A new species of frog of the genus Pristimantis from Tingo María National Park, Huánuco Department, central Peru (Anura, Craugastoridae)

Abstract A new species of Craugastoridae frog encountered from 1000–1700 m in elevation in the premontane forests of the Peruvian central Andes is described. The new species is similar in appearance to many other species of Pristimantis, but is easily distinguishable from these species by having bright red coloration on the groin, posterior surface of thighs, and shanks. The new species is only known for two localities 27 km apart in the Huánuco Region.


Introduction
Frogs of the genus Pristimantis (Craugastoridae) comprise one of the most striking, richest, and understudied groups in the Neotropics (Hedges et al. 2008;Padial and De la Riva 2009;Ortega-Andrade et al. 2015). Likewise, 131 species of Pristimantis are known to occur in Peru where they are distributed in many habitats on the western and eastern versants of the Andes (Frost 2016).
Although several species of Craugastoridae have been described from the eastern Andean slopes of central Peru over the last 15 years (Lehr et al. 2000;Lehr 2001;Lehr and Aguilar 2003;Duellman and Hedges 2005;Boano et al. 2008;Duellman and Chaparro 2008;Lehr and Oroz 2012;Chávez et al. 2015), amphibian taxonomic research in one of main drainages of the area: the Upper Huallaga river has focused on species of Bufonidae, Hylidae, Centrolenidae and Dendrobatidae (Duellman and Toft 1979;Aichinger 1991, Castroviejo-Fisher et al. 2009) with only one Pristimantinae (Craugastoridae) species recently described from this region (Duellman and Hedges 2007).
Tingo María National Park (TMNP) is a small protected area covering 4777 ha (SERNANP 2015) located in the Huallaga river basin on the eastern versant of the Peruvian central Andes. The landscape is dominated by a chain of small, isolated mountains commonly called "La Bella Durmiente" ("The sleeping beauty"), and belongs to lower montane forest (Gentry 1993). Few biological surveys have been conducted within this park, and its biological diversity is poorly known because terrorism and drug trafficking made the area inaccessible during the decades of the 80's and 90's. A rapid biological inventory was realized in 2014, 50 years after the creation by Peruvian law of TMNP, and resulted in an amphibian collection obtained by GC. Morphological analysis of collected specimens, and comparisons with similar species, led us to the discovery of a new species of frogs of the genus Pristimantis, which is described herein.

