AMERICAN MUSEUM NOVITATES

Number 3739: 17 pp. March 9, 2012

Copyright © American Museum of Natural History 2012

ISSN 0003-0082

A Second Species of the Family Allophrynidae (Amphibia: Anura)

SANTIAGO CASTROVIEJO-FISHER,[1] PEDRO E. PÉREZ-PEÑA,[2] JOSE M. PADIAL,[3] AND JUAN M. GUAYASAMIN[4]

ABSTRACT

We describe Allophryne resplendens, a new species from two localities in the Amazon rainforest of Loreto, Peru, of the family Allophrynidae, which was monotypic until this discovery. The new species can be readily differentiated from Allophryne ruthveni on the basis of dorsal and ventral coloration both in life and in preservative, transverse processes of presacral II oriented anterolaterally (oriented laterally in A. ruthveni), 19 nucleotide autapomorphies for 761 base pairs (bp) of the mitochondrial gene 16S, and 16 for 923 bp of 12S. Maximum parsimony analysis of the mitochondrial gene 12S and a fragment of up to 1060 bp of 16S supports the new species as sister to A. ruthveni.

INTRODUCTION

Discoveries and new descriptions of amphibian species have increased in recent years, with the number of nominal species growing of about 48.2% since 1985 (Frost et al., 2006) and 19.4% during the last decade (Köhler et al., 2005). The bulk of these new discoveries arise from applying recently developed advances in fields such as molecular biology, bioacoustics, and geography to clades from tropical regions (e.g., Meegaskumbura et al., 2002; Stuart et al., 2006; Fouquet et al., 2007; Vieites et al., 2009). Discovery of new species is, however, not randomly distributed across clades of amphibians. Some groups account for most of the newly described diversity (e.g., Ranidae alone includes 25% of the new species of amphibians described since 2004: AmphibiaWeb, 2011) whereas for other groups it may be decades before a second species within a genus is discovered. For example, Rhinatrema bivittatum (Guérin-Méneville, 1838) remained as the only known species of Rhinatrema Duméril and Bribon, 1841, for 169 years until Gower et al. (2010) described a new species, although that discovery was quickly followed by another (Wilkinson and Gower, 2010).

Allophryne ruthveni Gaige, 1926, has stood as the only member of this monotypic genus and the family Allophrynidae for 85 years. It is known to occur in the lowland rainforest of the Amazon and Orinoco basins, from Venezuela and the Guianas to southwestern Brazil and probably Bolivia (Frost, 2011). Despite its ample distribution, records are rare and sparse. Phylogenetically, the relationship Centrolenidae + Allophryne ruthveni is the most accepted hypothesis, supported by molecular phylogenetics (Austin et al., 2002; Faivovich et al., 2005; Wiens et al., 2005; Frost et al., 2006; Grant et al., 2006; Guayasamin et al., 2008) and morphological studies (Duellman, 2001; Burton, 2004; Wiens et al., 2005; but see Fabrezi, 2006). In this work, we describe the second species in the family Allophrynidae, found in the Amazon rainforest of Loreto, Peru.

MATERIALS AND METHODS

Nomenclature

Frost et al. (2006) placed Allophryne ruthveni in the monotypic subfamily Allophryninae, within Centrolenidae, whereas Guayasamin et al. (2009) argued to maintain the species in the monotypic family Allophrynidae. We followed the later arrangement (see Guayasamin et al., 2009, for argumentation).

Taxon Sampling and Morphology

Specimens were collected in the field according to approved animal use and care protocols (Heyer et al., 1994), and euthanized in Xylocaïne solution prior to fixation and preservation in ethanol 70%. Specimens studied are listed in appendix 1. Color characteristics are described according to digital photographs taken from life specimens in the field. External morphological characters were examined under a dissecting microscope. Measurements were taken with digital calipers on preserved specimens to the nearest 0.01 mm and rounded to 0.1 mm. Variables measured, as described in Duellman (2001), are as follows: snout-vent length (SVL); head length; head width; interorbital distance; eye diameter; upper eyelid; tympanum diameter; eye-nostril distance; distance from anterior margin of eye to tip of snout; internarial distance; eye-tympanum distance; length of forearm; hand length; finger I length; finger II length; width of disc of finger III; width of finger III; thigh length; shank length; foot length. Ontogenetic status was determined by examination of the development of gonads and presence of secondary sexual characteristics (e.g., vocal slits and sacs). Terminology for webbing is that described by Savage and Heyer (1967), as modified by Guayasamin et al. (2006). The osteological description was based on X-rays of the holotype; osteological terminology is that of Duellman and Trueb (1986), Fabrezi (1992, 1993), and Trueb (1973, 1993). Museum acronyms are: American Museum of Natural History (AMNH), Museo Historia Natural La Salle (MHNLS), and Museo de Zoología de la Universidad Nacional de la Amazonía Peruana (MZUNAP).

