Zootaxa 3095: 1–14 (2011) www.mapress.com / zootaxa/ Copyright © 2011 · Magnolia Press

ISSN 1175-5326 (print edition)

Article

ZOOTAXA ISSN 1175-5334 (online edition)

A new species of marsupial frog (Anura: Hemiphractidae: Gastrotheca) from the Andes of southern Peru WILLIAM E. DUELLMAN1,3, ALESSANDRO CATENAZZI2 & DAVID C. BLACKBURN1 1 Biodiversity Institute, University of Kansas, Lawrence, KS 66045-7561 USA. E-mail: [email protected]; [email protected] 2 Department of Biology, Gonzaga University, 502 E. Boone Ave., Spokane, WA 99201 USA. E-mail: [email protected] 3 Corresponding author

Abstract Gastrotheca nebulanastes sp. nov. from cloud forests in the upper Kosñipata Valley, Manu National Park, in the Andes of southern Peru is similar to G. excubitor, which inhabits grasslands in higher elevations than the cloud forests. The two species differ in relative lengths of the fingers, skin texture, coloration, and advertisement call. Although the new species has an elevational range of 2000–3300 m, it is most abundant at 2400–2800 m. A phylogenetic analysis of a previously defined clade of Gastrotheca based on a fragment of 16S mitochondrial gene provides strong support that the sister taxon to the new species is G. atympana, a species from farther north in the Cordillera Oriental in Peru. Key words: Anura, distribution, ecology, Gastrotheca, Hemiphractidae, new species, phylogenetic relationships

Resumen Gastrotheca nebulanastes sp. nov. de los bosques nublados de la parte alta del valle de Kosñipata, Parque Nacional del Manu en los Andes del sur de Perú se asemeja a G. excubitor, una especie que vive en la puna a elevaciones por encima del límite superior del bosque nublado. Las dos especies se diferencian por la longitud relativa de sus dígitos, textura de la piel, coloración y canto nupcial. A pesar de tener un rango de distribución altitudinal entre los 2000 y 3300 m, la nueva especie es más abundante a elevaciones entre 2400–2800 m. El análisis filogenético de un fragmento del gen mitocondrial 16S de un clado de Gastrotheca previamente definido apoya la hipótesis de que el taxon hermano de la nueva especie es G. atympana, una especie distribuida más al norte en la Cordillera Oriental en Perú. Palabras claves: Anura, distribución, ecología, Gastrotheca, Hemiphractidae, nueva especie, relaciones filogenéticas

Introduction In 1971 the senior author and Thomas H. Fritts discovered a distinctive, terrestrial species of Gastrotheca at the crest of Abra Acanacu (now Abra Acjanaco), a pass in the Cadena del Paucartambo, the northwestern part of the Cordillera de Carabaya, which is a part of the extensive Cordillera Oriental of the Andes of Peru and now protected as part of Manu National Park. They found the new species, G. excubitor (Duellman & Fritts, 1972) in wet puna also inhabited by the widespread G. marsupiata (Duméril & Bibron, 1841). Subsequently, both species were collected again at Abra Acanacu in 1975 and 1977, but during the latter trip specimens referred to G. excubitor were also found in humid montane forests at lower elevations northwest of Abra Acjanaco, as well as at other abras at elevations above 3500 m in Departamento (= Región) Cusco in southern Peru. More recent fieldwork (2007 and 2009) by one of us (AC) resulted in the collection of more individuals at localities in the humid montane forest, as well as the discovery that Hyla antoniiochoai De la Riva and Chaparro (2005) is an arboreal species of Gastrotheca in the upper Kosñipata (formerly Cosñipata) Valley (Catenazzi & Lehr 2009).

Accepted by S. Castroviejo: 12 Oct. 2011; published: 10 Nov. 2011

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Material and methods The 16 morphological measurements (Table 1), 25 external descriptive characters, and numbered diagnostic characters are those used by Duellman and Pyles (1980), Duellman and Hillis (1987), Duellman and Trueb (1988), and Duellman et al. (2001, 2004, 2006). All measurements are in millimeters; snout–vent length is abbreviated SVL. If sex could not be determined by the presence of a brood pouch or vocal sac, determination was made by examination of gonads. Fourteen morphological measurements were retained for Principal Component Analysis (Table 2) to maximize sample size of n =18 for G. nebulanastes and n = 23 for G. excubitor. Morphological data (non-transformed, after checking for normality) were analyzed with the princomp function using eigen on the correlation matrix in the stats package in R 2.10.1 (The R Foundation for Statistical Computing; http://www.R-project. org). Principal components 1 and 2 (representing 60.2% of variation) were used to produce a scatter plot. Photographs of frogs taken by Catenazzi and Duellman were used in the description of color in life; these photos are available at the Calphoto online database (http://calphotos.berkeley.edu). Specimens of other species examined and/or used in the phylogenetic analysis are listed in Appendix I. We recorded advertisement calls of male MUSM 28060 (SVL 42.0 mm) at the type locality between 1900 and 2030 on 4 April 2011. The air temperature during that time was 10.7–12.3ºC. We used a digital recorder (Song Meter SM1, Wildlife Acoustics, Inc., Concord, Massachusetts) and recorded advertisement calls in uncompressed .wav format. We recorded the advertisement call of several males (exact number unknown; males were not captured) of G. excubitor at Abra Acjanaco on 23–31 January 2009 (recorded between 2030 and 2230 and at temperatures of 7.5–9.0°C). We used Cool Edit version 96 (Syntrillium Software Corporation) and Raven Lite, version 1.0 (Cornell Laboratory of Ornithology) to resample recordings at a sampling frequency of 44 KHz, FFT width 512 points, and 16-bit resolution and analyze call variables. The Hamming window function for the spectrogram was set at 256 bands. Averages are reported ± SD. We analyzed a total of 20 calls for G. nebulanastes and 30 calls for G. excubitor. The following institutional abbreviations are used: AMNH = American Museum of Natural History, New York; FML = Fundación Miguel Lillo, Tucumán, Argentina; KU = Museum of Natural History, University of Kansas, Lawrence; LM = Linda Maxson tissue collection, Pennsylvania State University; MNCN = Museo Nacional de Ciencias Naturales, Madrid, Spain, MTD = Museum für Tierkunde Dresden; MHNC = Museo de Historia Natural, Universidad Nacional San Antonio Abad, Cusco, Peru; MUSM = Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru; MVZ = Museum of Vertebrate Zoology, University of California, Berkeley; TNHC = Texas Natural History Collection, University of Texas, Austin; and USNM = National Museum of Natural History, Washington. Spelling of Acjanaco and Kosñipata follows terms currently used in Peru and in recent publications; however, spellings in Carta Nacional “Calca,” Hoja 27-s (Instituto Geográfico Nacional, Lima) are Acanacu and Cosñipata. We determined the phylogenetic relationships of the new species through analysis of DNA sequence data for the mitochondrial 16S ribosomal RNA gene. We obtained data from three specimens of the new species of Gastrotheca, as well as topotypic specimens of G. excubitor (MUSM 26280–81), G. antoniiochoai (MUSM 27944), and the recently described G. pachachacae (MUSM 28492). We amplified the 16S gene using polymerase chain reaction (PCR) and the primers (16Sc and 16Sd) of Darst and Cannatella (2004), resulting in 810–880 bp of sequence per specimen. Genomic DNA extraction, as well as purification of PCR products and sequencing follows the methods of Esselstyn et al. (2008). Newly collected sequence data are accessioned in GenBank. Details on the taxa and the corresponding GenBank sequences used are provided in Appendix II. We generated a multiple alignment of 16S data using MAFFT v6.850 (Katoh et al., 2005) with minor adjustments made by eye; the alignment used for analysis is deposited in Dryad (doi:10.5061/dryad.bt760). We analyzed the 16S alignment as a single partition. Using the Akaike information criterion (AIC) and jModeltest v.0.1.1 (Posada, 2008), we selected the GTR + I +G model (lnL = –2648.47, AIC = 5388.93) as the best-fit model of sequence evolution (vs. next best, GTR + G: lnL = –2656.18, AIC = 5396.36). We estimated phylogenetic relationships using maximum-likelihood (ML) as implemented in RAxML v.7.0.4 (Stamatakis 2006). This analysis used a random starting tree, the faster rapid hill-climbing algorithm proposed by Stamatakis et al. (2007), and the GTR + I +G model of sequence evolution. The ML analysis used 100 search repetitions and we used the phylogenetic estimate with the smallest –ln likelihood score as the preferred ML phylogeny. We performed one thousand nonparametric bootstrap replicates in RAxML using the same model of sequence

