Search

Background Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. Results We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20–25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20–25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. Conclusion We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

The complex topography of the species-rich northern Andes creates heterogeneous environmental landscapes that are hypothesized to have promoted population fragmentation and diversification by processes such as vicariance or local adaptation. Previous phylogenetic work on the palm rocket frog (Anura: Aromobatidae: Rheobates spp.), endemic to midelevation forests of Colombia, suggested that valleys were important in promoting divergence between lineages. In this study, we first evaluated previous hypotheses of species-level diversity, then fitted an isolation-with-migration (IM) historical demographic model, and tested two landscape genetic models to explain genetic divergence within Rheobates: isolation by distance and isolation by environment. The data consisted of two mitochondrial and four nuclear genes from 24 samples covering most of the geographic range of the genus. Species delimitation by Bayesian Phylogenetics and Phylogeography recovered five highly divergent genetic lineages within Rheobates, among which few to no migrants are exchanged according to IM. We found that isolation by environment provided the only variable significantly correlated with genetic distances for both mitochondrial and nuclear genes, suggesting that local adaptation may have a role in driving the genetic divergence within this frog genus. Thus, genetic divergence in Rheobates may be driven more by variation among the local environments where these frogs live rather than by geographic distance.

PNAS 2022. Vol. 119 (4)e2115641119.https://doi.org/10.1073/pnas.2115641119

Boana platanera sp. nov. La Marca, Escalona, Castellanos, Rojas-Runjaic, Crawford, Señaris, Fouquet, Giaretta, and Castroviejo-Fisher (Figs. 2, 4–6, 8J, 10) Suggested common name: Banana tree dwelling frog (rana platanera, in Spanish). Boana xerophylla —populations north of the Orinoco River, Dubois (2017); Rojas-Runjaic & Infante-Rivero (2018); populations north of the Orinoco River, Barrio-Amorós et al. (2019); populations north of the Orinoco River, Escalona et al. (2019a); Cañizales (2020). Hypsiboas xerophyllus —populations north of the Orinoco River, Orrico et al. (2017). Hypsiboas crepitans —populations north of the Orinoco River, Faivovich et al. (2005); populations north of the Orinoco River, Köhler (2010); Lehtinen (2014); Guarnizo et al. (2015). Hyla crepitans — Lutz (1927); Shreve (1947); Alemán (1953); Ginés (1959); Röhl (1959); Rivero (1961); Fouquette (1966); Solano de Chacín (1968); Kenny (1969); populations north of the Orinoco River, Duellman (1970, 1997, 2001); populations north of the Orinoco River, Cochran & Goin (1970); Donoso-Barros & León (1972); Yústiz (1976); Kluge (1979); La Marca (1992); Kime et al. (2000); Lynch & Vargas Ramírez (2000); Lynch & Suárez-Mayorga (2001); Bernal et al. (2004); Nava (2005); populations from Colombia and Panama, Vanzolini & Myers (2015). Holotype. Adult male, ULABG 7758 (field number MES 018; Figs. 2, 4), from 2 km of La Soledad, on the road from Barinitas to Santo Domingo, Barinas state, Venezuela (08º48’26’’ N, 70º30’46’’ W [datum: WGS 84]; 947 m asl; Fig. 7A), collected by Enrique La Marca, Moisés Escalona, and Iván Mendoza, on June 13, 2012. Paratypes. Three adult male specimens, ULABG 7759–7761, topotypes, same data as holotype (Fig. 5). Three adult male specimens, ULABG 7762–7764, from La Soledad, Barinas state, Venezuela (08º48’35’’ N, 70º31’31’’ W [datum: WGS 84] 980 m asl), collected by Enrique La Marca, Moisés Escalona, and Iván Mendoza, on June 13, 2012. Referred specimens. Up to 497 specimens were used to assess the genetic and phenotypic variation, although they are not included in the type series. In addition, we analyzed the calls of eleven non-vouchered specimens. See Appendices 1–3 for details. Generic placement. The new species is assigned to