Molecular Analyses

We sequenced the complete mitochondrial gene 12S (962 bp) and a fragment of the gene 16S (1060 bp) from the specimen of Allophryne collected in Loreto, Peru, and three specimens of A. ruthveni from Delta Amacuro, Venezuela. Genomic DNA was isolated using a standard phenol-chloroform extraction protocol (Sambrook et al., 1989). The selected markers were amplified and sequenced using the same primers and PCR conditions as in Guayasamin et al. (2008) with the addition of primers 16L19 and 16H24 (Heinicke et al., 2007). Sequences from heavy and light strands were compared to generate a consensus sequence for each specimen using Sequencher 4.6 (Gene Codes Corp., 2006). We compared these sequences with those from GenBank for Allophryne ruthveni, as well as representative species of all other Neotropical families within Hyloidea, according to the taxonomy used in Frost (2011). We used sequences of Mantella madagascariensis to root phylogenetic trees. GenBank accession codes of all the sequences studied are provided in appendix 2.

Sequences of each marker were independently aligned in MAFFT (Katoh et al., 2005) under the E-INS-i option. The resulting multiple sequence alignments (Dryad Repository: doi:10.5061/dryad.2kp7q7vb) were concatenated and analyzed under the maximum parsimony criterion (MP) using TNT 1.1, Willi Hennig Society Edition (Goloboff et al., 2008) with gaps as a fifth character and using the “New Technology search” option at level 100, including sectorial searches, ratchet (see Nixon, 1999), drift, and tree fusing (see Goloboff, 1999). Bootstrap support (BS, Felsenstein, 1985) was evaluated through 1000 pseudoreplicates, with 10 RAS using TBR and 10 trees saved per replication. Uncorrected genetic distances were calculated from 761 bp of the 16S and 923 of the 12S (maximum length overlapping for all the Allophryne sequences, Dryad Repository: doi:10.5061/dryad.2kp7q7vb) in PAUP* 4.0b10 (Swofford, 1998).

Allophryne resplendens, new species

Figures 1A–C, 2A–D

Allophryne, sp. nov., Rodríguez and Knell, 2003: 244.

Holotype: MZUNAP-01-605 (field number PEPP 500), adult female from Lago Preto (4°27′35.0″S, 71°45′ 3.5″W; 120 m), Río Yavarí, Provincia Ramon Castilla, Departamento Loreto, Peru, collected by PEPP in November 2009. When collected, the distal section of the right hind limb of the specimen was missing; we cut a piece of tissue from the stump for molecular studies purposes.

Remarks: We are aware of three other records of the new species. One is a noncollected but photographed adult (fig. 2B) found in Lago Preto, Yavarí, Loreto, Peru, in July 2008 (photographs by anonymous volunteers of Earthwatch and deposited at Wildlife Conservation Society, WCS–Perú). The second is reported by Rodríguez and Knell (2003) also from Lago Preto (4°28′S, 71°46′W; 90 m), Yavarí, Loreto, Peru. Rodríguez and Knell (2003) cite this specimen as deposited at the MUSM. However, in a visit to the MUSM in March 2008, we were unable to locate the specimen or to get a voucher code for it. The third specimen, an adult male (fig. 2C, D), was collected on the south bank of Quebrada Hungurahui (4°14′42.97″S, 74°21′36.02″W; 114 m), an affluent of Río Tigre, approximately 2 km north of Comunidad Monteverde, Loreto, Peru, by William Lamar on 1 August 2006. This specimen was collected and preserved in ethanol; unfortunately, the jar did not seal properly and the specimen totally decomposed and could not be used as a type.

Generic Placement: The new species is assigned to the genus Allophryne because of the following combination of morphological characters: absence of metacarpal III process (its presence is a synapomorphy of centrolenid frogs), T-shaped terminal phalanges, tibiale and fibulare not fused, absence of neopalatines and quadratojugals, and protruding snout in lateral view; no phenotypic autoapomorphy is known for the genus. The molecular phylogeny also supports the placement of the new species in the genus Allophryne. The 10 most parsimonious trees (tree cost = 11,381 steps) resulting from 100 replicates (each including RAS + TBR + drift and tree fusing with the best tree hit 100 times) placed Allophryne resplendens, n. sp., as the sister species of Allophryne ruthveni (BS = 100; fig. 3).

Diagnosis: Allophryne resplendens, n. sp., is distinguished from A. ruthveni, the only other known species in the genus and family (character states of the latter are in parentheses), by (1) color pattern (cf. photos in figs. 1 and 2): dorsolateral surfaces in life black (cream to light brown) with large and bright glossy spots due to accumulation of iridophores (absent), most of which containing irregular yellow blotches (absent), dorsolateral surfaces in preservative dark brown (cream to light brown with dark brown stripes) with large and bright glossy spots due to accumulation of iridophores (absent), ventral surfaces in life black and opaque (not pigmented, translucent) with large and bright glossy spots due to accumulation of iridophores except in palms and plantar surfaces (absent or just a few off-white spots on the throat/chest and distal parts of legs), same character states for both species for ventral surfaces in preservative than in life, but coloration dark brown instead of black for A. resplendens; (2) osteology: transverse processes of presacral II oriented anterolaterally (oriented laterally; observed in two cleared and stained specimens; see appendix 1, and fig. 2C in Fabrezi and Langone, 2000); and (3) DNA sequences: uncorrected genetic distance > 2.5 % for 761 bp of mitochondrial gene 16S, and ≥ 1.7 % for 923 bp of 12S (table 1), 19 nucleotide autapomorphies for 761 bp of 16S, and 16 for 923 bp of 12S (table 2).

