A new species of Phrynopus (Amphibia, Anura, Craugastoridae) from upper montane forests and high Andean grasslands of the Pui Pui Protected Forest in central Peru

Abstract We describe a new species of Phrynopus from the upper montane forests and high Andean grasslands (puna) of the Pui Pui Protected Forest and its close surroundings (Región Junín, central Peru) and compare it morphologically and genetically with other species of Phrynopus. Phrynopus inti sp. n. is known from four localities outside and two localities inside the Pui Pui Protected Forest between 3350 and 3890 m a.s.l. Studied specimens of the new species are characterized by a snout-vent length of 27.2–35.2 mm in males (n = 6), and 40.4 mm in a single female, by having the skin on dorsum and flanks smooth with scattered tubercles, venter smooth, by lacking a tympanum, and males without vocal slits and nuptial pads. In life, the dorsum is pale grayish brown with or without dark brown blotches, or dorsum blackish brown with small yellow flecks, throat, chest and venter are pale grayish brown with salmon mottling, groin is pale grayish brown with salmon colored flecks, and the iris is golden orange with fine dark brown reticulations. The new species is morphologically most similar to Phrynopus kauneorum and P. juninensis. For the latter we describe the coloration in life for a specimen obtained at the type locality. A molecular phylogenetic analysis based on mitochondrial and nuclear DNA sequences inferred that the new species is most closely related to Phrynopus kauneorum, P. miroslawae, P. tautzorum, and an undescribed species distributed at high elevation in Región Pasco, central Peru.


Introduction
The Pui Pui Protected Forest (Bosque de Protección Pui Pui, hereafter PPPF; Figs 1, 2) is located in the Selva Central of Peru and covers 60,000 hectares (30% montane forest, 70% puna habitats) between 1700 and 4500 m a.s.l. (SERNANP 2010). We surveyed the herpetofauna of the PPPF in upper montane forests and high Andean grasslands (puna) between 2012 and 2013 in order to document the amphibian and reptile species richness and to evaluate their conservation status. Among the new amphibians were five new species of Pristimantis (Craugastoridae Hedges, Duellman, and Heinicke, 2008) (P. ashaninka ; P. attenboroughi P. bounides Lehr, von May, Moravec, & Cusi, 2017;P. humboldti Lehr, von May, Moravec, & Cusi, 2017;and P. puipui Lehr, von May, Moravec, & Cusi, 2017) and a new species of Phrynopus Peters, 1873. A phylogenetic analysis allowed us to examine the relationships among species of Phrynopus, including the new species, and to justify our generic assignment. Phrynopus currently contains 34 species (AmphibiaWeb 2017) distributed in montane forests and puna habitats between 2600 and 4490 m a.s.l. in northern and central Peru (Rodríguez andCatenazzi 2017, Duellman andLehr 2009). Herein we name and describe this new species of Phrynopus, supported by morphological and phylogenetic evidence, from upper montane forests and puna habitats.

Materials and methods
Fieldwork. The puna of the PPPF was reached by walking 1.5 days along a trail from Toldopama (11°30'15.4"S, 74°55'32.7"W, 3670 m a.s.l., two hours by car from Satipo) to Tarhuish (11°23'23.2"S; 74°57'02.5"W, 3783 m a.s.l.; Fig. 2) with the help of local guides. Fieldwork was conducted in puna and upper montane forests in 2012 between May 8 and 21 by EL and RvM, and in 2013 between June 21 and July 8 by EL, JM, and JCC. Collected specimens were preserved in 96% ethanol and stored in 70% ethanol.