Boana, within the B. faber group (sensu Faivovich et al. 2005), based on the results of the phylogenetic analyses (Fig. 1). Definition. The new species is defined by the combination of the following characters: (1) moderately large size, snout-vent length (SVL) 54.33–74.74 mm in females (n = 76) and 46.57–63.34 mm in males (n = 195); (2) head narrower than body, flat in dorsal view; (3) snout truncated (slightly rounded in some individuals) in dorsal and lateral view; nostrils protuberant; (4) horizontal pupil; first half of palpebral membrane densely pigmented with black dots; (5) supratympanic fold extents posteriorly from posterior eye corner and slopes downward to a point above arm insertion; (6) distance between tympanum and eye equal to or shorter than half tympanum diameter; (7) forearm relatively more robust than arm; fingers moderately long and slender with large rounded discs; (8) adult males with prepollical spine, protruding through distal end in some cases; (9) hand-web formula I 0–0 II (1–1 +)– (1–1 +) III 1.5–(2 - –2 +) IV; (10) foot-web formula I 1.5–1 + II (2 - –2 +)–(2–3 -) III 3–(2 - –3 -) IV (3–3 -)–(3 - –3 +) V; (11) cloacal opening at thighs upper level, with supracloacal fold directed posteriorly; pericloacal region dark-brown with a white-colored crenulated supracloacal fold; (12) skin areolate on throat, chest, belly, and ventral surfaces of thighs; (13) dorsal skin smooth, slightly granulated on flanks; (14) poorly developed ulnar fold extending along finger IV external side; (15) tarsal fold poorly evident; (16) calcars absent; (17) adult males with vocal sac single, median, subgular, and moderately distensible (Fig. 6H); (18) dorsal coloration is variable at night, from almost uniform pale yellowish ocher, reddish brown, to conspicuously marbled with more or less well-defined dark-brown marks, and an irregular or broad X-shaped on the mid dorsum (Fig. 6); (19) in preservative, palms and soles pale cream-colored, hand and foot webs cream with dark brown flecks, especially on foot. Diagnosis. Character states of compared species are given in parentheses. Boana platanera is sister of B. xerophylla (Fig. 1) and it can be diagnosed from the latter by the following combination of adult male characters: a pale orange-yellow or light brown coloration on dorsum (dark brown to green tones; Fig. 8I, J); presence of black or dark brown speckles in the anterior half of the palpebral membrane (absent; Fig. 9); pericloacal region dark brown (cream); advertisement call with shorter, 28−71 ms, first note length (79–124 ms in call type I and 85−110 ms in call type II), composed of five notes (one note in call type I). Although not diagnostic, the following characters may help to differentiate both species: the new species has a statistically significant larger body (46.57–63.34 mm) than B. xerophylla (45.13–56.71 mm) (Table 2 in Escalona et al. 2019a); in preservative, the gular region is cream with dark brown or black flecks near to the jaw border (cream and sometimes with few dark brown speckles on the border of the jaw; Fig. 9); advertisement call with statistically significant longer pulse interval (11−23 ms) than B. xerophylla (12–14 ms in call type I and 11−14 ms in call type II); statistically significant longer second phase length (137−312 ms) than B. xerophylla (73−150 ms in call type II). Boana platanera is sympatric with B. pugnax in lowland western and central Venezuela, as well as some regions in Colombia and Panama. The new species is distinguished from B. pugnax by having a pale orange-yellow or light brown coloration on dorsum (dark-brown to dark-grey tones; Fig. 8G, J); the anterior half of the palpebral membrane densely pigmented (poorly pigmented); dorsal surfaces of finger and toe discs usually cream, when pigmented, pale gray (dark); calcar absent (poorly developed); a complex advertisement call composed of five notes (simple call of one note). Although not diagnostic, B. platanera is distinguished from B. pugnax by having a statistically significant smaller body (males: B. platanera: 46.57–63.34 mm; B. pugnax: 55.85–74.80 mm [sample from western