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evolution with one search replicate per bootstrap replicate, a random starting tree, and optimizing branch lengths and model parameters during the bootstrap analysis. We calculated split support using SumTrees in DendroPy (Sukumaran & Holder 2010). Based on the analysis of Wiens et al. (2007), we rooted the resulting phylogeny using Gastrotheca zeugocystis.

Description of new species Gastrotheca nebulanastes new species Holotype: KU 173210, an adult female (Fig. 2A), from Buenos Aires (Fig. 5A), on Paucartambo–Pilcopata road, 2280 m, 13˚09'16" S, 71˚35'11"W, Distrito de Kosñipata, Provincia de Paucartambo, Región de Cusco, Peru, one of a series obtained on 19 January 1977, by W. E. Duellman and D. C. Cannatella. Paratypes: All on Paucartambo–Pilcopata road, Distrito de Kosñipata, Provincia de Paucartambo, Región de Cusco, Peru: KU 173209 (Fig. 2B), 173211–12, 173214–17, males; 173208, 173213, 173218 (now skeleton), females, collected with the holotype. AMNH 153080, male, from vicinity of Pillahuata, 2600 m, 13˚07'59.9" S, 71˚25'36.1" W, 28 August 1996, A. Catenazzi; AMNH 157005–06, males, from vicinity of Pillahuata, on Paucartambo—Pilcopata road, 2430 m, 13˚09'52" S, 71˚35'45" W, 15 January 1998, L. O. Rodríguez and A. Catenazzi; MUSM 20940, female, from Esperanza (Fig. 5B), 2780 m, 13˚10'44.07" S, 71˚36'33.10" W, 30 August 1996, A, Catenazzi; USNM 298184–90, males, 1.6 km (by road) E Pillahuata, 2350–2550 m, 13˚09'27.7" S, 71˚35'29.8" W, 16 May 1984, John E. Cadle; MVZ 265218, male, between Buenos Aires and Estrella, 2250 m, 13°8'39.60"S, 71°35'9.44"W, 13 February 2008, A. Catenazzi; MUSM 27888, juvenile, near Esperanza, 2750 m, 13°10'44.07"S, 71°36'33.10"W, 21 February 2008, A. Catenazzi; MUSM 27943, male, between Estrella and Rocotal, 2180 m, 13° 7'51.16"S, 71°34'37.65"W, 2 February 2009, A. Catenazzi. Referred specimens: All from Paucartambo–Pilcopata road, Región de Cusco, Peru: AMNH 153079, juvenile, from vicinity of Pillahuata, on Paucartambo—Pilcopata road, 2600 m, 13˚07'59.9" S, 71˚25'36.1" W, 28 August 1996, L. O. Rodríguez and A. Catenazzi; MUSM 20941–44, 20950–51; between Pillahuata, 2580 m and Esperanza, 2780 m, 13˚09'52" S, 71˚35'45" W, 30 August 1996, A. Catenazzi. Diagnosis. Placed in genus Gastrotheca by the unique feature of closed brood pouch on dorsum of female. A moderately small species (to 40.7 mm SVL) with (1) tibia length 49–56% SVL, slightly longer than foot; (2) interorbital distance about 1.5 times width of upper eyelid; (3) skin on dorsum moderately tubercular, not-co-ossified with skull, lacking transverse ridges; (4) supraciliary processes absent; (5) heel lacking calcar or tubercle; (6) tympanic annulus smooth; (7) Finger I longer than II with discs notably wider than digits; (8) fingers unwebbed; (9) webbing basal between Toes III, IV, and V; other toes unwebbed; (10) dorsum brown with darker brown markings; (11) head markings consisting dark brown interorbital bar; (12) pale dorsolateral stripe absent; (13) flanks tan with black spots; (14) venter dull creamy tan; (15) brood pouch single, dorsal. Gastrotheca nebulanastes is sympatric with G. antoniiochoai and parapatric with G. excubitor and G. marsupiata, which occur at higher elevations (above 3000 m, roughly corresponding to the lower limit of grasslands shown in Figure 4C). Univariate comparisons of measurements of male G. nebulanastes and G. excubitor reveal that male G. nebulanastes have longer tibia and Finger I (i.e., thumb), as well as some subtle differences in head proportions (Table 1), all variables load strongly and positively on the first Principal Component axis, PC1; the strongest loadings on PC1 are snout–vent length, tibia length, foot length, interorbital distance, and lengths of the first and third fingers (Table 2). The strongest loadings on PC2 indicate an inverse relationship between internarial distance and both eyelid width and the distance between the eye and nostril (Table 2). Thus, as in the univariate comparisons, the principal components analysis indicates subtle differences in head shape between G. nebulanastes and G. excubitor. Moreover, scatterplots of the first two principal components axes reveal that these two species occupy distinct regions of morphospace (Fig. 1). Other comparisons indicate that these two species differ in other proportions. The tibia length is less than 50% of SVL in G. excubitor and more than 50% of SVL in G. nebulanastes; the length of the tibia is less than the length of the foot in G. excubitor but greater than the length of the foot in G. nebulanastes. In G. excubitor the snout is bluntly rounded in profile, whereas it is truncate in G. nebulanastes, which has a coarsely granular dorsum compared to the smooth dorsum in G. excubitor. Other structural differences include relative

