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FIGURE 4 in A new cryptic species of glassfrog (Centrolenidae: Nymphargus) from Reserva Las Gralarias, Ecuador

FIGURE 4. Nymphargus lasgralarias sp. nov. (A, B, C) and Nymphargus griffithsi (D, E, F) in life. Photographs A and D were taken in-situ; Photographs B, C, E, and F taken ex–situ upon a white poster board background. Specimens represented: A = uncollected; B = MZUTI 094; C = MZUTI 094; D = MZUTI 102; E = MZUTI 099; F = MZUTI 099.

FIGURE 5 in A new cryptic species of glassfrog (Centrolenidae: Nymphargus) from Reserva Las Gralarias, Ecuador

FIGURE 5. Holotypes of Nymphargus griffithsi (A; BMNH 1940.2.20.4) and Nymphargus lasgralarias sp. nov. (B; MZUTI 096). Note dark dorsal spots on N. griffithsi. Photograph A by Martín Bustamante; photograph B by CRH.

FIGURE 3 in A new cryptic species of glassfrog (Centrolenidae: Nymphargus) from Reserva Las Gralarias, Ecuador

FIGURE 3. Measurement standards for Nymphargus lasgralarias sp. nov. (A) Call duration; A1 shows the start and A2 shows the end of the call. (B) Frequency; B1 shows where the initial frequency of the fundamental frequency was measured; B2 and B3 show where the upper and lower frequency of the fundamental frequency were measured, respectively.

FIGURE 2 in A new cryptic species of glassfrog (Centrolenidae: Nymphargus) from Reserva Las Gralarias, Ecuador

FIGURE 2. Map of the waterways in Reserva Las Gralarias and the surrounding region. The map indicates the observed localities of Nymphargus griffithsi and Nymphargus lasgralarias sp. nov., in addition to the type locality of N. griffithsi at the Saloya River, from Goin (1961).

FIGURE 1 in A new cryptic species of glassfrog (Centrolenidae: Nymphargus) from Reserva Las Gralarias, Ecuador

FIGURE 1. Map of Ecuador showing the type locality of Nymphargus lasgralarias sp. nov. (Reserva Las Gralarias; red circle).

FIGURE 16 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

FIGURE 16. Photographs of 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.

FIGURE 15 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 14 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 12 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 11 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 10 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 4 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 9 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 2 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

FIGURE 2. In situ set used for rearing embryos and tadpoles of Nymphargus pijao sp. nov. A. Recipient to capture hatchings. B. Plastic container used to rear tadpoles simulating natural conditions. C. Plastic container with water from the same creek, observe the presence of the inlet and outlet filters for facilitating water flow and D. Location and final disposition of the plastic container in the creek. Note the mesh and the barricade to protection to avoid the intrusion of predators.

FIGURE 7 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

FIGURE 7. Uncorrected pairwise genetic distances (%) among samples of Nymphargus pijao sp. nov. Percentages are inferred from mitochondrial markers 16S rDNA (red values) and 12S rDNA (blue values). The symbol "?" represents the unavailability of DNA sequences.

FIGURE 3 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 1. A in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 Central Andes 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 8 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

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 6 in A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus)

FIGURE 6. Uncorrected pairwise genetic distances (%) among closest related species of Nymphargus pijao sp. nov. inferred from mitochondrial 16S (red values) and 12S (blue values) rDNA gene evidence. Due to the unavailability of 16S sequences for N. humboldti, only percentages of genetic distances with the 12S marker are presented.