Morphological characters. The format for the description follows Lynch and Duellman (1997), except that the term dentigerous processes of vomers is used instead of vomerine odontophores (Duellman et al. 2006), and diagnostic characters are those of Duellman and Lehr (2009). Taxonomic classification follows Hedges et al. (2008), except that we followed Pyron and Wiens (2011) for family placement. Sex and maturity of specimens were identified by observing gonads through dissections. The senior author measured the following variables to the nearest 0.1 mm with digital calipers under a stereomicroscope: snout-vent length (SVL), tibia length (TL, distance from the knee to the distal end of the tibia), foot length (FL, distance from proximal margin of inner metatarsal tubercle to tip of Toe IV), head length (HL, from angle of jaw to tip of snout), head width (HW, at level of angle of jaw), horizontal eye diameter (ED), interorbital distance (IOD), upper eyelid width (EW), internarial distance (IND), eye-nostril distance (E-N, straight line distance between anterior corner of orbit and posterior margin of external nares), and egg diameter. Fingers and toes are numbered preaxially to postaxially from I-IV and I-V, respectively. We compared the lengths of toes III and V by adpressing both toes against Toe IV; lengths of fingers I and II were compared by adpressing these fingers against each other. All drawings were made using a stereomicroscope and a camera lucida. Photographs of live specimens were used for descriptions of coloration in life and for evaluation of morphological characters that might have been impacted by the preservation process. Information on species for comparative diagnoses was obtained from Duellman and Lehr (2009) and from original species descriptions. For specimens examined see Appendix I. Codes of collections are: MUSM -Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru; NMP6V -National Museum, Prague, Czech Republic; UMMZ -University of Michigan Museum of Zoology, Ann Arbor, USA. Field number code is: IWU -Illinois Wesleyan University, Bloomington, USA. Threat status was evaluated using the IUCN criteria (IUCN Standards an Petitions Subcommittee 2016).
Maps. Maps were made with ArcGIS 10.0 (ESRI 2011). The estimated area was calculated by a minimum convex polygon using known sites of occurrence of the species as defined by IUCN (2012).
Molecular phylogenetic analysis. Our analysis included DNA sequence data from Phrynopus species that were available in GenBank (as of 1 August 2017; Table 1) as well as sequences from other closely related genera (Lynchius, Oreobates) and more distantly related ones (Ischnocnema guentheri, Hypodactylus brunneus, and H. dolops) as outgroups following the results of Padial et al. (2014). Newly produced sequences include those obtained from seven specimens of the new species and one specimen of Phrynopus juninensis collected near Hacienda Cascas, Junín, the type locality of this species (Table 1). Our analysis also included sequences from three mitochondrial and two nuclear genes for several species of Phrynopus included in a recent study (De la Riva et al. 2017). The mitochondrial genes were a section of the 16S rRNA gene, a section of the 12S rRNA gene, and the protein-coding gene cytochrome c oxidase subunit I (COI). The nuclear genes were the recombination-activating protein 1 (RAG1) and Tyrosinase precursor (Tyr).
Extraction, amplification, and sequencing of DNA followed protocols previously used for Neotropical terrestrial breeding frogs (Lehr et al. 2005, Hedges et al. 2008. Primers used are listed in Appendix II. We employed the following thermocycling conditions to amplify DNA from each gene using the polymerase chain reaction (PCR). For 16S, we used: 1 cycle of 96 °C/3 min; 35 cycles of 95 °C/30 s, 55 °C/45 , and obtained sequence data by running the purified reaction products in an ABI 3730 Sequence Analyzer (Applied Biosystems). We deposited the newly obtained sequences in GenBank (Table 1). We used Geneious R6, version 6.1.8 (Biomatters 2013; http://www.geneious. com/) to align the sequences with the built-in multiple alignment program. Prior to conducting phylogenetic analysis, we used PartitionFinder, version 1.1.1 (Lanfear et al. 2012) to select the appropriate models of nucleotide evolution and used the Bayesian information criterion (BIC) to determine the best partitioning scheme and substitution model for each gene. The best partitioning scheme included five subsets (BIC value: 27719.16). The first partition subset included both the 12S and 16S sequences and the best fitting substitution model was GTR+I+G. The remaining four subsets were partitioned according to codon positions as follows (substitution model in parenthesis): one set including the 1 st codon position of COI and the 3 rd codon position of both RAG1 and Tyr (K80+G); one set with only the 2 nd codon position of COI (HKY); one set with only the 3 rd codon position of COI (HKY); one set including the 1 st and 2 nd codon position of RAG1 and the 1 st and 2 nd codon position of Tyr (HKY+I).