Description of the Holotype: Adult female, SVL 28.4 mm. Head wider than long (HL = 85.7% of HW); snout protruding in lateral profile and broadly rounded in dorsal view (fig. 1A, C); canthus rostralis indistinct, straight; loreal region flat; lips not flared; nostril small, not protuberant, closer to tip of snout than to eye. Eye of moderate size (ED = 10.6% of SVL). Tympanic membrane and tympanic annulus differentiated; tympanum relatively small (TD = 3.9% of SVL). Dentigerous process of vomer indistinguishable. Ulnar tubercles absent; relative lengths of fingers: III > IV > II > I webbing on hands basal. Discs expanded, truncate; subarticular tubercles round, conspicuous; few supernumerary tubercles; palmar tubercle oval, simple. Posterior limbs relatively long (FL = 40.8% of SVL; TL = 43.3% of SVL); tarsal tubercles absent; feet about 3/4 webbed, formula: I 0+–2 II 1––2 III 1–2 IV 3––2– V; discs on toes elliptical; inner metatarsal tubercle ovoid; outer metatarsal tubercle small, barely evident; subarticular tubercles round, conspicuous; supernumerary tubercles absent. Skin on dorsal surfaces of head and body shagreen with hemispherical pustules having a central spicules; venter areolate. Cloacal opening directed posteriorly at midlevel of thighs, covered by a slightly developed cloacal sheath; cloacal tubercles absent.

Color in Life: This description is based on photographs of the holotype and the other three mentioned specimens (fig. 2; Rodríguez and Knell, 2003: fig. 5C). Dorsolateral surfaces with large and bright glossy spots due to accumulation of iridophores, most of which contain yellow irregular blotches, set in a washed black reticulum; reticulum carries smaller and less bright oval to pentagon-shaped accumulations of iridophores from which brown spicules arise. Ventral surfaces black and opaque with large and bright glossy spots due to accumulation of iridophores except in palms and plantar surfaces. Iris dark bronze with dark reticulations, pupillary ring absent, pupil black. There seems to be sexual dichromatism; the female has fewer spicules and more ventral glossy spots than the male, which is also the case in Allophryne ruthveni.

Color in Preservative: This description is based exclusively on the holotype (fig. 1A–C). Dorsolateral surfaces with white large and bright irregular spots due to accumulation of iridophores, yellow irregular blotches absent, set in a dark brown reticulum that carries smaller and less bright oval to pentagon-shaped accumulations of iridophores from which brown spicules arise. Ventral surfaces dark brown with large and bright glossy spots due to accumulation of iridophores except in palms and plantar surfaces. Iris brown and pupil white.

Osteology (fig. 4): The skull is widest posterior to the orbit and at the level of the articulation of the maxilla with the squamosal. The neopalatines and quadratojugals are absent. The anterior ramus of the pterygoid articulates with the posterior end of the maxilla. The vertebral column has eight presacral vertebrae that have transverse processes except presacral I. The vertebral profile in decreasing order of overall width of bony parts is: sacrum > III > IV > V ≈ VI ≈ VII > II > I. The orientations of the transverse processes of presacral VI are lateral, whereas those of presacrals II, VII, and VIII are anterolateral, and those of presacral III–V are posterolateral. The bony sacral diapophysis is broadly expanded distally, and has a slender base that is approximately a third the width of the distal margin. The leading edge of each diapophysis is slightly concave with an anterolateral orientation, whereas the posterior margin is nearly straight and oriented posterolaterally. The urostyle is slender and shorter than the length of the presacral portion of the vertebral column; the urostyle bears a dorsal crest on its anterior half. The tibiale and fibulare are not fused medially. The phalangeal formulae for the hand and foot are standard, 2-2-3-3 and 2-2-3-4-3, respectively. In increasing order of length, the order of the digits on the hand is I < II < IV < III and that of the foot is: I < II < III < V < IV. The terminal phalanges of hands and feet are T-shaped.

Measurements: The morphometric data for the female holotype are (in mm): SVL = 28.4; head length = 7.8; head width = 9.1; thigh length = 11.6; shank length = 12.3; foot length = 11.5; interorbital distance = 3.6; upper eyelid width = 2.0; internarial distance = 1.7; eye-to-nostril distance = 2.5; snout-eye distance = 3.3; eye diameter = 3.0; tympanum diameter = 1.1; eye-tympanum distance = 1.1; forearm length = 5.7; hand length = 8.7; finger I length = 6.1; finger II length = 6.5; and width of disc of finger III = 1.6.