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lengths of Fingers I and II (Finger I equal to, or shorter than Finger II in G. excubitor and longer than Finger II in G. nebulanastes), inner tarsal fold weak on distal half of tarsus in G. excubitor and absent in G, nebulanastes), and toes about one-third webbed in G. excubitor, in contrast to only basal webbing in G. nebulanastes. The two species also differ in coloration (Fig. 2A–D); a dark canthal stripe and pale labial stripe are present in G. excubitor but absent in G. nebulanastes, which as dark brown vertical bars on the anterior surfaces of the thighs in contrast these surfaces being uniform tan or tan with brown mottling in G. excubitor. Finally, the number of pulses and the amplitude modulation of long notes differ between the two species (Table 3; Fig. 3). In G. nebulanastes, long notes have a higher number of pulses (F1,42 = 11.9, p = 0.001) and pulse amplitude is variable among calls than in G. excubitor. The dominant frequency of the secondary (short) note is lower than the fundamental frequency of the long note in G. nebulanastes but higher than the fundamental frequency of the long note in G. excubitor. TABLE 1. Univariate statistics for adults of Gastrotheca excubitor and Gastrotheca nebulanastes. Females of G. nebulanastes were not used for between-sexes and between-species statistics.

Character Snout-vent length Tibia length Foot length Head length Head width Thumb length Third finger length Interorbital distance Internarial distance Eye-nostril Eye diameter Tympanum diameter Eyelid width Orbit-jaw Nostril-jaw Disc width

G. excubitor (23 ♂, 15 ♀) Sex Mean Range

SD

G. nebulanastes (18 ♂, 5 ♀) Mean Range

SD

♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀

3.47 2.78* 1.11** 1.33 1.17 1.43 0.70** 1.02 0.70 1.15 0.46** 0.51 0.74 0.87* 0.33** 0.64 0.15 0.16* 0.41** 0.45 0.26** 0.49* 0.25** 0.26 0.28 0.42 0.33** 0.37 0.35 0.50 0.20** 0.34

35.4 37.7 18.3 20.3 16.6 18.1 12.0 13.5 12.9 14.0 6.6 6.9 11.5 12.6 4.7 4.7 2.4 1.8 3.1 3.3 3.4 3.6 1.8 1.8 3.1 3.2 1.4 1.2 2.7 2.6 1.7 2.1

1.74 1.81 0.68 0.73 1.02 0.51 0.37 1.68 0.48 0.95 0.49 0.26 0.47 0.48 0.47 0.26 0.48 0.29 0.13 0.19 0.24 0.14 0.25 0.22 0.08 0.08 0.20 0.09 0.30 0.14 0.17 0.15

33.7 35.8 16.9 17.5 16.5 17.2 11.6 11.9 12.8 13.3 6.2 6.3 11.1 11.7 4.4 4.3 2.2 2.4 3.4 3.5 3.3 3.5 1.6 1.6 3.2 3.1 1.8 1.7 2.7 2.7 1.6 1.7

20.9–38.7 32.6–40.8 15.0–19.5 15.2–19.1 14.6–18.9 14.9–19.3 10.6–13.1 10.6–14.1 11.3–14.0 12.0–15.3 5.2–7.2 5.5–7.5 9.7–12.4 10.4–13.2 3.8–5.0 3.3–5.4 2.0–2.5 2.1–2.8 2.2–3.9 2.9–4.3 2.8–3.7 2.9–4.3 1.0–2.0 1.2–2.0 2.8–3.8 2.4–3.6 1.2–2.8 1.3–2.5 1.7–3.3 2.0–3.7 1.3–2.0 1.4–2.8

32.3–38.3 35.8–40.7 17.0–19.7 19.4–21.4 13.8–18.5 17.5–18.8 11.4–12.7 12.2–16.4 12.4–13.5 12.5–14.9 5.6–7.2 6.6–7.3 10.3–11.9 12.0–13.3 3.9–5.4 4.4–5.0 1.5–2.9 1.4–2.2 2.8–3.3 3.1–3.6 2.7–3.8 3.5–3.8 1.4–2.1 1.6–2.1 2.9–3.2 3.1–3.3 1.1–1.6 1.1–1.3 2.0–3.0 2.5–2.8 1.4–2.1 2.0–2.3

* Differences between means of sexes in G. excubitor significant (ANOVA, P≤ 0.01). ** Differences between means for males of the two species significant (ANOVA, P≤ 0.01).

Gastrotheca marsupiata, which differs from all of the species considered here in the diagnosis by having eggs that hatch as tadpoles instead of direct development into froglets, also differs from G. nebulanastes in several structural features—more acutely rounded snout in dorsal view, skin on dorsum smooth with low rounded granules (coarsely granular in G. nebulanastes), Finger I shorter than Finger II (longer than Finger II G. nebulanastes), tibia length less than 50% of SVL and shorter than foot (greater than 50% of SVL and longer than foot G. nebulanastes),

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and distinct tarsal fold on distal half of tarsus (fold absent G. nebulanastes). The dorsum is pale tan to pale green with irregular brown or green markings and a dark canthal stripe and pale labial stripe in G. marsupiata (Fig. 2F), whereas the dorsum is much darker and canthal and labial stripes are absent in G. nebulanastes.

FIGURE 1. Principle components analysis of 14 meristic characters (nostril–jaw and disc width not included) of adults of two populations of Gastrotheca.