Venezuela]; Table 4); and a statistically significant longer advertisement call (B. platanera: 200−451 ms; B. pugnax: 156–258 ms). The new species differs from Boana crepitans by having a pale orange-yellow or light brown coloration on dorsum (gray-cream tones to dark brown; Fig. 8H, J); advertisement call composed of five notes (two notes). Although not diagnostic, the new species has a statistically significant shorter ETD (eye to tympanum distance; B. platanera: 0.96–2.78 mm) than B. crepitans (1.99–3.29 mm) (Tables 1–2 in Escalona et al. 2019a); statistically significant shorter advertisement call (B. platanera: 200−451 ms; B. crepitans: 368−473 ms), with a statistically significant shorter second phase (B. platanera: 137−312 ms; B. crepitans: 278−367 ms) (Tables 2, 3 in Escalona et al. 2019a). Boana platanera differs from B. rosenbergi by having a smooth dorsum, thighs, and flanks (rough or with tubercles in at least one of these regions; Fig. 8F, J); smaller body size (males: B. platanera: 46.57–63.34 mm; B. rosenbergi: 75.55–83.98 mm); presence of black dots on anterior half of the palpebral membrane (absent); ulnar fold smooth (slightly crenulated); and a complex advertisement call (simple in B. rosenbergi) with shorter duration (B. platanera: 200−451 ms; B. rosenbergi: 551–759 ms) (Table 2). The new species differs from Boana exastis by having a smooth dorsum, thighs, and flanks (rough or with tubercles in at least one of these regions; Fig. 8D, J); smaller body size (males: B. platanera: 46.57–63.34 mm; B. exastis: 81.1–99.0 mm); presence of black dots on anterior half of the palpebral membrane (absent); poorly-defined dermal fringes on limbs (crenulated dermal fringes), less webbing in hands and feet (more extensive webbing in hands and feet), absence of cloacal plate (presence); absence of calcars (presence); complex advertisement call (simple in B. exastis), with shorter duration (B. platanera: 200−451 ms; B. exastis: 807–1229 ms) (Table 2). Boana platanera differs from B. lundii by having a smooth dorsum, thighs, and flanks (rough or with tubercles in at least one of these regions; Fig. 8E, J); presence of black dots on anterior half of the palpebral membrane (absence); poorly-defined dermal fringes on limbs (well defined); less webbing in hands and feet (more webbing in hands and feet); absence of calcars (presence); complex advertisement call (simple), with shorter duration (B. platanera: 200−451 ms; B. lundii: 834–1228 ms) (Table 2). The new species differs from Boana pardalis by having a smooth dorsum, thighs, and flanks (rough or with tubercles in at least one these regions; Fig. 8C, J); presence of black dots on first half of the palpebral membrane (absence); poorly-defined dermal fringes on limbs (crenulated); less webbing in hands and feet (more webbing in hands and feet); absence of cloacal plate (presence); absence of calcars (presence); complex advertisement call (simple), with longer duration (B. platanera: 200−451 ms; B. pardalis: 117–143 ms), composed of 4–6 notes (one) (Table 2). Boana platanera differs from B. faber by having a smaller body size (males: B. platanera: 46.57–63.34 mm; B. faber: 77.90–97.08 mm); presence of black dots on anterior half of the palpebral membrane (absence); ulnar fold present (absent); cream gular coloration with dark-brown flecks near to the jaw border (dark); complex advertisement call (simple), longer advertisement call (B. platanera: 200−451 ms; B. faber: 75–121 ms), composed of 4–6 notes (one note), with higher fundamental frequency (B. platanera: 542.7−1125.0 Hz; B. faber: 344.5–447.9 Hz) (Table 2). Boana platanera is readily distinguishable from B. albomarginata by having a smooth dorsal skin (slightly granular in B. albomarginata), a pale orange-yellow coloration with dark-brown marks, irregular or broad X-shaped on the dorsum and dark-brown bars on flanks and thighs (green with some white spots on the dorsum; Fig. 8A, J); supratympanic fold slopes downward to a point above arm insertion (supratympanic extends posteriorly until half of the laterodorsal region); absence of calcar (presence); complex advertisement call (simple), with longer duration (B. platanera: 200−451 ms; B. albomarginata: 166–185 ms), with 4–6 notes (one note), and lower fundamental frequency (B. platanera: 542.7−1125.0 Hz; B. albomarginata: 1136.9–1231.7 Hz) (Table 2).Although not diagnostic, the new species is larger bodied (males: B. platanera: 51.97–59.08 mm; B. albomarginata: 44.68–53.6 mm). Holotype description. Adult male in a good state of preservation (Fig. 4), with a piece of tissue sampled from the right thigh. Morphometric measurements of the holotype are presented in Table 5. Body slender; head wider than long (HW/HeL = 1.06); snout truncated in lateral and dorsal views; nostrils protuberant, oblique and directed dorsolaterally; internarial distance smaller (66 %) than eye-to-nostril distance; eye diameter, upper eyelid width and interorbital distance approximately equal to tympanum diameter; canthus rostralis rounded, almost indistinct; loreal region concave; lips not flared; eyes large (ED/HeL = 0.28), protruding; interorbital region and dorsal surface of snout slightly concave. Tympanum conspicuous, nearly circular; tympanic membrane differentiated; tympanic annulus well-defined, with upper border concealed by the tympanic membrane. Vocal sac single, subgular; vocal slits large, located at the posterolateral edge of the mouth floor. Tongue wider than long, cordiform, slightly notched behind; almost completely attached to mouth floor. Choanae elliptical, widely separate. Vomerine teeth massive; vomerine processes arched, slightly separate and between choanae. Arms slender; forearms more robust than upper arms; an inconspicuous crenulated fringe along forearm ventroexternal surface and prolateral side finger IV (more conspicuous on right forearm). Hand with an elliptical and flat palmar tubercle and an inconspicuous thenar tubercle; prepollex well-developed, with a single curved spine (Fig. 4); numerous small supernumerary tubercles scattered on the palm; relative fingers length: III>IV>II>I; subarticular tubercles single, rounded to conical and prominent; fingers discs large, expanded and nearly circular; disc on first finger slightly smaller than those of other fingers; third finger disc diameter about 50 % of eye diameter and approximately 60 % of tympanum diameter; hand-webbing formula: I 0–0 II 1 + –1 III 1.5–2 + IV. Legs long and slender; thigh length slightly longer than shank length (FL/TL = 1.01); without calcars—the protrusion of the skin fold on the right heel, which resembles a calcar, is caused by the fixation position of the hind limbs, with the feet oriented posteriorly; tarsal fringe not evident; foot with an elliptical inner metatarsal tubercle; outer metatarsal tubercle not evident; subarticular tubercles single, rounded and prominent; supernumerary tubercles scarce, small, and flat; toes extensively webbed, with rounded and expanded disks; foot-web formula: I 1.5–1 + II 2 + –2 III 3–3 - IV 3–3 + V. Skin on dorsal surfaces smooth; throat, chest, belly and ventral surface of thighs areolate; supracloacal fold crenulated; pericloacal region tuberculate. Color of euthanized holotype. The holotype was photographed two minutes after anesthesia and the following description was based in those photographs. Dorsum with a large dark brown irregular blotch over an olive background; flanks olive with vertical narrow brown bands; groin and postero-ventral part of flank orange; throat and chest dark gray; chin olive with dark brown markings; upper part of venter gray; middle and lower part of venter reddish orange, with gray tubercles; upper parts of extremities gray to olive, with brown bands or markings; foot web reddish; iris light greenish gray with fine dark venation. Color of holotype in preservative. Dorsum pale brown with dark-brown irregular blotches without a defined pattern, darker on dorsum than on head (Fig. 4); upper surface of arms pale brown, with one dark-brown incomplete band on forearm, wider on the external edge; dorsal surface of hands with one dark-brown incomplete band (more evident on the right hand); third and fourth fingers with the same coloration of arms; first and second fingers pale