We employed a Bayesian approach using MrBayes, version 3.2.0 (Ronquist and Huelsenbeck 2003) to infer a molecular phylogeny. Our analysis included 44 terminals and a 2684-bp concatenated partitioned dataset. We performed an MCMC Bayesian analysis that consisted of two simultaneous runs of 8 million generations, and we set the sampling rate to be once every 1000 generations. Each run had three heated chains and one "cold" chain, and the burn-in was set to discard the first 25% samples from the cold chain. At the end of the run, the average standard deviation of split frequencies was 0.002257. Following the completion of the analysis, we used Tracer 1.6 (Rambaut and Drummond 2003) to verify convergence. Subsequently, we used FigTree (http:// tree.bio.ed.ac.uk/software/figtree/) to visualize the majority-rule consensus tree and the posterior probability values to assess node support. Additionally, we used the R package 'APE' (Paradis et al. 2004) to estimate uncorrected p-distances (i.e., the proportion of nucleotide sites at which any two sequences are different).

Molecular phylogenetic analysis.
Placement of the new species in the genus Phrynopus was strongly supported by this analysis. We recovered a well-supported tree (Figure 3) that was generally congruent with previous trees (Padial et al. 2014) and sup-  Lehr & Aguilar, 2002, and an undescribed species. Based on the available data, the new species is most closely related to an undescribed species of Phrynopus distributed at high elevation (3600-3850 m a.s.l., Lehr et al. 2005) in Región Pasco. This newly identified cryptic species was previously recognized as P. juninensis Shreve, 1938 given their similar morphology and coloration (Lehr et al. 2005, Padial et al. 2014) and will be formally named and described in a future paper. The uncorrected p-distances between the new species and all other species of Phrynopus ranged between 4.5 and 14.1% (Table 2). The shortest distance occurs between the new species and the undescribed species (uncorrected p-distance 1.5-2.8%) while the uncorrected p-distances between P. kauneorum and the new species vary between 3.7 to 4.8% (Table 2). Our analysis also suggests that P. nicoleae Chaparro, Padial, & De la Riva, 2008 and P. tribulosus Duellman & Hedges, 2008 might represent one species.   Generic placement. We assign this species to Phrynopus based on molecular evidence (Fig. 3).
Diagnosis. A species of Phrynopus having the following combination of characters: (1) Skin on dorsum and flanks shagreen with scattered, low tubercles, more dense on dorsum; skin on venter smooth; discoidal fold absent, thoracic fold present; prominent supratympanic fold; dorsolateral folds absent; (2) tympanic membrane and tympanic annulus absent; (3) snout rounded in dorsal and lateral views; (4) upper eyelid without enlarged tubercles; width of upper eyelid narrower than IOD; cranial crests absent; (5) dentigerous processes of vomers minute or absent; (6) vocal slits and nuptial pads absent; (7) Finger I shorter than Finger II; tips of digits bulbous, rounded; (8) fingers without lateral fringes; (9) ulnar and tarsal tubercles absent; (10) heel without tubercles; inner tarsal fold absent; (11) inner metatarsal tubercle rounded, about three times as large as ovoid outer metatarsal tubercle; supernumerary plantar tubercles absent; (12) toes without lateral fringes; basal webbing absent; Toe V slightly longer than Toe III; toe tips bulbous, rounded, about as large as those on fingers; (13) in life, dorsum pale grayish brown with or without dark brown blotches or blackish brown with small yellow flecks; throat, chest and venter pale grayish brown with salmon mottling, groin pale grayish brown with salmon colored flecks; iris golden orange with fine dark brown reticulations; (14) SVL 27.2-35.2 mm in males (n = 6), and 40.4 mm in single female.