Gastrotheca antoniiochoai is essentially sympatric with G. nebulanastes, and differs from G. nebulanastes by having the skin on the dorsum weakly shagreened (coarsely granular in G. nebulanastes), a dark canthal stripe (absent in G. nebulanastes), Finger I shorter than Finger II (I longer than II in G. nebulanastes), and the interorbital distance less than the width of the upper eyelid (much greater than width of upper eyelid in G. nebulanastes) (Fig. 2E). Moreover, dominant frequencies range between 2544 and 3445 Hz in the advertisement call of G. antoniiochoai, but only between 1653 and 1730 Hz in G. nebulanastes (Table 3). Gastrotheca excubitor has a single, median brood pouch, whereas G. antoniiochoai has paired lateral brood pouches, a feature otherwise known only in G. zeugocystis Duellman, Lehr, May, and Rodríguez in Región de Huánuco in the Andes in central Peru. Gastrotheca nebulanastes might be confused with G. ochoai Duellman and Fritts, a slightly smaller species, which is like G. nebulanastes in having the interorbital space being much greater than the width of the upper eyelid and discs on the fingers much wider than the adjacent digit, but G. ochoai differs from G. nebulanastes by having an acuminate snout in dorsal view (bluntly rounded in G. nebulanastes), smooth to finely granular skin on the dorsum (coarsely granular in G. nebulastes), and Finger I shorter than Finger II (longer than Finger II in G. nebulanastes). The only other small species of Gastrotheca on the humid slopes of the Andes in Central and Southern Peru are G. atympana Duellman, Lehr, May, and Rodríguez from Región de Junín, G. rebeccae Duellman and Trueb from Región de Ayacucho, and G. pachachacae Catenazzi and von May from Región de Apurímac. Gastrotheca atympana is unique in the genus by lacking a tympanum; G. rebeccae is like G. nebulanastes in having a broad interorbital area, discs on the fingers that are much wider the digits proximal to the disc, and a tibia longer than the foot, but differs from G. nebulanastes by having Finger I shorter than Finger II (longer than Finger II in G. nebulanastes), snout acutely rounded in dorsal view and bluntly rounded in profile (bluntly rounded in dorsal view and truncate in profile in G. NEW GASTROTHECA FROM PERU

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nebulanastes), skin on dorsum smooth to weakly granular (coarsely granular in G. nebulanastes), and by having a dark canthal stripe and pale labial stripe (stripes absent in G. nebulanastes). Gastrotheca pachachacae differs from G. nebulanastes in lacking a dark interorbital bar (present in G. nebulanastes), and in having a narrower interorbital area, shorter tibia, Finger I slightly longer than Finger II, and discs on the fingers that are slightly wider than digits proximal to discs (much wider than digits in G. nebulanastes).

FIGURE 2. A. Gastrotheca nebulanastes, KU 173210, female, 37.3 mm SVL. B. G. nebulanastes, KU 173209, male, 36.0 mm SVL. C. G. excubitor, KU 193200, female, 35.1 mm SVL. D. G. excubitor, KU 139199, male, 33.9 mm SVL. E. G. antoniiochoai, MUSM 27949, female, 32.5 mm SVL. F. G. marsupiata, KU 139188, male, 34.9 mm SVL.

Description of holotype. Body robust; SVL 37.3 mm; head slightly wider than long; snout rounded in dorsal view and nearly truncate in profile, extending slightly beyond margin of lower lip; canthus rostralis round in section; loreal region slightly concave; lips round; top of head flat; interorbital distance 155% of width of upper eyelid; internarial area flat; nostrils barely protuberant, directed laterally at terminus of canthus rostralis slightly posterior to level of anterior margin of lower jaw; diameter of eye about equal to its distance from nostril; tympa-

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num round, separated from eye by distance half again length of tympanum; tympanic annulus distinct, smooth; supratympanic fold weak, extending from posterior corner of orbit to point posterodorsal to tympanum. TABLE 2. Results from the Principal Component Analysis of 14 meristic characters (nostril–jaw and disc width not included) of adults of two populations of Gastrotheca. The three highest loadings for each component are in boldface. Component

PC1

PC2

PC3

PC4

PC5

Snout–vent length

0.309

0.010

–0.274

0.263

0.082

Tibia length

0.324

–0.058

–0.254

0.061

–0.101

Foot length

0.314

0.195

–0.161

–0.286

0.123

Head length

0.285

0.044

–0.416

–0.186

–0.229

Head width

0.287

0.281

–0.150

0.279

–0.145

Interorbital distance

0.306

–0.140

0.184

0.175

–0.164

Internarial distance

0.235

–0.388

0.286

–0.421

0.106

Eye–nostril

0.058

0.510

0.492

–0.278

-0.511

Eye diameter

0.283

0.007

0.319

0.513

–0.135

Eyelid width

0.089

0.610

0.093

–0.012

0.659

Tympanum width

0.280

–0.225

0.412

0.190

0.321

Thumb length

0.328

–0.143

0.012

–0.330

0.167

Third finger length

0.334

0.059

0.026

–0.197

–0.102

Standard deviation

2.574

1.554

1.183

0.976

0.856

Proportion of variance

0.442

0.161

0.093

0.063

0.049

Cumulative proportion

0.442

0.603

0.696

0.756

0.808

Loadings

Importance of components

Arm slender; row of ulnar tubercles absent; hand moderately large; fingers long, unwebbed; discs moderately large, rounded, width of disc on Finger III equal to length of tympanum; relative lengths of fingers I > II < IV < III; subarticular tubercles prominent, round, none bifid; supernumerary tubercles minute, subconical, numerous on proximal segments; palmar tubercle low, flat, ovoid; prepollical tubercle elliptical. Hind limb robust; tibia length 54.1% SVL; foot length 49.1% SVL; calcar, tarsal tubercles, and inner tarsal fold absent; outer metatarsal tubercle minute, round; inner metatarsal tubercle large, elliptical, barely visible from above; toes long; relative length of toes I < II < III < V < IV; toes not webbed except for basal webbing between Toes IV and V; subarticular tubercles small, round; supernumerary tubercles minute, round, numerous on proximal segments. Skin on dorsum granular; two large granules posteroventral to tympanum; skin on flanks areolate; skin on throat, belly, and ventral surfaces of thighs granular, on other surfaces of smooth; cloacal tubercles and folds absent; pouch opening V-shaped with anterior border at level of posterior edge of sacrum. Vomerine odontophores inclined posteromedially, narrowly separated medially, between round choanae, bearing four teeth each. Color in preservative: Dorsum pale brown with darker brown markings consisting of an interorbital triangle bifurcated posteriorly; left bifurcation connected to large irregularly shaped dark brown mark on dorsum of body; irregular dark brown spots postsacrally. Canthal and labial stripes absent. Flanks creamy tan with three large dark brown to black vertical marks; anterior surfaces of thighs tan with dark brown transverse bars extending over dorsal surfaces to dark brown posterior surfaces of thighs; three transverse dark brown bars on each shank and tarsus; cloacal region dark brown with small white spot posteroventral to opening. Venter uniform pale brown. Color in life: Dorsum dull green with dark brown to black markings (Fig. 2A); flanks colored like dorsum except with orange-brown suffusion; hidden surfaces of limbs orange-brown; venter metallic tan; tympanum brown; single small white spot on left side of dorsum; iris pale bronze tan with black reticulations. Measurements (in mm): SVL 37.3, tibia length 20.2, foot length 18.3, head length 12.9, head width 14.2, interorbital distance 4.8, internarial distance 1.7, eye–nostril distance 3.4, diameter of eye 3.5, diameter of tympanum