Material and methods
Format for diagnosis and description of the new species follow those of Lynch and Duellman (1997). For systematic of Craugastoridae we follow Hedges et al. (2008), Pyron andWiens (2011), andPadial et al. (2014). Specimens collected were sacrificed with a 20% benzocaine solution and fixed in 10% formalin, then stored in 70% ethanol and deposited in the herpetological collection at Centro de Ornitologia y Biodiversidad (COR-BIDI). The following variables were taken as described in Duellman and Lehr (2009) and were measured to the nearest 0.1 mm with digital calipers under a stereoscope: snoutvent length (SVL); eye-nostril distance (EN); head length (HL); head width (HW); interorbital distance (IOD); internarial distance (IND); tibia length (TL); foot length (FL); eye diameter (ED); upper eyelid width (EW).
Fingers and toes are numbered preaxially to postaxially from I-IV and I-V respectively. We determined comparative lengths of toes III and V by adpressing both toes against Toe IV; lengths of fingers I and II were determined by adpressing the fingers against each other. Specimens were sexed based on external sexual characteristics (e.g., presence of vocal sacs in males), all specimens were collected when they were calling, thus are considered adult males. Photographs were taken in the field and laboratory by GC, and used for descriptions of coloration in life and in preserved condition respectively. In addition to the type series of the new species, specimens examined are listed in Appendix I.
The advertisement calls of a chorus of males (CORBIDI 15563-68, 15577-78) were recorded at the type locality on 21 November 2014 (T air = 23.0 °C; taken with a digital thermo hygrometer to the nearest 0.1 °C). A digital recorder (Marantz PM-D661MK2) and unidirectional microphone (Sennheiser ME64) were used for field recording, and Raven Pro version 1.4 (Cornell Laboratory of Ornithology, Ithaca, NY) to analyze call variables. A total of 48 calls were analyzed. The following variables were measured from oscillograms: note, duration, and rate, interval between notes or calls, number of pulses, and presence of amplitude modulation. Variables measured from spectrograms included dominant frequency, and presence of frequency modulation or harmonics. Spectral parameters were calculated through fast Fourier transform (FFT) set at a length of 512 points (Hann window, 50% overlap). Averages are reported ± SD.
Diagnosis. The new species is distinguished by the following combination of characters: (1) skin on dorsum finely shagreen, that on venter areolate, discoidal fold absent, dorsolateral folds absent; (2) tympanic membrane and tympanic annulus distinct, weak supratympanic fold covering dorsal and posterior edges of tympanum, horizontal diameter of eye 3x the diameter of tympanum; (3) snout acuminate in dorsal view, truncated and posteroventrally inclined in lateral view, canthus rostralis weakly concave in dorsal view, angular in lateral view, loreal region concave, rostral papilla absent; (4) upper eyelid lacking tubercles, cranial crests absent; (5) dentigerous process of vomers absent; (6) males with vocal sacs and vocal slits, nuptial excrescences absent; (7) finger I and finger II of equal length, fingers II and III bearing rounded discs about 1.5 times wider than digits, finger IV bearing a rounded disc about twice as wide as its digit; (8) fingers with narrow lateral fringes; (9) antebrachial tubercle absent; (10) ulnar and tarsal tubercles absent (11) inner metatarsal tubercle oval twice as long as round outer metatarsal tubercle, low supernumerary plantar tubercles at the base of toes I, II, and III; (12) toes with narrow lateral fringes, webbing absent, toe V longer than toe III; (13) in life, males with dorsum creamy yellow or yellowish brown with dark blotches; canthal stripe creamy white extending to the orbits; throat yellow; belly creamy white; groins, posterior surfaces of thighs, and shanks bright red; iris cream with brown flecks; (14) SVL in adult males 19.1-21.9 mm; SVL in females unknown.
The uncorrected genetic distances (Table 2) support the generic placement of the new species and its distinctiveness with respect to superficially similar species. According to these analyses, the most closely related species is P. pluvialis (Shepack et al. 2016), which despite sharing a similar body shape can easily be distinguished from P. pulchridormients by the presence of a rostral papilla, larger size, and coloration patterns.
The new species is also similar to the recently described Pristimantis ardyae (Reyes-Puig et al. 2013) from Ecuador in having red groins (red or orange in P. ardyae), but can be distinguished by the following characters (condition for P. ardyae in parentheses): upper eyelid lacking tubercles (bearing two small rounded tubercles), low ulnar tubercles present (absent), and iris cream with brown flecks (orange with fine black reticulations).
Description of the holotype. An adult male (CORBIDI 15578) with a SVL of 21.9 mm, head as wide as long; snout subacuminate in dorsal view and truncated in lateral view, relatively short (eye-nostril distance 12% of SVL); canthus rostralis distinct in lateral view; loreal region concave; nostrils protuberant, directed anteriorly; interorbital area flat, broader than upper eyelid (upper eyelid width 59% of interorbital distance); cranial crests absent; upper eyelid lacking tubercles; tympanic membrane distinct, differentiated of surrounding skin; tympanic annulus distinct, round with weak supratympanic fold obscuring upper and posterodorsal edges of annulus (Fig. 2); tympanum diameter 31% of eye diameter; postrictal tubercles absent. Choanae small, rounded, not concealed by palatal shelf of maxillary; tongue longer than wide and granular. Skin texture on dorsum and flanks finely shagreen; dorsolateral folds absent; venter areolate; thoracic fold present, discoidal fold absent, cloacal sheath absent. Forearm slender; ulnar tubercles low, ulnar fold absent; radio-ulnar length 23% of SVL; fingers with narrow lateral fringes; relative lengths of fingers I ≤ II < IV < III; palmar tubercle bilobed, thenar tubercle oval (Fig. 3); subarticular tubercles round, prominent; supernumerary palmar tubercles present at the base of all fingers; disc cover finger I slightly expanded, those of fingers III and IV extensively expanded (Fig. 3), outer discs of fingers as wide as those of toes; discs covered with elliptical ventral pads defined by circummarginal grooves. Hind limbs slender; tibia length 50% of SVL; foot length 85% of tibia length; tarsal fold absent, tarsal tubercles low; heel lacking tubercles; toes with narrow lateral fringes; subarticular tubercles round, prominent; inner metatarsal tubercle oval, about 2.4 times the size of subconical outer tubercle; supernumerary plantar tubercles low at the base of all toes; discs covers slightly expanded; toes with defined pads; discs pads nearly elliptical; relative length of toes I < II < III < V < IV; tip of toe V reaching proximal border of distal subarticular tubercle IV; tip of toe II reaching distal border of medial subarticular tubercle of Toe IV.  of thighs are pinkish-gray with irregular red blotches. Iris golden with fine dark flecks. In daytime, yellowish-brown coloration turns into pale yellow.