Distribution: Allophryne resplendens new species is currently known only from two localities in Peru (fig. 5). The type locality is in the Yavarí River drainage. This drainage includes other rivers, such as the Yavarí Mirín and Quebrada Esperanza. The region is dominated by the geological formation that covers much of northeastern Peru: the Pebas formation, a thick slab of clays and sands deposited in ancient lakes and rivers (Räsaänen et al., 1998; Sánchez et al., 1999; de la Cruz et al., 1999). The southern sector, close to Angamos, is associated with an uplifted geological structure known as the Iquitos Arch, which stretches hundreds of kilometers across Loreto and into Colombia. The vegetation of the area includes upland (terra firma) forest concentrated along the Iquitos Arch, flooded (varzea) forest and palm swamps. The second known locality is the western side of the Amazon River, Quebrada Hungurahui (an affluent of Río Tigre), approximately 2 km north of Comunidad Monteverde, Loreto, Peru. Allophryne resplendens, n. sp., is likely present in other varzea forests at least around and between the two known localities. Thus, it is possible that this species also occurs in Brazil.

Natural History: This is an arboreal species that has been found perched on leaves and branches below 2 m. The holotype was found on a Lepidocaryum tenue (Arecaceae). These medium-size palm trees form particular forests known locally as irapayales. The specimen from Quebrada Hungurahui was also found in an irapayal forest. Allophryne resplendens, n. sp., is very difficult to find, which could reflect very low densities, at least in the lower strata of the forest outside the breeding season (the sister species is a explosive breeder). After 49.45 hours of search only one specimen was collected (table 3), and Rodríguez and Knell (2003) found only a single specimen in 200 hours of search. William Lamar (personal commun.) has visited the locality in Quebrada Hungurahui during the last 10 years in different months and has not been able to locate by sight or sound any other specimens. At least 45 species of amphibians have been reported from the type locality and surroundings, of which 23 have been found in swamp palm forest.

Etymology: The specific name resplendens is derived from the Latin verb resplendo meaning “to glitter,” which we used in allusion to the bright and ornate coloration of the frog.

FIG. 1. Photographs of preserved specimens of Allophryne. A–C, Allophryne resplendens, new species, adult female holotype (SVL = 28.4 mm); D–E, Allophryne ruthveni, adult female, Surinam, AMNH 87687 (SVL = 27.0 mm). Photographs by JMP (A–C) and SCF (D–E).

FIG. 2. Photographs of live specimens of Allophryne. A, Allophryne resplendens, n. sp., adult female holotype (SVL = 28.4 mm); B, A. resplendens, n. sp., specimen not collected, sex and size unknown, topotype; C–D, A. resplendens, new species, adult male, Quebrada Ungurahue (SVL = 25.0 mm); E–F, A. ruthveni, adult male, Venezuela, MHNLS 20231 (SVL = 23.5 mm). Photographs by Mark Bowler (A), WCS–Perú (B), William Lamar (C–D), and SCF (E–F).

FIG. 3. Strict consensus of the 10 most parsimonious trees inferred from 12S and 16S sequences (tree cost = 11,381 steps). Bootstrap support values are indicated above branches when ≥ 70.

FIG. 4. X-ray of the holotype of Allophryne resplendens, n. sp. Arrow points to the anterolaterally oriented transverse processes of presacral II.

FIG. 5. Known distribution of Allophryne resplendens, n. sp. Star represents type locality.

TABLE 1. Uncorrected genetic distances between specimens of Allophryne inferred from 923 bp of the mitochondrial genes 12S (above the diagonal) and 761 bp of the 16S (below the diagonal). Dryad Repository: doi:10.5061/dryad.2kp7q7vb.

1

2

3

4

5

6

7

8

1 A. resplendens

*

2.0%

2.1%

1.7%

2.1%

–

–

2.1%

2 A. ruthveni 3301

2.6%

*

0.1%

0.3%

0.1%

–

–

0.1%

3 A. ruthveni 3302

2.6%

0.0%

*

0.2%

0.0%

–

–

0.0%

4 A. ruthveni 3303

3.0%

0.7%

0.7%

*

0.2%

–

–

0.2%

5 A. ruthveni AY843564

2.9%

0.5%

0.5%

0.9%

*

–

–

0.0%

6 A. ruthveni EU662973

2.9%

0.4%

0.4%

1.1%

0.4%

*

–

–

7 A. ruthveni AF364512

3.4%

0.9%

0.9%

1.4%

1.5%

1.4%

*

–

8 A. ruthveni AY819328

–

–

–

–

–

–

–

*

TABLE 2. Autapomorphies for Allophryne resplendens new species with respect to A. ruthveni inferred from a fragment of 761 bp and 923 bp of the mitochondrial genes 16S and 12S respectively. Dryad Repository: doi:10.5061/dryad.2kp7q7vb.

Marker

Position

A. ruthveni

A. resplendens

16S

91

G

A

92

T

C

101

G

A

153

T

C

174

C

T

240

T

C

280

A

–

282

A

T

333

–/T

C

342

T

G

464

A

G

542

C

T

544

T

C

558

A

G

584

T

C

604

A

C

624

T

C

628

T

C

638

A

T

12S

59

T

C

91

A

G

299

G

A

341

T

C

370

T

C

375

T

C

390

T

C

459

T/-

C

583

A

G

625

A

T

724

G

A

748

C

T

851

T

C

852

T

C

861

T

C

922

A

G

TABLE 3. Search effort distributed by forest type and season in the Yavarí river.