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1.9, orbit–jaw distance 1.2, nostril–jaw distance 2.5, length of Finger I 7.0 mm, length of Finger III 12.8 mm, width of disc on Finger III 2.0. Variation. Males are slightly larger than females, but the proportions of the two sexes do not seem to be different (Table 1).Eighteen other adults were captured and measured in the field. The SVLs were 36.3 and 40.9 mm in two females and 27.8–35.8 mm in males ( x = 32.4 ± 2.2 mm, n = 14). Two females weighed 4.8 and 5.0 g and 14 males weighed 1.6–3.9 g ( x = 2.4 ± 0.6 g). The skin on the dorsum is variously granular, even in a juvenile 20.4 mm SVL (AMNH 153979), and weakest in a male 34.4 mm SVL (AMNH 157006). All adults have 2–5 prominent, rounded, white tubercles ventral and ventrolateral to the tympanum. Basal webbing is present between Toes IV and V in all, and between Toes III–IV in six, adults. Each vomer has four or five teeth. In preserved specimens, the dorsum is brown with darker brown markings consisting of an interorbital bar that usually is connected to a middorsal or roughly bifurcate mark that extends to the sacral region or beyond. The flanks are tan with dark brown to black markings—prominent spots or vertical bars. There are three dark brown transverse bars on the dorsal surfaces of the thighs than are continuous with a dark brown longitudinal mark on the posterior surface of the thigh. There are three diagonal dark brown bars on each shank and three or four such bars on each foot. A faint creamy white supracloacal stripe and short heel stripes are discernable in most specimens. Except for the dark brown interorbital bar, head markings, if present, are indistinct. Color notes on living individuals comprising the type series: Dorsum pale grayish tan, yellowish tan, or olivetan with dark brown or metallic green markings. Cream spots present on some. Flanks, groin, anterior and posterior thighs creamy tan to reddish brown with blue, bluish green, or dark brown spots. Webbing tan; fingers and toes tan. Venter metallic bronze-brown. Metallic creamy bronze tubercles along posterior upper jaw. Iris pale bronze with fine black reticulations (WED field notes, 19 January 1977). Details of coloration of living individuals from the vicinity of Pillahuata and Esperanza are as follow: The dorsal coloration varies from beige to dark brown, gray or green with numerous green flecks and a darker pattern of two continuous or interrupted longitudinal marks connected to the interorbital bar. In some individuals the darker pattern is most visible and green flecks are minute or concealed by the background dorsal coloration, whereas in other individuals the green flecks are predominant and interrupt or largely cover the darker longitudinal marks. The dorsal coloration extends onto the dorsal surfaces of digits and limbs, with transverse bars on thighs barely noticeable in some individuals and presenting a distinct contrast in other individuals. In all individuals, the flanks are pale and have green and bluish flecks; in some individuals the bluish flecks form a continuous longitudinal stripe from the point of arm insertion to the middle of the body. The tubercles ventrolateral to the cloaca are white. The throat and chest are cream or dark brown without marks. Dark brown canthal and supratympanic stripes are diffuse and bordered with cream. The tympanum is brown. The margin of the upper lip is colored like the snout (brown to pale brown) with green flecks. The margin of the upper eyelid is cream. The iris is bronze with black reticulations, and the periphery of the eye is black. The bones are white. In juveniles the dorsum is brown with bronze tones, and green or bluish green flecks are predominant over the darker longitudinal markings. The throat and chest are yellow or cream, whereas the ventral surfaces of abdomen and limbs are vermillion. TABLE 3. Comparison of call variables between species of Gastrotheca from the upper Kosñipata valley. Species

G. antoniiochoai1 G. excubitor G. nebulanastes

*

Dominant frequency (Hz)* Long note Short note

N (pulses)* (long note)

Duration (ms) Long note

Short note

2915 (2544–2968) 1730 (1615–1884) 1730 (1653–1962)

33.6 ± 1.9 (26–38) 36.9 ± 5.4 (30–45) 41.6 ± 3.2 (37–47)

544.8 ± 27.5 (412–601) 992.7 ± 153.7 (666–1267) 818.2 ± 70.0 (689–916)

21.3 ± 1.8 (17–22) 27.6 ± 2.4 (22–31) 30.9 ± 7.8 (24–54)

3286 (3180–3445) 1769 (1653–1846) 1692 (1653–1730)

Indicates variables that vary little with temperature (Sinsch & Joermann, 1989). Recorded at Tair = 20.0°C (Catenazzi & Lehr 2009).

**

Vocalization and reproduction. Males call from grasses, shrubs, trees, terrestrial and arboreal bromeliads, and the ground in the montane scrub or understory of the cloud forest. The advertisement call consists of a long

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note 818.2 ± 70.0 ms (range 689–916 ms) in duration with 41.6 ± 3.2 pulses (range 37–47 pulses), and one or more secondary notes (Table 3; Fig. 3). Secondary notes have a duration of 30.9 ± 7.8 ms (range 24–54 ms), emphasized frequencies of 1653–1730 Hz (median 1692 Hz), and typically are single-pulsed. Pulse rate in long notes average 51.0 ± 1.2 Hz (range 49.5–53.7 Hz) at temperatures of 10.7–12.3°C. Long notes have some frequency modulation: dominant frequencies range between 1461–1615 Hz (median 1500 Hz) for the third pulse, 1538–1884 Hz (median 1634 Hz) for a central pulse and 1653–1769 Hz (median 1730 Hz) for the third-to-last pulse. Pulses emitted at highest amplitude in long notes have dominant frequencies of 1653–1962 Hz (median 1730 Hz) and are generally located in the second half of the note (after the 20th pulse), although amplitude modulation vary among calls and does not always increase constantly during the long note (see Fig. 3). Males can be heard calling on most nights on rock walls bordering the road at elevations between 2700–2800 m. Field notes accompanying USNM 298184–90 indicate that males were calling on 16 May 1984.