Comparisons. Phrynopus inti sp. n. is readily distinguished from its 34 congeners in Peru (AmphibiaWeb 2017), by its relatively large SVL (except for P. juninensis and P. kauneorum) of up to 40.4 mm, by having the groin pale grayish brown with salmon colored flecks, the venter pale grayish brown with salmon mottling and the iris golden orange with fine dark brown reticulations. Phrynopus inti sp. n. is most similar to the large central Peruvian species P. juninensis (SVL up to 43.1 mm, Duellman andLehr 2009) andP. kauneorum (SVL up to 56.4 mm, Lehr et al. 2002b), Fig. 4. All three species share a gray ground coloration and dark brown canthal and supratympanic stripes, lack dorsolateral folds and males lack vocal slits and nuptial pads, but can be distinguished as follows: Phrynopus inti sp. n. has weak postocular folds (absent in both P. juninensis and P. kauneorum), has dentigerous processes of vomers (absent in P. juninensis, present in P. kauneorum), skin on dorsum shagreen with scattered, low tubercles (smooth to weakly areolate in P. juninensis, smooth in P. kauneorum), skin on venter smooth (areolate in P. juninensis, smooth in P. kauneorum), dorsum pale grayish brown with or without dark brown blotches or blackish brown with small yellow flecks (dorsum grayish brown with dark brown markings in P. juninensis, dorsum pale brown to tan with dark brown markings in P. kauneorum), venter pale grayish brown with salmon mottling (pale brown with gray blotches in P. juninensis, pinkish to grayish tan in P. kauneorum), and the iris is golden orange (copper in P. juninensis, dark brown in P. kauneorum).
Description of the holotype. Head as wide as body, wider than long, HW 110% of HL; HW 38% of SVL; HL 35% of SVL; snout short, rounded in dorsal and lateral views (Figs 5A, B), ED larger than E-N distance (ED 148% of E-N); nostrils protuberant, directed dorsolaterally; canthus rostralis slightly curved in dorsal view, rounded in profile; loreal region slightly concave; lips rounded; upper eyelid without enlarged tubercles; EW slightly narrower than IOD (EW 94% of IOD); postocular folds low, extending from posterior margin of upper eyelid to level of upper arm insertion (Fig.  5B); supratympanic fold broad, extending from posterior corner of eye to level of upper arm insertion; tympanic membrane and tympanic annulus absent, tympanic region without postrictal tubercles. Choanae small, ovoid, close to but not concealed by palatal shelf of maxilla; dentigerous processes of vomers minute, embedded in mucosa of mouth, widely separated; tongue broad, about twice as long as wide, not notched posteriorly, posterior half free; vocal slits absent.
Skin on dorsum shagreen with scattered, low tubercles, more dense on posterior half of body, dorsolateral folds absent (Fig. 5B); skin on flanks shagreen with few scattered, low tubercles; skin on throat, chest and belly smooth (Fig. 5D); discoidal fold absent, thoracic fold present; cloacal sheath not distinct; cloacal region without tubercles. Outer surface of forearm without tubercles; outer palmar tubercle barely visible, low, ovoid, slightly smaller than ovoid inner palmar tubercle; supernumerary tubercles absent; subarticular tubercles low, ovoid, most prominent on base of fingers; fingers without lateral fringes; Finger I shorter than Finger II; tips of digits rounded, bulbous, lacking circumferential grooves; nuptial pads absent (Fig. 6A).
Hind limbs long and slender, TL 39% of SVL; FL 43% of SVL; dorsal surface of hind limbs shagreen with few low tubercles; anterior surfaces of thighs shagreen, posterior surfaces of thighs weakly areolate; heel without a conical tubercle; outer surface of tarsus without tubercles; outer metatarsal tubercle rounded, weakly conical, about four times as large as prominent ovoid inner metatarsal tubercle; supernumerary plantar tubercles absent; subarticular tubercles low, ovoid in dorsal view, most distinct on base of toes; toes without lateral fringes; basal webbing absent; toe tips bulbous, rounded, lacking circumferential grooves, about as large as those on fingers; relative lengths of toes: 1 < 2 < 3 < 5 < 4; Toe V slightly longer than Toe III (Fig. 6B).
