Gastrotheca nebulanastes, Duellman, William E., Catenazzi, Alessandro & Blackburn, David C., 2011

Duellman, William E., Catenazzi, Alessandro & Blackburn, David C., 2011, A new species of marsupial frog (Anura: Hemiphractidae: Gastrotheca) from the Andes of southern Peru, Zootaxa 3095, pp. 1-14 : 3-12

Gastrotheca nebulanastes new species

Holotype: KU 173210 , an adult female ( Fig. 2A ), from Buenos Aires ( Fig. 5 A), 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. 2 B), 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 Paucar-tambo—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. 5 B), 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 4 C). 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. n e b u l a n a s t e s; 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 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 .

G. excubitor (23 3, 15 Ƥ) G. nebulanastes (18 3, 5 Ƥ)

Character Sex Mean Range SD Mean Range SD

Snout-vent 3 33.7 20.9–38.7 3.47 35.4 32.3–38.3 1.74 length Ƥ 35.8 32.6–40.8 2.78* 37.7 35.8–40.7 1.81 Tibia length 3 16.9 15.0–19.5 1.11** 18.3 17.0–19.7 0.68

Ƥ 17.5 15.2–19.1 1.33 20.3 19.4–21.4 0.73 Foot length 3 16.5 14.6–18.9 1.17 16.6 13.8–18.5 1.02

Ƥ 17.2 14.9–19.3 1.43 18.1 17.5–18.8 0.51 Head length 3 11.6 10.6–13.1 0.70** 12.0 11.4–12.7 0.37

Ƥ 11.9 10.6–14.1 1.02 13.5 12.2–16.4 1.68 Head width 3 12.8 11.3–14.0 0.70 12.9 12.4–13.5 0.48

Ƥ 13.3 12.0–15.3 1.15 14.0 12.5–14.9 0.95 Thumb length 3 6.2 5.2–7.2 0.46** 6.6 5.6–7.2 0.49

Ƥ 6.3 5.5–7.5 0.51 6.9 6.6–7.3 0.26 Third finger 3 11.1 9.7–12.4 0.74 11.5 10.3–11.9 0.47 length Ƥ 11.7 10.4–13.2 0.87* 12.6 12.0–13.3 0.48 Interorbital 3 4.4 3.8–5.0 0.33** 4.7 3.9–5.4 0.47 distance Ƥ 4.3 3.3–5.4 0.64 4.7 4.4–5.0 0.26 Internarial 3 2.2 2.0–2.5 0.15 2.4 1.5–2.9 0.48 distance Ƥ 2.4 2.1–2.8 0.16* 1.8 1.4–2.2 0.29 Eye-nostril 3 3.4 2.2–3.9 0.41** 3.1 2.8–3.3 0.13

Ƥ 3.5 2.9–4.3 0.45 3.3 3.1–3.6 0.19 Eye diameter 3 3.3 2.8–3.7 0.26** 3.4 2.7–3.8 0.24

Ƥ 3.5 2.9–4.3 0.49* 3.6 3.5–3.8 0.14 Tympanum 3 1.6 1.0–2.0 0.25** 1.8 1.4–2.1 0.25 diameter Ƥ 1.6 1.2–2.0 0.26 1.8 1.6–2.1 0.22 Eyelid width 3 3.2 2.8–3.8 0.28 3.1 2.9–3.2 0.08

Ƥ 3.1 2.4–3.6 0.42 3.2 3.1–3.3 0.08 Orbit-jaw 3 1.8 1.2–2.8 0.33** 1.4 1.1–1.6 0.20

Ƥ 1.7 1.3–2.5 0.37 1.2 1.1–1.3 0.09 Nostril-jaw 3 2.7 1.7–3.3 0.35 2.7 2.0–3.0 0.30

Ƥ 2.7 2.0–3.7 0.50 2.6 2.5–2.8 0.14 Disc width 3 1.6 1.3–2.0 0.20** 1.7 1.4–2.1 0.17

Ƥ 1.7 1.4–2.8 0.34 2.1 2.0–2.3 0.15

* 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 ), 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. 2 F), whereas the dorsum is much darker and canthal and labial stripes are absent in G. nebulanastes .

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. 2 E). 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.

nebulanastes ), skin on dorsum smooth to weakly granular (coarsely granular in G. n e b u l a n a s t e s), 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 ).

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; tympanum 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.

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 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.

Species Dominant frequency (Hz)* N (pulses)* Duration (ms)

Long note Short note (long note) Long note Short note

G. antoniiochoai 1 2915 3286 33.6 ± 1.9 544.8 ± 27.5 21.3 ± 1.8 (2544–2968) (3180–3445) (26–38) (412–601) (17–22)

G. excubitor 1730 1769 36.9 ± 5.4 992.7 ± 153.7 27.6 ± 2.4 (1615–1884) (1653–1846) (30–45) (666–1267) (22–31)

G. nebulanastes 1730 1692 41.6 ± 3.2 818.2 ± 70.0 30.9 ± 7.8 (1653–1962) (1653–1730) (37–47) (689–916) (24–54)

*Indicates variables that vary little with temperature ( Sinsch & Joermann, 1989). ** Recorded at T air = 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 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, T air = 10.7°C) and Gastrotheca excubitor (male not collected, Abra Acjanaco, 24 January 2009, T air = 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 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.

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 T min was 11.7–13.1˚C, average T max was 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. 5 A). 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 ( 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. 5 B). There is sharp transition from the cloud forest to the puna characteristic of the Abra Acjanaco that is inhabited by G. excubitor ( Fig. 5 C); from the abra on a clear day one can glimpse the forested upper Kosñipata Valley ( Fig. 5 D).

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 T min was 5.5–7.0˚C, and the average T max 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. 5 C), which continues to lower elevations in the Río Kosñipata Valley ( Fig. 5 D). 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).

Figures

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

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

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.

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.

Tables

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 PC 1 PC 2 PC 3 PC 4 PC 5 Loadings

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
Importance of components
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