Flooding season (November)

Dry season (June–July)

Palm swamps

Varzea

Terra firma

Palm swamps

Varzea

Terra firma

Search hours

2.5

7.42

12.28

8.75

10.50

8.00

Number of people

2

2

1

2

2

2

Specimens found

1

0

0

0

0

0

DISCUSSION

Taxonomy

Monotypic supraspecific taxa can result problematic. For example, different Linnean ranks convey the same information (Allophrynidae = Allophryne = Allophryne ruthveni) and phylogenetic definitions are repetitive (see Guayasamin et al.’s [2009] attempt to define Allophrynidae and Allophryne). Our discovery of a new species of Allophryne partly solves this problem. Allophryne is no longer equivalent to the species Allophryne ruthveni and we provide a new phylogenetic definition of the family and genus: a clade stemming from the most recent common ancestor of Allophryne ruthveni Gaige, 1926, and Allophryne resplendens Castroviejo-Fisher et al., 2011.

Phylogenetic Relationships

Our MP tree shows the sequences of A. resplendens, n. sp., as sister to those of A. ruthveni. However, the MP analysis does not support the relationship (Leptodactylidae, (Allophrynidae, Centrolenidae)), which has been suggested in other phylogenetic studies (e.g., Frost et al., 2006; Guayasamin et al., 2008; Heinicke et al., 2009; Pyron and Wiens, 2011) although with low support. We explain this lack of resolution by the fact that our dataset was primarily designed to evaluate whether A. resplendens, n. sp., was more closely related to Allophrynidae than to representatives of all other families of Neotropical Hyloidea. Our results are therefore not comparable to studies using much larger taxon and character sampling (e.g., Frost et al., 2006; Pyron and Wiens, 2011).

The unique combination of phenotypic characters exhibited by Allophryne ruthveni has long puzzled herpetologists, resulting in different and conflicting phylogenetic hypotheses placing Allophrynidae as the sister taxon of the families Bufonidae (Gaige, 1926; Laurent, 1980, 1986), Hylidae (Lynch and Freeman, 1966; Hoogmoed, 1969; Duellman, 1975; Duellman and Trueb, 1986), or Centrolenidae (Noble, 1931; Lutz, 1968; Duellman, 2001; Burton, 2004; Wiens et al., 2005). Although no unique autapomorphy has been described for A. ruthveni, the following combination of characters has been proposed as diagnostic: intercalary elements absent, biaxial articulation between last two phalanges, T-shaped terminal phalanges, tibiale and fibulare not fused, absence of neopalatines and quadratojugals, prepollex and prehallux formed by one proximal element each (Fabrezi and Langone, 2000, 2001; Fabrezi, 2006). Unfortunately, we have been able to secure only one specimen of A. resplendens, n. sp., so we could investigate only three of the characters mentioned above (i.e., T-shaped terminal phalanges, tibiale and fibulare not fused, absence of neopalatines and quadratojugals). The next logical step in the study of Allophryne would be to gather more information on these and other phenotypic characters and study them in combination with other relevant information and taxa in a cladistic framework.

ACKNOWLEDGMENTS

We thank Mark Bowler, Miguel Antúnez, Maria Soledad Riveros, Bray Torres, Franco Santana, Claudia Rios, Ciro Pinedo, Richard Bodmer, Pablo Puertas, and the volunteers of Earthwatch Institute for supporting the study in Lago Preto; Juan Carlos Chaparro for his support and help in the description of this species; and William Lamar for photographs and data on the new locality. The Wildlife Conservation Society–Perú (WCS–Perú) and AmazonEco provided financial support for fieldwork. Collection permits in Peru (authorization N° 320-2009) were issued by AG-DGFFS-DGEFFS. This study is included in the “Contrato Marco de Acceso a Recursos Genéticos N° 0001, 11 Enero 2007” subscribed between Fundacion La Salle de Ciencias Naturales and the Ministerio del Ambiente, Venezuela. The work of SCF was financed by a Fulbright/Ministry of Education postdoctoral research contract. The Universidad Tecnológica Indoamérica supports J.M.G.’s research. J.M.P.’s research is funded by a Gerstner Postdoctoral Fellowship at the American Museum of Natural History.

REFERENCES

AmphibiaWeb. 2011. Information on amphibian biology and conservation. Berkeley, CA. Internet resource (http://amphibiaweb.org/), accessed June 14, 2011.

Austin, J.D., et al. 2002. A molecular perspective on the evolutionary affinities of an enigmatic neotropical frog, Allophryne ruthveni. Zoological Journal of the Linnean Society 134: 335–346.