Coloration of the holotype in life (Fig. 5). Dorsum pale grayish brown with dark brown blotches, a dark brown X-shaped marking on shoulder region and an irregular shaped dark brown interorbital blotch. Flanks paler than dorsum with few pale brown flecks. Canthal and supratympanic stripes dark brown. Upper lip with few pale brown flecks. Arms and legs dorsally with few pale and dark brown blotches and flecks. Throat, chest and venter pale grayish brown with salmon mottling, denser on posterior half of belly and thighs. Groin, posterior surfaces of thighs, posterior surfaces of tibias and dorsal surfaces of feet vibrant salmon colored. Iris golden orange with fine dark brown reticulations.  Coloration of the holotype in preservative. Dorsum tan with dark brown blotches and dark brown X-shaped marking on shoulder region and an irregular shaped dark brown interorbital blotch. Flanks paler than dorsum, with few pale brown flecks. Canthal and supratympanic stripes dark brown. Upper lip with few pale brown flecks. Arms and legs dorsally tan with few pale and dark brown blotches and flecks. Groin creamy white. Throat, chest and venter creamy white and pale gray mottled. Ventral surfaces of hand and feet creamy white. Iris pale gray.
Distribution, natural history, and threat status. Phrynopus inti sp. n. is known from four localities outside and two localities inside the Pui Pui Protected Forest between 3350 and 3890 m a.s.l., covering an estimated area of 101.3 km 2 (Figs 1, 2).
The type locality, Quebrada Tasta (Fig. 2), is outside the PPPF. The holotype and three paratypes (MUSM 31184, UMMZ 245218, 245219) were found in the afternoon in a forest patch under rocks near the house of Evaristo Bórquez Quintana, on 9 May   2012 at 3609 m a.s.l. (Fig. 10C). The vegetation at the type locality consists of Polylepis trees, small bushes, ferns, moss, and Peruvian feather grass. No sympatric anurans were recorded. One specimen (MUSM 31203) was found in the afternoon under moss in a Polylepis forest patch near the trail from Tasta to Tarhuish at 3886 m a.s.l. Three specimens (MUSM 31968, 31969, UMMZ 245220) were collected in the morning under rocks and in moss in the mountain slopes of the Toldopampa valley close to Toldopampa at 3670 m a.s.l. (Fig. 10B). Specimens were found under rocks and in moss. Sympatric anurans include Gastrotheca griswoldi Shreve, 1941. One specimen (NMP6V 75584) was collected under moss in the early afternoon at the Satipo-Toldopampa Road at km 134 on the left side of the road coming from Satipo at 3350 m a.s.l. (Fig. 10A). Sympatric anurans here include Pristimantis bounides (MUSM 31970, 31971) and Gastrotheca griswoldi (MUSM 31972). Three specimens (MUSM 31974, 31976, NMP6V 75585) were found under rocks and in moss in Antuyo at 3700 m a.s.l. (Fig. 10D). Sympatric anurans here include Pristimantis attenboroughi (MUSM 31975) and Gastrotheca griswoldi (IWU 290). Four specimens (MUSM 31984, 31985, NMP6V 75586, 75587) were found in the puna in the afternoon in moss close to the Laguna Sinchon at 3890 m a.s.l. (Fig. 10E). Sympatric anurans here include Pristimantis puipui (MSUM 31981-83).
One male specimen (MUSM 31203) had as ectoparasites five trombiculid mites on the right side in the area of the upper arm insertion. Such parasites are not uncommon in Andean frogs (e.g., Quinzio andGoldberg 2015, Lehr et al. 2017).
The IUCN Red List criteria (IUCN 2001) consider that if a species occurs in fewer than 10 threat-defined locations and the extent of occurrence (EOO) is < 20,000 km 2 , it should be classified as Vulnerable or Endangered. Phrynopus inti sp. n. is known from six localities distributed in the PPPF and its buffer zone (Fig. 10), with an estimated EOO of 101.3 km 2 . As such, this new species might be classified as Vulnerable if we take into account these criteria. However, given that the PPPF may host a greater number of locations (two of them are inside the protected area), we propose that Phrynopus inti sp. n. should likely be categorized as Near Threatened (NT). Despite that two locations of the known distribution of Phrynopus inti sp. n. are within the PPPF (Fig. 10) and formally protected, other factors such as fungal infections, climate change, pollution, and manmade fires (used to expand grazing areas for livestock) continue to be threats for many Andean amphibians even inside protected areas (Catenazzi and von May 2014). Agriculture and cattle raising are more acute in the Toldopampa valley than in the Tasta valley.