Measurements and proportions of the holotype (in millimeters
Coloration in preservative. As described above, but yellowish-brown coloration turns creamy-yellow with tiny dark flecks on dorsum, limbs and ventral surfaces of hands and feet; red coloration turned pinkish-white, and venter creamy-yellow; iris gray. Advertisement call. A chorus of several males (CORBIDI 15563-68, 15577-78) was recorded at a distance of 1 meter from the microphone; thus the description refers to such context. The general structure of calls in this chorus (2'45'' recording) is that calls include a variable number of single-pulse notes (Fig. 5). It is possible, considering advertisement calls in similar Pristimantis species, that males emit simpler vocalizations, i.e., single notes separated by longer durations, outside of choruses. Males in the chorus produced calls with 3.3 ± 0.7 notes (range 2-5 notes). Note duration averaged 45.9 ± 12.6 ms (range 31-75 ms). Fundamental frequency averaged 2763 ± 133 ms (range 2531-3094 Hz) and did not vary within or among notes; likely the main source of variation in fundamental frequency was among individuals. Within single notes, much of the energy was concentrated in the first half of the note.
Variation. Measurements and proportions of the specimens examined are given in Table 1. Dorsal coloration pattern is paler in CORBIDI 15563-64, 15568 than the holotype. Specimen CORBIDI 15565 (Figure 4, D-F) has a minute dorsolateral yellow spot. Specimens CORBIDI 15563-66 have a darker yellow throat.
Etymology. The name is composed of two words in Latin, "pulcher" which means beautiful, and "dormientes" = sleeping, in reference to the chain of mountains located within Tingo María National Park, above the city of Tingo Maria, locally known as Sleeping Beauty (Bella Durmiente), because it looks like a sleeping reclined woman ( Figure 6A). Table 1. Measurements and morphological proportions of Pristimantis pulchridormientes sp. n. Range is followed by mean value and standard deviation in parenthesis (n = 9 adult males).

Discussion
The new species is not assigned to any taxonomic group despite the presence of morphological characters (i.e. head and body dorsoventrally compressed; skin on dorsum finely shagreen and that on venter areolate) suggesting a possible inclusion in the Pristimantis lacrimosus group. Group assignment is currently avoided because of the unclear taxonomic status of the P. lacrimosus group, as shown by recent phylogenetic studies (Padial et al. 2014;Ortega-Andrade et al. 2015). Further hindering group assignments among frogs of the genus Pristimantis are high species diversity (Hedges et al., 2008), small genetic distances (i.e. P. acuminatus complex; Ortega-Andrade et al. 2015), and limitations of morphological characters in defining species groups (Elmer and Cannatella 2008;Hedges et al. 2008;Padial and De la Riva 2009). Furthermore, phylogenetic relationships proposed by Hedges et al. (2008) for species in the putative P. lacrimosus group are not confirmed by recent studies, such as the strongly supported phylogeny published by Padial et al. (2014). In fact, Hedges et al. (2008) placed P. acuminatus outside of the P. lacrimosus group, but Padial et al. (2014) showed that this species is closely related to species of the putative P. lacrimosus group, evidencing the complicated taxonomy of this clade.
The analysis of genetic distances (uncorrected p-distances) shows that Pristimantis pulchridormientes sp. n. is closely related to both P. pluvialis and an undescribed species (Pristimantis sp.) from southeastern Peru (Table 2), which both show clear morphological differences with P. pulchridormientes (Shepack et al. 2016). In contrast, other species such as P. acuminatus, P. bromeliaceus, and P. omeviridis, show higher genetic distances exceeding 10%.
At both localities the new species was found in arboreal microhabitats, frequently calling perched on leaves 2 m above the ground. The only other species sharing a similar microhabitat and presumably ecological niche (also nocturnal) was P. aff. mendax, but this frog was not found in sympatry with P. pulchridormientes, because its altitudinal range of 100-900 m asl does not overlap with the range of P. pulchridormientes from 1095-1700 m asl.
With only two known localities, it is difficult to predict the potential distribution range of this species. Although the type locality had a large population and was located inside a protected area, the locality where paratype CORBIDI 16606 was collected had a very fragmented habitat surrounded by orange plantations and corn cropland. Considering that the upper Huallaga drainage is highly disturbed by agriculture activities which fragments the submontane and montane forests (Catenazzi and von May 2014), the distribution of P. pulchridormientes is considered to be highly fragmented with the only known protected population living on an isolated slope of Tingo Maria NP. On the basis of our limited knowledge of its distribution, and according to the IUCN Red List guidelines (2016) we recommend the species be placed in the Data Deficient category of the IUCN Red List of Threatened species.