Burton, T.C. 2004. Muscles of the pes of hylid frogs. Journal of Morphology 260: 209–233.

de la Cruz, B., et al. 1999. Geología de los cuadrángulos de: Lagunas, Río Cauchío, Santa Cruz, Río Sacarita, Río Samiria, Bretaña, Requena, Remoyacu, Angamos, Santa Isabel, Tamanco, Nueva Esperanza, Buenas Lomas, Laguna Portugal, Puerto Rico, Tabalosos, Curinga, Quebrada Capanahua, Quebrada Betilia y Río Yaquerana. Boletín del Instituto Geológico, Minero y Metalúrgico (INGEMMET), Carta Geológica Nacional, Sector Energía y Minas 132, Serie A: 1–372.

Duellman, W.E. 1975. On the classification of frogs. Occasional Papers of the Museum of Natural History, University of Kansas 42: 1–14.

Duellman, W.E. 2001. Hylid frogs of Middle America. Ithaca, NY: Society for the Study of Amphibians and Reptiles.

Duellman, W.E., and L. Trueb. 1986. Biology of amphibians. New York: McGraw-Hill.

Duméril, A.M.C., and G. Bibron. 1841. Erpétologie générale ou histoire naturelle complète des reptiles. Vol. 8. Paris: Librairie Encyclopédique de Roret.

Fabrezi, M. 1992. El carpo de los anuros. Alytes 10: 1–29.

Fabrezi, M. 1993. The anuran tarsus. Alytes 11: 47–63.

Fabrezi, M. 2001. A survey of prepollex and prehallux variation in anuran limbs. Zoological Journal of the Linnean Society 131: 227–248.

Fabrezi, M. 2006. Morphological evolution of Ceratophryinae (Anura, Neobatrachia). Journal of Zoological Systematics and Evolutionary Research 44: 153–166.

Fabrezi, M., and J.A. Langone. 2000. Los caracteres morfológicos del controvertido Neobatrachia arborícola Allophryne ruthveni Gaige, 1926. Cuadernos de Herpetología 14: 47–59.

Faivovich, J., et al. 2005. Systematic review of the frog family Hylidae, with special reference to Hylinae: phylogenetic analysis and taxonomic revision. Bulletin of the American Museum of Natural History 294: 1–240.

Fouquet, A., et al. 2007. Underestimation of species richness in Neotropical frogs revealed by mtDNA analyses. PLoS ONE 2 (10): e1109. [doi: 10.1371/journal.pone.0001109]

Frost, D.R. 2011. Amphibian species of the world: an online reference. Version 5.5 (31 January, 2011). New York: American Museum of Natural History, electronic database (accessible at http://research.amnh.org/vz/herpetology/amphibia/)

Frost, D.R., et al. 2006. The amphibian tree of life. Bulletin of the American Museum of Natural History 297: 1–370.

Gaige, H.T. 1926. A new frog from British Guiana. Occasional Papers of the Museum of Zoology, University of Michigan 176: 1–3.

Gene Codes Corporation. 2006. Sequencher Version 4.1. Ann Arbor, Michigan.

Goloboff, P.A. 1999. Analyzing large data sets in reasonable times: solutions for composite optima. Cladistics 15: 415–428.

Goloboff, P.A., et al. 2008. TNT, a free program for phylogenetic analysis. Cladistics 24: 774–786.

Gower, D.J., M. Wilkinson, E. Sherratt, and P.J.R. Kok. 2010. A new species of Rhinatrema Duméril & Bibron (Amphibia: Gymnophiona: Rhinatrematidae) from Guyana. Zootaxa 2391: 47–60.

Grant, T., et al. 2006. Phylogenetic systematics of dart-poison frogs and their relatives (Amphibia: Athesphatanura: Dendrobatidae). Bulletin of the American Museum of Natural History 299: 1–262.

Guayasamin, J.M., M.R. Bustamante, D. Almeida-Reinoso, and C.W. Funk. 2006. Glass frogs (Centrolenidae) of Yanayacu Biological Station, Ecuador, with the description of a new species and comments on centrolenid systematics. Zoological Journal of the Linnean Society of London 147: 489–513.

Guayasamin, J.M., S. Castroviejo-Fisher, J. Ayarzagüena, L. Trueb, and C. Vilà. 2008. Phylogenetic relationships of glassfrogs (Centrolenidae) based on mitochondrial and nuclear genes. Molecular Phylogenetics and Evolution 48: 574–595.

Guayasamin, J.M., et al. 2009. Phylogenetic systematics of Glassfrogs (Amphibia: Centrolenidae) and their sister taxon Allophryne ruthveni. Zootaxa 2100: 1–97.

Guérin-Méneville, F.-É. 1838. Iconographie du règne animal de G. Cuvier où représentation d’après nature de l’une des espèces les plus remarquables et souvent non encore figurées, de chaque genre d’animaux, avec un texte descriptif mis au courant de la science. Vol. 3 (Part Reptiles). Paris: J.B. Ballière.

Heinicke, M.P., W.E. Duellman, and S.B. Hedges. 2007. Major Caribbean and Central American frog faunas originated by oceanic dispersal. Proceedings of the National Academy of Sciences of the United States of America 104: 10092–10097.