cream colored; dorsal surface of thighs pale brown with dark brown bars that form an irregular reticular pattern; dorsal surface of shanks, metatarsus and toes pale brown with dark brown blotches without a defined pattern; webbing on hands cream colored with dark brown flecks. Flanks cream with dark-brown vertical lines. Pericloacal region dark brown surrounded by white. Ventral surfaces of arms, forearms and hand palms pale cream; pattern on dorsal surface of thigh partially extended to ventral surface; lower edge of thighs pale cream; shanks pale brown with dark brown blotches; metatarsus, feet and foot-web cream with numerous dark brown flecks; throat cream with dark brown flecks, mostly in borders of the mouth; chest and belly pale cream. Variation in morphology. The variation of external measurements, as well as hand and foot webbing formula of some type specimens and from all the specimens examined is shown in Table 5. There is sexual dimorphism in body size, with males generally smaller than females (males: 46.57–63.34 mm; females: 54.33–74.74 mm), and by the presence of a prominent prepollical spine in males (Figs. 5, 6). Color variation in life. When active at night, adult specimens have a yellow, tan, to light brown coloration, with brown patterns (irregular in shape or with a broad X-shaped blotch near the head; some specimens have a brown vertebral line) on dorsum (Figs. 6A, 7C). Brown dots are spread on the flanks, above the upper lip, on the limbs, and on the webbings. Some specimens have a brown line delimiting the supratympanic fold. Some specimens have apparent scars on the dorsum. There is a brown blotch with a white line on the upper margin on heels and cloacal region. There are several vertical brown bars on flanks and transversal brown bars on the thighs. White coloration predominates along all the ventral surfaces, with strong orange or pinkish tones on the belly, arms, and thighs. In some specimens, the dorsal brown bars of the thighs are projected on the internal portion. The tarsus has two colorations delimited by the tarsal fold. The outer portion of the tarsus has a brown coloration with orange tones, while the internal portion has orange coloration. Some specimens have some brown spots around the mandible, on the upper portion of the thigh, and/or the webbing at the hands and foots. During the day, when normally are inactive, they have a pale cream coloration (Fig. 6E). The brown patterns and spots, when visible, are pale gray, and the bars on the flanks and thighs are dark brown almost black. Juvenile specimens have a pale green coloration with black diffuse dots on the dorsum (Fig. 6F). During the night, the iris around the pupil is metallic gray color and the outer area is pale emerald (Fig. 6A, C, D). Color variation in preservative. Coloration pattern of paratopotypes in preservative is depicted in Fig. 5. Specimens from localities other than the type locality show some color variation, as follows: dorsum occasionally may be uniformly pale-brown (e.g., ULABG 2950, 2951, 7755, 7803), sometimes bearing two scapular dark brown spots (e.g., ULABG 5338), or few scattered small dots on sacral region (e.g., ULABG 5337, 5338) or dorsum (e.g., ULABG 7745). Some specimens tend to have an ashy background with irregular markings (e.g., ULABG 5060, 6902, 7753) and, very rarely, have a lichen aspect (e.g., ULABG 7756, 7757). More often, dorsum bears large blotches that may be irregular, Published as part of Escalona, Moisés, Marca, Enrique La, Castellanos, Michelle, Fouquet, Antoine, Crawford, Andrew J., Rojas-Runjaic, Fernando J. M., Giaretta, Ariovaldo A., Señaris, J. Celsa & Castroviejo-Fisher, Santiago, 2021, Integrative taxonomy reveals a new but common Neotropical treefrog, hidden under the name Boana xerophylla, pp. 401-448 in Zootaxa 4981 (1) on pages 413-423, DOI: 10.11646/zootaxa.4981.3.1, http://zenodo.org/record/4921029, {"references":["Dubois, A. (2017) The nomenclatural status of Hysaplesia, Hylaplesia, Dendrobates and related nomina (Amphibia, Anura), with general comments on zoological nomenclature and its governance, as well as on taxonomic databases