl), on 02 June 2018 by Fernando Vargas-Salinas. Adult males ( ARUQ 1360 ) collected at creek El Impes , Bellavista reserve , municipality of Pijao , department of Quindío, Colombia (4º19′31.8′′N, 75º42′0.36′′W, 1926 m asl), on 15 February 2021 by Fernando Vargas-Salinas. Tadpoles ( ARUQ 1235–1245 , MPUJ ANFB 10967 ) collected at creek El Impes, reserve Bellavista, municipality of Pijao, department of Quindío, Colombia (4º19′31.8′′N, 75º42′0.36′′W, 1926 m asl), between June 2018 and January 2019 by Fernando Vargas-Salinas. Adult males ( ICN 30139 , 30145 ) collected at road near to Corregimiento of San Antonio del Chamí, municipality of Mistrató, department of Risaralda, Colombia (5°25′12″N, 75°56′18″W, 1550 m asl), on 06 April 1992 by Pedro M. Ruiz-Carranza. Adult female ( ICN 30818 ) collected at stream Santa Helena, vereda La Trinidad, municipality of Pueblo Rico, department of Risaralda, Colombia (1540 m asl), on 01 September 1991 by Maria Cristina Ardila. Adult female ( ICN 31313 ) collected at stream Santa Helena, vereda Los Planes, municipality of Santuario, department of Risaralda, Colombia (2430 m asl), on 01 January 1992 by Maria Cristina Ardila. Adult male ( ICN 31323 ) collected at stream Risaralda, vereda La Cumbre, municipality of Apia, department of Risaralda, Colombia (5°8′50″N, 76°0′33″W, 2150 m asl), on 01 January 1992 by Maria Cristina Ardila. Adult male ( ICN 28787 ) collected at Corregimiento El Boquerón, municipality of El Cairo, department of Valle del Cauca, Colombia (1900 m asl), on 22 June 1991 by John D. Lynch. Adult male ( ICN 41394 ) collected at stream La Seca, 8 km near Corregimiento Queremal, municipality of Dagua, department of Valle del Cauca, Colombia (1940–2050 m asl), on 13 July 1995 by John D. Lynch (also see Appendix V for species and specimens examined) .

Characterization. Nymphargus pijao is diagnosed by the following characteristics: (1) vomerine teeth absent or present (one or two vomerine teeth) on very reduced dentigerous processes of the vomer; (2) truncate snout in dorsal and lateral view ( Figs. 9A and 10A–B ); (3) tympanum visible (tympanic membrane and annulus differentiated) with supratympanic fold present; (4) dorsal skin in reproductive males smooth with abundant microspicules, low warts associate with yellow spots (in life); microspicules absent in females ( Fig. 8A–D ); (5) ventral skin and thighs granular and translucent, pair of large, round, flat tubercles on ventral surfaces of the thighs; (6) parietal peritoneum covered by iridophores, more than 50% (condition P3, sensu Cisneros-Heredia & McDiarmid 2007); visceral peritoneum translucent with the exception of the pericardium that is covered by iridophores (condition V 1, sensu Cisneros-Heredia & McDiarmid 2007) ( Fig. 8B–C ); (7) liver tetra-lobed (Condition H0, sensu Cisneros-Heredia & McDiarmid 2007); (8) humeral spine absent (crista ventralis very low; Fig. 11B ); (9) webbing between fingers I–II absent; basal between fingers II– IV: II (2 - – 31/2)–(3 + – 31/2) III (2 - –3 +)–(1 + –3 -) IV ( Fig. 12A ); (10) webbing between toes I (1 + –2 +)–(2 - – 21/2) II (1 - – 21/2)–(1 + – 21/2) III (1 - –2 +)–(1 + – 31/2) IV (11/4 – 31/2)–(1 + –2) V ( Fig. 12B ); (11) ulnar folds present, translucent and low; (12) nuptial excrescence type I, concealed prepollex; (13) Finger I shorter than Finger II (Finger I length 70–95% of Finger II); (14) diameter of eye larger than width of disc on Finger III (disc of Finger II corresponds to a 31.5–61.8% of the diameter of the eye); (15) in life, dorsum green with tiny clusters of melanophores forming small flecks and yellow or green-yellowish dots; green bones ( Figs. 8A and 13D ); (16) color in preservative, dorsum lavender or pale lavender with or without clusters of melanophores forming small flecks and cream-colored dots ( Figs. 9 and 10A–B ); (17) iris coloration in life, silver white or faint gold with fine reticulations and small flecks on the upper and lower side of the iris; (18) melanophores present and abundant at the base and along fingers III and IV, less dense on Finger II, and rarely present on Finger I, present and abundant along toes IV and V, less dense in the other fingers; (19) males call from the upper surfaces of leaves ( Fig. 13D ) ( Arcila-Pérez et al. 2017); (20) the advertisement call is one pulsed note with a duration of 0.023 ± 0.007 s (0.013 – 0.037 s, n = 7) and a dominant frequency of 3.68 ± 0.09 kHz (3.62–3.79 kHz, n = 7) ( Arcila-Pérez et al. 2017); (21) unknown combat behavior, (22) egg masses deposited on the underside of leaves, although one egg clutch was found on the upper side of a leaf ( Arcila-Pérez et al. 2017); clutch size: 35– 50 eggs (42 ± 6.6, n = 11) ( Fig. 13A–B ); (23) tadpole snout rounded in lateral and dorsal view; spiracle sinistral, located posterolaterally; tail long, low; dorsal fin slightly higher than ventral fin; mouth anteroventral; labial tooth row formula 2(2)/3(1) and occasionally 2(2)/3(1)(2) or 2(2)/2(1); upper jaw sheath arch-shaped; lower jaw sheath U-shaped; tadpoles are almost translucent, coiled intestines visible through the skin, liver orange-pink, heart, sinus hyobranchialis and other parts of the circulatory system pink ( Figs. 14 and 15 ); (24) SVL in adult males 24.6 ± 1.4 (22.1–27.3 mm, n = 27), and in adult females 24.7 ± 1.5 (24.6–28.6 mm, n = 7) ( Table 1); (25) insertion of musculus pectoralis abdominalis deep (see pectoral and humeral musculature section); (26) insertion of musculus deltoideus episternalis ventrolateral (see pectoral and humeral musculature section) ( Fig. 3E ); (27) small testis size (relative length with respect to that of kidneys in reproductive adults) ( Fig. 4A ).