Heinicke, M.P., et al. 2009. A new frog family (Anura: Terrarana) from South America and an expanded direct-developing clade revealed by molecular phylogeny. Zootaxa 2211: 1–35.

Heyer, W.R., M.A. Donnelly, R.W. McDiarmid, L.-A.C. Hayek, and M.S. Foster (editors). 1994. Measuring and monitoring biological diversity standard methods for amphibians. Biological Diversity Handbook Series. Washington, DC: Smithsonian Institution Press.

Hoogmoed, M.S. 1969. Notes on the herpetofauna of Surinam II. On the occurrence of Allophryne ruthveni Gaige (Amphibia, Salientia, Hylidae) in Surinam. Zoologische Mededelingen 44: 76–81.

Katoh, K., K. Kuma, H. Toh, and T. Miyata. 2005. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059–3066.

Köhler, J., et al. 2005. New amphibians and global conservation: a boost in species discoveries in a highly endangered vertebrate group. Bioscience 55: 693–696.

Laurent, R.F. 1980 “1979.” Esquisse d’une phylogenèse des anoures. Bulletin de la Société Zoologique de France 104: 397–422.

Laurent, R.F. 1986. Souss classe des lissamphibiens: Lissamphibia systématique, In P.-P. Grassé and M. Delsol (editors), Traité de zoologie: anatomie, systématique, biologie: 14, fasc. 1B (batraciens): 594–798. Paris: Masson.

Lutz, B. 1968. Taxonomy of the Neotropical Hylidae. Texas Memorial Museum, Pearce-Sellards Series 11: 3–26.

Lynch, J.D., and H.L. Freeman. 1966. Systematic status of a South American frog, Allophryne ruthveni Gaige. University of Kansas Publications, Museum of Natural History 17: 493–502.

Meegaskumbura, M., et al. 2002. Sri Lanka: an amphibian hot spot. Science 298: 379.

Nixon, K.C. 1999. The parsimony ratchet, a new method for rapid parsimony analysis. Cladistics 15: 407–414.

Noble, G.K. 1931. The biology of the Amphibia. New York: McGraw-Hill.

Pyron, R.A., and J.J. Wiens. 2011. A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution 61 (2): 543–583. [doi:10.1016/j.ympev.2011.06.012]

Räsaänen, M., A. et al. 1998. Geología y geoformas de la zona de Iquitos. In S. Kalliola and R. Flores-Paitán (editors), Geoecología y desarrollo amazónico: estudio integrado en la zona de Iquitos, Perú. Annales Universitatis Turkuensis (Series A2) 144: 59–137.

Rodríguez, L.O., and G. Knell. 2003. Amphibians and Reptiles. In N. Pitman, C. Vriesendorp, and D. Moskovits (editors), Perú: Yavarí. Rapid Biological Inventories Report 11: 147–150, 244. Chicago: Field Museum.

Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press.

Sánchez F., et al. 1999. Geología de los cuadrángulos de Puerto Arturo, Flor de Agosto, San Antonio del Estrecho, Nuevo Perú, San Felipe, Río Algodón, Quebrada Airambo, Mazán, Francisco de Orellana, Huanta, Iquitos, Río Manití, Yanashi, Tamshiyacu, Río Tamshiyacu, Buen Jardín, Ramón Castilla, Río Yavarí Mirin y Buena Vista. Boletín Instituto Geológico, Minero y Metalúrgico (INGEMMET), Carta Geológica Nacional, Sector Energía y Minas 132, Serie A: 1–372.

Savage, J.M., and W.R. Heyer. 1967. Variation and distribution in the tree-frog genus Phyllomedusa. Beiträge zur Neotropischen Fauna 2: 111–131.

Stuart, B.L., R.F. Inger, and H.K. Voris. 2006. High level of cryptic species diversity revealed by sympatric lineages of Southeast Asian forest frogs. Biology Letters 2: 470–474.

Swofford, D.L. 1998. PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4.0b1. Sunderland, MA: Sinauer Associates.

Trueb, L. 1973. Bones, frogs, and evolution. In J.L. Vial (editor), Evolutionary biology of the anurans: contemporary research on major problems: 65–132. Columbia, MO: University of Missouri Press.

Trueb, L. 1993. Patterns of cranial diversity among the Lissamphibia. In J. Hanken and B.K. Hall (editors), The skull, vol. 2: Patterns of structural and systematic diversity: 255–343. Chicago: University of Chicago Press.

Vieites, D.R., et al. 2009. Vast underestimation of Madagascar’s biodiversity evidenced by an integrative amphibian inventory. Proceedings of the National Academy of Sciences of the United States of America 106: 8267–8272.

Wiens, J.J., J.W. Fetzner, C.L. Parkinson, and T.W. Reeder. 2005. Hylid frog phylogeny and sampling strategies for speciose clades. Systematic Biology 54: 719–748.

Wilkinson, M., and D.J. Gower. 2010. A new species of Rhinatrema Duméril & Bibron (Amphibia: Gymnophiona: Rhinatrematidae) from Amazonas, Brazil. Zootaxa 2650: 63–68.