and websites. Bionomina, 11, 1 - 48.","Rojas-Runjaic, F. J. & Infante-Rivero, E. E. (2018) Inventario rapido de los anfibios y reptiles del bosque ombrofilo del cerro Las Antenas, en la vertiente venezolana de la Sierra de Perija. Memoria de la Fundacion La Salle de Ciencias Naturales, 76 (184), 29 - 60.","Barrio-Amoros, C. L., Rojas-Runjaic, F. J. M. & Senaris, J. C. (2019) Catalogue of the amphibians of Venezuela: Illustrated and annotated species list, distribution, and conservation. Amphibian and Reptile Conservation, 13 (1), e 180), 1 - 198.","Escalona, M., Junca, F., Giaretta, A., Crawford, A. & La Marca, E. (2019 a) Contrasting genetic, acoustic, and morphological differentiation in two closely related gladiator frogs (Hylidae: Boana) across a common Neotropical landscape. Zootaxa, 4609 (3), 519 - 547. https: // doi. org / 10.11646 / zootaxa. 4609.3.8","Canizales, I. (2020 \" 2019 \") Comunidad de anuros en ambiente de sabana de la Cordillera de la Costa de Venezuela. Acta Biologica Venezuelica, 39 (1), 107 - 123.","Orrico, V. G., Nunes, I., Mattedi, C., Fouquet, A., Lemos, A. W., Rivera-Correa, M., Lyra, M., Loebman, D., Pimenta, B., Caramaschi, U., Rodrigues, M. T. & Haddad, C. F. B. (2017) Integrative taxonomy supports the existence of two distinct species within Hypsiboas crepitans (Anura: Hylidae). Salamandra, 53, 99 - 113.","Faivovich, J., Haddad, C. F. B., Garcia, P. C. D. A., Frost, D. R., Campbell, J. A. & Wheeler, W. C. (2005) Systematic review of the frog family Hylidae, with special reference to Hylinae: a phylogenetic analysis and taxonomic revision. Bulletin of the American Museum of Natural History, 294, 1 - 240. https: // doi. org / 10.1206 / 0003 - 0090 (2005) 294 [0001: srotff] 2.0. co; 2","Lehtinen, R. M. (2014) Confirmation of nest building in a population of the gladiator frog Hypsiboas crepitans (Anura, Hylidae) from the island of Tobago (West Indies). Herpetology Notes, 7, 227 - 229.","Guarnizo, C. E, Paz, A., Munoz-Ortiz, A., Flechas, S. V., Mendez-Narvaez, J. & Crawford, A. J. (2015) DNA Barcoding survey of anurans across the Eastern Cordillera of Colombia and the impact of the Andes on cryptic diversity. PLoS ONE, 10, e 0127312. https: // doi. org / 10.1371 / journal. pone. 0127312","Lutz, A. (1927) Notas sobre batrachios da Venezuela e da Ihla de Trinidad. Notes on batrachians from Venezuela and Trinidad. Memorias do Instituto Oswaldo Cruz, 20 (1), 35 - 65.","Shreve, B. (1947) On Venezuelan reptiles and amphibians collected by Dr. H. G. Kugler. Bulletin of the Museum of Comparative Zoology, 99 (5), 517 - 537. [https: // www. biodiversitylibrary. org / page / 4322505 page / 555 / mode / 1 up]","Aleman, G. C. (1953) Contribucion al estudio de los reptiles y batracios de la Sierra de Perija. Memoria de la Sociedad de Ciencias Naturales La Salle, 13, 2058 - 225.","Gines, Hno. (1959) Familias y generos de anfibios - Amphibia de Venezuela. Memoria de la Sociedad de Ciencias Naturales La Salle, 19 (53), 84 - 146.","Rohl, E. (1959) Fauna Descriptiva de Venezuela (Vertebrados). 4 th Edicion. Nuevas Graficas, Madrid, 516 pp.","Rivero, J. A. (1961) Salientia of Venezuela. Bulletin of the Museum of Comparative Zoology, Harvard, 126 (1), 1 - 207.","Fouquette, M. J. (1966) Some hylid frogs of the Canal Zone, with special reference to call structure. Caribbean Journal of Science, 6, 167 - 172.","Solano de Chacin, H. (1968) Anfibios comunes del Valle de Caracas. In: Crema, M. (Ed.), Estudio de Caracas. Vol. 1. Ecologia vegetal y fauna. Universidad Central de Venezuela, Caracas, pp. 259 - 294.","Kenny, J. S. (1969) The amphibia of Trinidad. Studies on the fauna of Curacao and other Caribbean Islands, 29, 1 - 62. [https: // www. repository. naturalis. nl / document / 549896]","Duellman, W. E. (1970) The hylid frogs of Middle America. University of Kansas Monograph, Museum of Natural History, 1, 1 - 427.","Duellman, W. E. (1997) Amphibians of La Escalera region, southeastern Venezuela: taxonomy, ecology, and biogeography. Scientific Papers, Natural History Museum, The University of Kansas, 2, 1 - 52. https: // doi. org / 10.5962 / bhl. title. 