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.

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.

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).

Distribution. Based on morphology and DNA sequences, Nymphargus pijao is endemic to Colombia ( Fig. 1A ). This species is distributed on both slopes of the cordillera Occidental and the western slope of the cordillera Central in the departments of Antioquia, Cauca, Chocó, Quindío, Risaralda, and Valle del Cauca. Frogs were found between 1480–2430 m asl.

Etymology. The specific epithet is a noun in apposition, and is dedicated to the Pijaos, an indigenous ethnic group known for their strong opposition to Spanish colonization during the American conquest ( Salmoral 1963). The Pijaos people originally inhabited the central Andes mountains of Colombia, including the upper basins of the Cauca and Magdalena rivers in the departments of Valle del Cauca, Tolima and Quindío ( Salmoral 1963). Additionally, the epithet of the new species also honors the municipality of Pijao in the department of Quindío, where the species is commonly found.

Figures

FIGURE 1. A. Geographic distribution of Nymphargus pijao sp. nov. The yellow dot denotes the type locality (Parque Nacional Natural Munchique, municipality of El Tambo, department of Cauca, Colombia; 1890 m asl); the blue dot indicates the study area in which tadpoles were reared and most natural history features for the new species have been recorded (Bellavista reserve, municipality of Pijao, department of Quindío, western slope of the CentralAndes of Colombia); the red dots indicates historical records based on literature and collections; the magenta triangle corresponds to the record of N. griffithsi from La Planada reserve, Nariño. B. Image of the study area: creek El Impes at Bellavista reserve Pijao, Quindío, Colombia.

FIGURE 3. Difference in pectoral musculature among genera of Centrolenidae. A. Diagram of a ventral view for Rulyrana mcdiarmidi (adult male, QCAZ 42049) showing the musculus deltoideus clavicularis (D. c), m. deltoideus episternalis (D. ep), m. supracoracoideus superficialis (Spc. s), m. supracoracoideus profundus (Spc. p), m. pectoralis sternalis (P. es), m. pectoralis abdominalis (P. a) and coracoradialis tendon (Cr. t). B. Detail of humeral muscles of “Centrolene’’ acanthidiocephalum, adult male (ICN 5278). C. Teratohyla spinosa, adult male (ICN 19437). D. Nymphargus griffithsi (DHMECN 4164). E. N. pijao sp. nov. adult male (ICN 60228). Note the development of the crista ventralis of the humerus in males of B, C and D (i.e., humeral spine present), and the unnoticed crista ventralis (covered = humeral spine absent) in A and E. Character state codification is indicated in each of the species. Scale bars = 2 mm.

FIGURE 4. Variation on testis size in centrolenids. Characters indicated with states in parenthesis and color dots: testicle (t) and blue dots, and kidney (k) and orange dots. A. Small-sized testicle in Nymphargus pijao sp. nov. (ICN 26014). B. Medium-sized testicle in Centrolene sanchezi (QCAZ 22387). C and D. Large-sized testicles in N. oreonympha (ICN 20779) and N. megista (ICN 27767), respectively. Scale bars = 2 mm.

FIGURE 5. Phylogenetic relationships of Nymphargus pijao sp. nov. (green branches) and its most closely related species (black lines). A skeletal topology of glassfrogs on the left is shown, indicating the position of the genus Nymphargus and highlighting in a green rectangle the expanded section. Numbers above branches are Goodman-Bremer support values. The code besides the name of the species refers to institutional or field numbers. Illustration by Ana María Ospina-L.