APPENDIX 1

Additional Specimens Studied

Allophryne ruthveni: VENEZUELA: Delta Amacuro: Reserva Forestal Rio Grande, Coderforce (08°16′00.8″N, 61°45′16.3″W; 192 m), MHNLS 20231–6 (adult males). Amazonas: Base camp of Tapirapeco expedition, upper rio Mavaca (150 m), AMNH 131351 (adult female, cleared and stained). SURINAM: Saramacca: Raleigh Cataracts, Coppename river (50 m), AMNH 87687 (adult female), 876888 (adult male). GUYANA: Iwokrama: Pakatau creek, AMNH 163990 (adult female). Marudi creek, AMNH 44749 (male). Unknown Locality: AMNH 70108 (adult female), AMNH 70110 (unknown sex, cleared and stained).

APPENDIX 2: Species, with Their Corresponding Sequences and Genbank Codes, Used in the Study.

Sequences newly generated for this study are in boldface.

Family

Genus

Species

12S

16S

Allophrynidae

Allophryne

resplendens

JQ436697

JQ436698

Allophryne

ruthveni

AY819328

—

Allophryne

ruthveni

JQ436702

JQ436700

Allophryne

ruthveni

JQ436703

JQ436699

Allophryne

ruthveni

JQ436704

JQ436701

Allophryne

ruthveni

AY843564

AY843564

Allophryne

ruthveni

—

AF364512

Allophryne

ruthveni

—

EU662973

Aromobatidae

Allobates

talamancae

170026605

170026605

Rheobates

palmatus

170026602

170026602

Bufonidae

Atelopus

peruensis

77176491

77176491

Dendrophryniscus

minutus

61697193

61697193

Rhaebo

nasicus

77176549

77176549

Rhinella

arenarum

61697184

61697184

Centrolenidae

Celsiella

revocata

EU663379

EU663019

Centrolene

savagei

EU663380

187729151

Chimerella

mariaelenae

EU663350

EU662991

Cochranella

nola

EU663375

EU663015

Espadarana

andina

EU663335

EU662976

Hyalinobatrachium

taylori

EU663420

EU663056

Ikakogi

tayrona

EU663358

EU662999

Nymphargus

bejaranoi

EU663422

EU663059

Rulyrana

spiculata

EU663382

EU663022

Sachatamia

albomaculata

EU663362

EU663003

Teratohyla

pulverata

EU663416

EU663053

APPENDIX 2 (continued)

Family

Genus

Species

12S

16S

Vitreorana

gorzulae

EU663348

EU662989

Ceratophryidae

Atelognathus

patagonicus

61697182

61697182

Batrachyla

leptopus

61697183

61697183

Ceratophrys

cranwelli

61697186

61697186

Lepidobatrachus

sp

37699560

37699560

Telmatobius

sp

61697380

61697380

Cycloramphidae

Alsodes

gargola

61697176

61697176

Eupsophus

calcaratus

61697198

61697198

Dendrobatidae

Ameerega

trivittata

170026722

170026722

Dendrobates

auratus

61697192

61697192

Epipedobates

tricolor

37624303

37624303

Silverstoneia

flotator

170026684

170026684

Hemiphractidae

Gastrotheca

fissipes

61697203

61697203

Stefania

schuberti

61697379

61697379

Hylidae

Anotheca

spinosa

61697177

61697177

Aparasphenodon

brunoi

61697178

61697178

Argentohyla

siemersi

61697181

61697181

Bokermannohyla

martinsi

61697252

61697252

Bromeliohyla

bromeliacia

61697223

61697223

Charadrahyla

taeniopus

61697290

61697290

Duellmanohyla

soralia

61697195

61697195

Ecnomiohyla

miotympanum

61697256

61697256

Hyla

arborea

61697212

61697212

Hyloscirtus

charazani

61697229

61697229

Hypsiboas

geographicus

61697239

61697239

Isthmohyla

rivularis

61697270

61697270

Osteocephalus

taurinus

61697320

61697320

Leptodactylidae

Leptodactylus

ocellatus

61697299

61697299

Lithodytes

lineatus

37699553

37699553

Paratelmatobius

poecilogaster

159139110

159139110

Scythrophrys

sawayae

159139126

159139126

Mantellidae

Mantella

madagascariensis

89255435

89255435

Strabomantidae

Lynchius

flavomaculatus

166155972

166155972

Oreobates

cruralis

166155971

166155971

Pristimantis

diadematus

166155973

166155973

Pristimantis

galdi

166155975

166155975

Yunganastes

pluvicanorus

61697197

61697197

[1]Division of Vertebrate Zoology (Department of Herpetology), American Museum of Natural History.

[2]Wildlife Conservation Society, Programa Loreto, Perú.

[3]Division of Vertebrate Zoology (Department of Herpetology), American Museum of Natural History.

[4]Centro de Investigación de la Biodiversidad y Cambio Climático, Universidad Tecnológica Indoamérica, Av. Machala y Sabanilla, Quito, Ecuador.