16166","Duellman, W. E. (2001) The Hylid Frogs of Middle America. Second edition. Society for the Study of Amphibians and Reptiles, Ithaca, New York, 1170 pp.","Cochran, D. M. & Goin, C. J. (1970) Frogs of Colombia. Bulletin of the United States National Museum, 288, 1 - 655.","Donoso-Barros, R. & Leon, J. (1972) Desarrollo y evolucion larval de Hyla crepitans (Amphibia-Salientia). Boletin de la Sociedad de Biologia de Concepcion, 44, 117 - 127.","Yustiz, E. (1976) Salientios (Amphibia) de Barquisimeto. Tarea Comun, 3, 137 - 145.","Kluge, A. G. (1979) The gladiator frogs of Middle America and Colombia - A reevaluation of their systematics (Anura: Hylidae). Occasional Papers of the Museum of Zoology, University of Michigan, 688, 1 - 24.","La Marca, E. (1992) Catalogo Taxonomico, Biogeografico y Bibliografico de las Ranas de Venezuela. In: Cuadernos Geograficos. Vol. 9. Universidad de Los Andes, Merida, pp. 1 - 197.","Kime, N. M., Turner, W. R. & Ryan, M. J. (2000) The transmission of advertisement calls in Central American frogs. Behavioral Ecology, 11 (1), 71 - 83.","Lynch, J. D. & Vargas Ramirez, M. (2000) Lista preliminar de especies de anuros del Departamento del Guainia, Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Fisicas y Naturales, 24 (93), 579 - 590.","Bernal, M. H., Montealegre, D. P. & Paez, C. A. (2004) Estudio de la vocalizacion de trece especies de anuros del municipio de Ibague, Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Fisicas y Naturales, 28 (108), 385 - 390.","Nava, F. (2005) Estudio filogeografico de las ranas plataneras (Anura: Hylidae), en la region occidental de Venezuela, a traves de electroforesis de aloenzimas. Licentiate thesis. Departamento de Biologia, Universidad de Los Andes, Merida, Venezuela, 51 pp.","Vanzolini, P. E. & Myers, C. W. (2015) The herpetological collection of Maximilian, Prince of Wied (1782 - 1867), with special reference to Brazilian materials. Bulletin of the American Museum of Natural History, 395, 1 - 155.","Ewel, J. J., Madriz, A. & Tosi, J. A. (1976) Zonas de Vida de Venezuela. Memoria Explicativa sobre el Mapa Ecologico. 2 nd Edition. Ministerio de Agricultura y Cria, Fondo Nacional de Investigaciones Agropecuarias, Caracas, 270 pp, 1 map.","Monasterio, M. & Reyes, S. (1980) Diversidad ambiental y variacion de la vegetacion en los paramos de los Andes Venezolanos. In: Monasterio, M. (Ed.), Estudios Ecologicos en los Paramos Andinos. Universidad de Los Andes, Merida, pp. 47 - 91.","Sanchez, D., Chacon-Ortiz, A., Leon, F., Han, B. A. & Lampo, M. (2008) Widespread occurrence of an emerging pathogen in amphibian communities of the Venezuelan Andes. Biological Conservation, 141 (11), 2898 - 2905. https: // doi. org / 10.1016 / j. biocon. 2008.08.009","Flechas, S. V., Paz, A., Crawford, A. J., Sarmiento, C., Acevedo, A. A., Arboleda, A., Bolivar-Garcia, W., Echeverry-Sandoval, C. L., Franco, R., Mojica, C., Munoz, A., Palacios-Rodriguez, P., Posso-Terranova, A. M., Quintero-Marin, P., Rueda- Solano, L. A., Castro-Herrera, F. & Amezquita, A. (2017) Current and predicted distribution of the pathogenic fungus Batrachochytrium dendrobatidis in Colombia, a hotspot of amphibian biodiversity. Biotropica, 49 (5), 685 - 694. https: // doi. org / 10.1111 / btp. 12457","Marquez, M., Nava-Gonzalez, F., Sanchez, D., Calcagno, M. & Lampo, M. (2010) Immunological clearance of Batrachochytrium dendrobatidis infection at a pathogen-optimal temperature in the hylid frog Hypsiboas crepitans. EcoHealth, 7 (3), 380 - 388. https: // doi. org / 10.1007 / s 10393 - 010 - 0350 - x","IUCN (2012) IUCN Red List Categories and Criteria: Version 3.1. 2 nd Edition. IUCN, Gland, and Cambridge, iv + 32 pp. [https: // portals. iucn. org / library / node / 10315]","La Marca, E. (1996) First record of Hyla pugnax (Amphibia: Anura: Hylidae) in Venezuela. Bulletin of the Maryland Herpetological Society, 32 (2), 35 - 42.","Escalona, M., Prieto-Torres, D. & Rojas-Runjaic, F. J. M. (2017) Unveiling the geographic distribution of Boana pugnax (Schmidt, 1857) (Anura, Hylidae) in Venezuela: new state records, range extension, and potential distribution. Check List, 13, 671 - 681."]}