FIGURE 8. Photographs of Nymphargus pijao sp. nov. in life. A. Dorsolateral view of a male (holotype ARUQ 1377). B. Ventral view of a froglet (not collected) from the creek El Impes, reserve Bellavista, municipality of Pijao, department of Quindío, Colombia; 1926 m asl. C. Ventral view of a male (ICN 59946) from the municipality of Frontino, department of Antioquia, Colombia; 2000 m asl. D. Dorsolateral view of a female (ICN 59949) from the municipality of Frontino, department of Antioquia, Colombia; 2000 m asl. Note the chromatic variation in Nymphargus pijao sp. nov. in life. E. Adult male (ICN 60227) from the municipality of Ciudad Bolivar, department of Antioquia, Colombia; 1850 m asl. F. Adult male (ICN 19728) from the municipality of Urrao, department of Antioquia, Colombia; 1770–1800 m asl. G. Adult male (ICN 24933) from the reserve of Bremen, municipality of Filandia, department of Quindío, Colombia; 1930 m asl. Photos not to scale. Photograph credits, A: Gustavo González-Durán, E: Taran Grant, F: J. D. Lynch and G: Pedro M. Ruiz-Carranza (r.i.p.).

FIGURE 9. Holotype of Nymphargus pijao sp. nov., in dorsal and ventral view (ARUQ 1377) collected in the stream Sopladero, at 40 km northwest of Corregimiento Uribe, cabaña La Romelia, Parque Nacional Natural Munchique, municipality of El Tambo, department of Cauca, Colombia on 17 September 2017 by Gustavo González-Durán.

FIGURE 10. Lateral and dorsal snout shapes of some glassfrog species of genus Nymphargus. A and B. Adult male of Nymphargus pijao sp. nov., holotype (ARUQ 1377) from the municipality of El Tambo, department of Cauca, Colombia; 1890 m asl. C and D. Adult male of N. siren (QCAZ 37975) from the biological station Yanayacu, province of Napo, Ecuador; 2120 m asl. E and F. Adult male of N. griffithsi, (DHMECN 4164) from the biological station Pahuma Orchid, province of Pichincha, Ecuador; 2200 m asl. Scale bar = 2 mm

FIGURE 11. Hand and ventral view of the humeri of adult males of A. Nymphargus griffithsi (ICN 12163) from Reserva Natural La Planada, Ricaurte, Nariño, and B. N. pijao sp. nov. (JAV361) from Hacienda La Argelia, Vereda la Tulia, Bolivar, Valle del Cauca. Scale bar = 5 mm.

FIGURE 12. Hand and foot webbing of some species of the genus Nymphargus. A and B. adult male of Nymphargus pijao sp. nov., holotype (ARUQ 1377) from the municipality of El Tambo, department of Cauca, Colombia; 1890 m asl. C and D. adult male of N. siren (QCAZ 37975) from the biological station Yanayacu, province of Napo, Ecuador; 2120 m asl. D and E. adult male of N. griffithsi, (DHMECN 4164) from the biological station Pahuma Orchid, province of Pichincha, Ecuador, 2200 m asl. Scale bar A, C, E = 2 mm and B, D, F = 5 mm.

FIGURE 13. Egg clutch, embryos, froglet, and adult male of Nymphargus pijao sp. nov. A. Egg clutch (not collected) on the underside of a leaf. Note the unpigmented eggs in a clear jelly. B. Embryos developed at Stage 21 (not collected). C. Froglet (not collected) recently emerged from the water and D. Calling adult male dorsum (not collected), note the green-yellowish dots on the dorsum. All photos were taken in the municipality of Pijao, department of Quindío, Central Andes of Colombia; 1926 m asl. Photos not to scale.

FIGURE 14. Images of a preserved tadpole of Nymphargus pijao sp. nov. at Stage 35 (ARUQ 1243). A. Lateral view, B. Dorsal view C. Oral disc of Nymphargus pijao tadpole at Stage 28 (ARUQ 1239). Scale bar A, C = 2 mm, and B = 0.5 mm.

FIGURE 15. Images of a living tadpole of Nymphargus pijao sp. nov. at Stage 37 (paratype MPUJ ANFB 10967). A. Lateral view. B. Dorsal view. C. Ventral view. Scale bar A = 5 mm, and B, C = 2 mm.

FIGURE 16. Photographsof males of the genus Nymphargus of some of the species from Colombia and Ecuador.A. Nymphargus pijao sp. nov. (ICN 26013). B. N. griffithsi (ICN 12149). C. N. cristinae (ICN 18643). D. N. buenaventura (DHMECN 3562). E. N. siren (KU 146610). F. N. humboldti (QCAZ 41073). G. N. lasgralarias (QCAZ 48132). H. N. sucre (MUZTI 1421). I. N. laurae (DHMECN 15383). J. N. chancas (CORBIDI 14148). K. N. pluvialis (KU 173224). L. N. spilotus (JD 060). Photos not to scale. Photograph credits. A—C: Pedro M. Ruiz-Carranza (r.i.p). D: Mario Yánez-Muñoz. E: Martin R. Bustamante F: Santiago Ron. G: Luis A. Coloma. H: Alejandro Arteaga. I: H. Mauricio Ortega-Andrade. J: Santiago Castroviejo-Fisher. K: Juan Manuel Guayasamin. L: Jesse Delia.