Discussion
With a snout-vent length of up to 40.4 mm, Phrynopus inti sp. n. represents one of the largest species of the genus. Usually, Phrynopus species are characterized by a small robust body, short limbs, narrow or only slightly expanded tips of toes and fingers, and absence of a tympanum. These morphological features seem to be associated with a life in moss layers and grass bunches at elevations between 2600 and 4400 m a.s.l. (Rodríguez andCatenazzi 2017, Duellman andLehr 2009). In the PPPF, however, this niche is widely occupied by several small Pristimatis species (Pristimantis attenboroughi, P. bounides, P. humboldti, and P. puipui), all of which exhibit a similar body form and lifestyle as most species of Phrynopus. In particular, two species of Pristimantis in the PPPF, P. attenboroughi, P. puipui, appear to have adapted to similar niches in upper montane forests and puna that are typically occupied by species of Phrynopus with small robust bodies, short limbs, and discs without circumferential groves. Ad-ditionally, like in most species of Phrynopus, both P. attenboroughi and P. puipui lack a tympanum. The use of genetic characters in such cases of convergence is necessary to determine the proper generic placement and phylogenetic relationships . The inclusion of P. juninensis from its type locality in our phylogeny (Fig. 2) revealed the existence of a cryptic species (Phrynopus sp.) that was previously thought to be Phrynopus juninensis. This new species, which is found in an area located >50 km away from the type locality of P. juninensis, will be described in the near future.
Our phylogenetic analysis suggested that Phrynopus nicoleae, Chaparro, Padial & De la Riva, 2008 is a junior synonym of Phrynopus tribulosus Duellman & Hedges, 2008. The high genetic similarity between P. nicoleae and P. tribulosus was originally identified by De la Riva et al. (2017), who suggested a possible synonymy, but no formal taxonomic action was proposed. Additionally, new evidence suggests that one other species (not included in the tree presented here) is also genetically similar to both P. nicoleae and P. tribulosus (von May, unpublished). The synonymy among these three species will be discussed in more detail in an upcoming paper.
De la Riva et al. (2017) pointed out an underestimated radiation of craugastorid frogs in the Eastern Andes of Peru and Bolivia and described five new species and a new genus (Microkayla). Ten years earlier, De la Riva (2007) described 12 new species from Bolivia and new amphibians are discovered in similar quantities from Andean Peru. The Andes are indeed a hotspot for biodiversity (Myers et al. 2000); five of the six anuran species recorded by us in upper montane and puna habitats of the PPPF represented new species (see  and this paper), and descriptions of other new anuran as well as reptile taxa are expected. Herpetological surveys conducted by us between 2012 and 2014 demonstrate that the PPPF houses unique amphibian assemblages associated with cloud forest and puna habitats. Therefore, the protection of the PPPF and its native flora and fauna in central Andean Peru is of great importance. The beneficial role of any protected area stands out in light of ongoing habitat loss caused by development and land use changes in neighboring areas including the buffer zone surrounding the PPPF.
a Northern European Explorers Grant (GEFNE13-11) from National Geographic Society Science and Exploration Europe. Illinois Wesleyan University provided a Junior Faculty Leave in 2012. RvM thanks the National Science Foundation Postdoctoral Research Fellowship in Biology (DBI-1103087) and the National Geographic Society Committee for Research and Exploration (Grant # 9191-12). The work of JM was financially supported by the Ministry of Culture of the Czech Republic (DKRVO 2013/14, 2015/15, 2016/15 and 2017. Collecting permits (N° 001-2012-SERNANP-JEF, N°-0120-2012-AG-DGFFS-DGEFFS, N°-064-2013-AG-DGFFS-DGEFFS, R.D._N°_359-2013-MINAGRI-DGFFS-DGEFFS) and export permits were issued by the Ministerio del Ambiente, Lima, Peru. We thank the University of Michigan Museum of Zoology (UMMZ) for providing funds to cover the publication costs and G. Schneider (UMMZ) for providing museum numbers.