FIGURE 3. Advertisement calls of Gastrotheca nebulanastes (MUSM 28060, Esperanza, 4 April 2011, Tair = 10.7°C) and Gastrotheca excubitor (male not collected, Abra Acjanaco, 24 January 2009, Tair = 9.5°C).

One female (KU 173218) having a SVL of 40.7 mm gave birth to 17 young on 27 January 1977. Fourteen of the froglets (KU 173521–22) have SVLs of 9.5–9.8 mm ( x = 9.64 ± 0.21). Immediately after birth the dorsum was creamy tan with grayish brown transverse bars. Two days after birth they assumed the color pattern of adults. The dorsum was brown with dark brown markings. The throat was white; the belly was transparent, and the rest of the venter was yellow. The iris was deep bronze. Another female (MUSM 20940) having a SVL of 35.8 mm contains eight oviductal eggs. Phylogenetic relationships. In order to evaluate the phylogenetic relationships of Gastrotheca nebulanastes, we analyzed our newly collected DNA sequence data together with data from previous studies (Darst & Cannatella, 2004; Wiens et al., 2007; Lehr et al., 2005). We restricted our analysis to taxa previously demonstrated to form a strongly supported clade (Wiens et al., 2007) and to which the new species is likely closely related; analyses including all of hemiphractid diversity further supports that the new species is a member of this clade (data not

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shown). This clade consists of 12 species: G. atympana, christiani, chrystosticta, excubitor, gracilis, griswoldi, marsupiata, ochoai, peruana, pseustes, psychrophila, and stictopleura. We included all of these species in our analysis except for G. gracilis for which 16S is not available. Wiens et al. (2007) also found strong support that G. zeugocystis is the sister taxon of this clade. Because of the morphological similarities between G. zeugocystis and G. antoniiochoai, we also included G. zeugocystis in our analysis.

FIGURE 4. Maximum-likelihood phylogram estimated from mitochondrial DNA sequences (16S ribosomal RNA genes) depicting the phylogenetic relationships of G. nebulanastes, with nonparametric bootstrap values greater than 70% indicated.

We find strong support that Gastrotheca nebulanastes is the sister taxon of G. atympana, a species known only from Pampa Hermosa (1540 m) in the Río Chanchamayo Valley in the Cordillera Oriental of Peru (Fig. 4). Both G. nebulanastes and G. atympana form a strongly supported clade with G. excubitor, G. ochoai, and the recently described G. pachachacae (Catenazzi & von May, 2011). Wiens et al. (2007) also found strong support for a clade containing G. atympana, G. excubitor, and G. ochoai, yet our analysis including topotypic material of G. excubitor indicates that the specimen of G. excubitor included by Wiens et al. (2007) may represent another taxon. Distribution and ecology. Gastrotheca nebulanastes is known only from elevations of 2000–3300 m in the upper Río Kosñipata Valley on the northeast slope of Abra Acjanaco in the Cordillera Oriental in Región de Cusco in the Parque Nacional Manu in southern Peru. All localities are in cloud forest along the road between Paucartambo and Pilcopata. At 2100 m (Rocotal), during the period 2000–2007, average Tmin was 11.7–13.1˚C, average Tmaxwas 20.5–22.3˚C, and average precipitation was 369–710 mm for the month of February (Servicio Nacional de Metereología e Hidrología, Lima, Peru). The type locality was a small truck stop on the southeast side of the road in 1977, which is bounded by cloud forest on steep slopes with tree ferns, moss-covered cliffs, and cascading streams. At the type locality eight individuals were sitting on rocks on a mossy cliff and three were perched on sticks or bases of ferns no more than 10 cm from the cliff at night (Fig. 5A). Elevational records for juvenile and adult specimens captured in the field (n = 33) indicates that this species is most abundant between 2600 and 2800 m

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(Fig.6) where it inhabits the montane scrub, the ridges of steep slopes covered with Andean alder, shrubs, and abundance of terrestrial mosses, lycophites, and lichens (Fig. 5B). There is sharp transition from the cloud forest to the puna characteristic of the Abra Acjanaco that is inhabited by G. excubitor (Fig. 5C); from the abra on a clear day one can glimpse the forested upper Kosñipata Valley (Fig. 5D).

FIGURE 5. Upper Kosñipata Valley. A. Habitat of Gastrotheca nebulabastes at the type locality, 2400 m, with David C. Cannatella in the foreground. January 1977. B. Forested slopes near Esperanza, 2780 m, where G. nebulanastes is abundant. 19 January 1998. C. Abra Acjanaco, 3520 m, showing transition to cloud forest on the northeastern slope. January 1977. D. View northeast down the upper Río Kosñipata Valley as seen from Abra Acjanaco at 3520 m. The sinuous road to Pilcopata is visible in the foreground and on the left. January 1977.

FIGURE 6. Elevational distribution of Gastrotheca nebulanastes from a sample of juveniles and adults (n = 33) captured between 2100 and 3000 m in the upper Kosñipata Valley.

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The following species of anurans also are known to occur at the type locality: Hyloscirtus armatus (Boulenger), Nymphargus pluvialis (Cannatella & Duellman), Oreobates lehri (Padial, Chaparro, & De la Riva), Pristimantis pharangobates (Duellman), P. usurpator De la Riva, Chaparro, and Padial, and Telmatobius sp. At localities at higher elevations Gastrotheca nebulanastes also occurs in sympatry with Bryophryne hanssaueri Lehr and Catenazzi, B. nubilosus Lehr and Catenazzi, Centrolene sp., and Noblella pygmaea Lehr and Catenazzi. Gastrotheca nebulanastes is sympatric with G. antoniiochoai at elevations of 2800–3300 m. Both species occur in the montane scrub and cloud forest, where G. antoniiochoai seems to be restricted to water-filled epiphytic bromeliads. The elevational distribution of G. nebulanastes marginally overlaps with the lower end of the elevational range of G. excubitor, a species primarily found in grassland habitats (puna) dominated by bunchgrasses (Stipa sp., Calamagrostis sp., etc), elfin forest, and other habitats at the transition between the cloud forest and the puna. Etymology.The specific name is derived from the Latin nebula meaning fog and the Greek nastes meaning dweller. The name is applied because the upper Kosñipata Valley inhabited by this species commonly is enshrouded in fog. Remarks. Presently, several populations in the Cordillera Oriental in Región de Cusco are assigned to Gastrotheca excubitor—3080–3580 m on the slopes of Abra Amparaes, 3160–4080 m on the slopes of Abra Málaga, and 3100–3200 m on the slopes of Abra Marcapata. Not all of these populations may represent G. excubitor. As noted in Figure 3, KU 173171 from 1.5 km SW Amparaes, 3580 m is genetically different from topotypic G. excubitor. A juvenile (USNM 298182) having a SVL of 19.8 mm is tentatively assigned to G. nebulanastes supposed from an elevation of 1469 m on the Paucartambo–Pilcopata road may have incorrect locality data, inasmuch as the elevation is more than 500 m lower than any other localities where the species is known. Although Table 1 suggests that SVL is similar in G. excubitor and G. nebulanastes, data from individuals captured and released in the field indicate that G. excubitor can grow to a much larger size than G. nebulanastes. In G. excubitor, the largest male had a SVL of 46.3 mm and weighed 6.4 g (vs. 35.8 mm and 3.4 g in G. nebulanastes), whereas the largest brooding female had a SVL of 47.3 mm and weighed 9.0 g (vs. 40.9 mm and 5.0 in G. nebulanastes). The vegetation at Abra Acjanaco consists primarily of puna grass (Stipa sp. and Calamagrostis sp.) with some ferns and mosses on the steeper slopes, some small bushes (Baccharis), and a variety of low herbs, including orchids. Three days in February 1975, temperatures were 4–16˚C with slight rainfall (trace to 4.0 mm daily). During the period 2000–2007, the range of average Tmin was 5.5–7.0˚C, and the average Tmax was 12.0–17.6˚C; the average precipitation was 235–330 mm for the month of February (Servicio Nacional de Metereología e Hidrología, Lima, Peru). At this locality, Gastrotheca excubitor is common; individuals were found under rocks by day and were heard calling at night. The ratio of G. excubitor to G. marsupiata is 25:1; tadpoles of this species were found in a small temporary pond. Other species of anurans collected at Abra Acjanaco are Bryophryne cophites (Lynch), B. hanssaueri, Noblella pygmaea,Psychrophrynella usurpator, Telmatobius timens De la Riva, Aparicios & Ríos. The small gymnophthalmid lizard, Proctoporus bolivianus Werner is extremely abundant. Between 3150 and 3200 m on the northeast slope of the abra there is a rapid transition from puna grassland to cloud forest (Fig. 5C), which continues to lower elevations in the Río Kosñipata Valley (Fig. 5D). All known specimens of Gastrotheca antoniiochoai and G. nebulanastes are from the montane scrub or cloud forest in this valley, in which neither G. excubitor or G. marsupiata occur. The amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis has been reported from the Kosñipata Valley and is the probable cause of severe population declines in amphibian assemblages of the upper Parque Nacional del Manu (Catenazzi et al. 2011). Infection with this fungus has been confirmed in one juvenile specimen of G. nebulanastes (MUSM 27942) obtained at 2110 m on 31 January 2009 (Catenazzi et al. 2011). Fourteen other specimens captured in July 2007, February and October 2008, and February 2009 were negative for the fungus using a PCR-based test (Catenazzi et al. 2011).

Acknowledgments For the loan of specimens from their respective institutions, we are indebted to J. Córdova, W. R. Heyer, and D. R. Frost. In 1977, collecting permits were provided by C. P. del Prado, Dirección de Conservación, Lima. Logistic support was provided by O. Ochoa of Cusco; WED was assisted in the field by D. C. Cannatella; the field work was supported by a grant (DEB 76-09986) from the National Science Foundation, USA. Specimens collected in

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2007 were exported with permit 010778-AG-INRENA issued by the Ministerio de Agricultura, Lima. We thank Manu NP for research permits and Wayqecha Biological Station for logistic support. Fieldwork between 2007 and 2010 was supported by grants from the Amazon Conservation Association, the Rufford Foundation, the Chicago Board of Trade Endangered Species Fund and the Amphibian Specialist Group to A.C. A.C. was assisted in the field by J. C. Jahuanchi, J. Carrillo, W. Qertehuari, A. Machaca, I. Chinipa, R. Sotelo, C. Quispe. AC. was supported by a post-doctoral fellowship from the Swiss National Science Foundation (116305).

References Catenazzi, A. Lehr, E., Rodríguez, L.O. & Vredenburg, V.T. (2011) Batrachochytrium dendrobatidis and the collapse of montane anuran communities in the upper Manu National Park, southeastern Peru. Conservation Biology, 25, 382–391. Catenazzi, A. & Lehr, E. (2009) The generic allocation of “Hyla antoniiochoai De la Riva & Chaparro, 2005 (Anura), with description of its advertisement call and ecology. Zootaxa, 2304, 61–68. Catenazzi, A. & von May, R. (2011) New species of marsupial frog (Hemiphractidae: Gastrotheca) from an isolated montane forest in Peru. Journal of Herpetology 45, 161–166. Da la Riva, I. & Chaparro, J. C. (2005) A new species of tree frog from the Andes of southeastern Peru (Anura: Hylidae: Hyla). Amphibia-Reptilia, 26, 515–521. Darst, C.R. & Cannatella, D. C. (2004). Novel relationships among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 31, 462–475. Duellman, W.E. & Fritts, T.H. (1972) A taxonomic review of the southern Andean marsupial frogs (Hylidae: Gastrotheca). Occasional Papers Museum of Natural History, University of Kansas, 9, 1–37. Duellman, W.E. & Hillis, D.M. (1987) Marsupial frogs (Anura: Hylidae: Gastrotheca) of the Ecuadorian Andes: resolution of taxonomic problems and phylogenetic relationships. Herpetologica, 43, 141–173. Duellman, W.E., Lehr, E. & Aguilar, C. (2001) A new species of marsupial frog (Anura: Hylidae: Gastrotheca) from the Cordillera Azul in Peru. Scientific Papers Natural History Museum University of Kansas, 22, 1–10. Duellman, W.E., Lehr, E., Rodríguez, D. & von May, R. (2004). Two new species of marsupial frogs (Anura: Hylidae: Gastrotheca) from the Cordillera Oriental in central Peru. Scientific Papers Natural History Museum University of Kansas, 32, 1–10. Duellman, W.E. & Pyles, R. A. (1980) A new marsupial frogs (Hylidae: Gastrotheca) from the Andes of Ecuador. Occasional Papers Museum of Natural History, University of Kansas, 84, 1–13. Duellman, W.E. & Trueb, L (1988) Cryptic species of hylid marsupial frogs in Peru. Journal of Herpetology, 22, 159–179. Duellman, W.E., Trueb, L. & Lehr, E. (2006) A new species of marsupial frog (Anura: Hylidae: Gastrotheca) from the Amazonian slopes of the Cordillera Oriental in Peru. Copeia, 2006, 595–603. Esselstyn, J.A., Garcia, H.J.D., Saulog, M.G. & Heaney, L. R. (2008). A new species of Desmalopex (Pteropodidae) from the Philippines, with a phylogenetic analysis of the Pteropodini. Journal of Mammalogy 89, 815–825. Katoh, K., Kuma, K.-I., Toh, H. & Miyata, T. (2005). MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research, 33, 511–518. Lehr, E., Fritzsch, G. & Müller, A. (2005). Analysis of Andes frogs (Phrynopus, Leptodactylidae, Anura) phylogeny based on 12S and 16S mitochondrial rDNA sequences. Zoologica Scripta, 34, 593–603. Posada, D. (2008). jModelTest: phylogenetic model averaging. Molecular Biology and Evolution 25, 1253–1256. Sinsch, U. & Joermann, G. (1989). Vocalization and mating behaviour of the marsupial frog, Gastrotheca marsupiata (Duméril and Bibron, 1841) (Amphibia, Hylidae). Copeia, 1989, 755–760. Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics,22, 2688–2690. Stamatakis, A., Blagojevic, F., Nikolopoulos, D. & Antonopoulos, C. (2007). Exploring new search algorithms and hardware for phylogenetics: RAxML meets the IBM cell. Journal of VLSI Signal Processing, 48, 271–286. Sukumaran, J. & Holder, M.T. (2010). DendroPy: a Python library for phylogenetic computing. Bioinformatics, 26, 1569–1571. Wiens, J. J., Kuczynski, C.A., Duellman, W.E. & Reeder, T.W. (2007). Loss and re-evolution of complex life cycles in marsupial frogs: does ancestral trait reconstruction mislead? Evolution 61, 1886–1899.

APPENDIX I. Additional material examined. Numbers in bold face indicate that tissues were used in the phylogenetic analysis. Gastrotheca antoniiochoai, Peru: Cusco: near Quebrada Toqoryuoc, Wayqecha Research Center, Kosñipata Valley, 2845 m, MHNC 0068 (photo of living holotype); near Quebrada Huancohumanio, Esperanza, Kosñipata Valley, 2817 m, MNCN 42013; Paucartambo–Shintuya road, Wayqecha Research Center, 2950 m, MUSM 27944, 27945–49. Gastrotheca atympana, Peru: Junín: Pampa Hermosa, 1540 m, MUSM 18692

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Gastrotheca christiani, Argentina: Jujuy: Abra de Cañas, FML 2881. Gastrotheca chrysosticta, Argentins: LM 58. Gastrotheca excubitor, Peru: Cusco: North slope Abra Acjanaco, 3270–3520 m, KU 139193–201, MUSM 26280–26281; 1.5 km SW Amparaes, 3580 m, KU 11731790, KU 173171, 173172–73, 2,5 KM SW Amparaes, 3780 m, KU 173174–77. Gastrotheca griswoldi, Peru: 6 km NE Paccha, 3840 m, KU 181701. Gastrotheca marsupiata, Peru: Cusco: Abra Acjanaco, 25 km NNE Paucartambo, 3520 m, KU 163187, 164047 (tadpoles); Abra Huancarane, 10 km E. Huancarane, 3790 m, KU 163188-215; San Jerónimo, 10.7 km ESE Cuzco, 3150 m, KU 214813. Gastrotheca nebulanastes. Peru: Cusco: Esperanza, 2750 m, MUSM 27288; between Buenos Aires and Estrella, 2250 m, MCZ 265218; between Esperana, 2780 m and Pillahuata, 2580 m, MUSM 20940–44, 20950–51; Km 152 on Paucartamno–pilcopata roads, 1469 m, USNM 298182; Rocotal, 2180 m, MUSM 27943. Gastrotheca ochoai, Peru: Cusco:Chilca, 10 km N Ollantaytambo, 2760 m, KU 138628–65, 139202–09,), 148572, KU 173499. Gastrotheca pachachacae. Peru: Apurímac: Ccerabamba–Andina Chinchay cloud forest, 3050 m, MUSM 28489–91, 28492. Gastrotheca peruana Peru: Cajamarca: 23 km SW Celendín,3050 m,KU 181740 Gastrotheca pseustes, Ecuador: Chimborazo: Tixan, 2990 m, TNHC 62492. Gastrotheca psychrophila, Ecuador: Loja–Zamora-Chinchipe: 13–15 km E Loja, 2770–2850 m, KU 142634 Gastrotheca rebeccae, Peru: Ayacucho: Ccarpa, below Tambo on Valle de Apurimac trail, 2470 m, KU 196806–11 Gastrotheca stictopleura, Peru:Huánuco: Paugmaray, Provincia de Huachon, 2600 m, MTD 45230 Gastrotheca zeugocystis, Peru: Huánuco: Cordillera de Carpish, 2920 m, MUSM 18685.

APPENDIX II. GenBank accession numbers for DNA sequences of species used in the phylogenetic analysis. Species of Gastrotheca

Collection #

16S

G. antoniiochoai

MUSM 27944

JN157620

G. atympana

MUSM 18692

DQ679384

G. christiani

FML 2881

DQ679386

G. chrysosticta

LM 58

DQ679387

G. excubitor

KU 173171

DQ679391

MUSM 26280

JN157523

MUSM 26281

JN157624

G. griswoldi

KU 181701

AM039648

G. marsupiata

KU 214813

DQ679397

G. nebulanastes

MCZ 265218

JN157625

MUSM 27943

JN157621

MUSM 27888

JN157626

G. ochoai

KU 173499

DQ679400

G. peruana

KU 181740

DQ679402

G. pseustes

TNHC 62492

AY326051

G. psychrophila

KU 142634

DQ679404

G. stictopleura

MTD 45230

DQ679407

G. zeugocystis

MUSM 18675

DQ679411

G. pachachacae

MUSM 28492

JN157620

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