A new minute species of Pristimantis ( Amphibia : Anura : Craugastoridae ) with a large head from the Yanachaga-Chemillén National Park in central Peru , with comments on the phylogenetic diversity of Pristimantis occurring in the Cordillera Yanachaga

We describe a new minute species of the genus Pristimantis, P. boucephalus sp. nov., from the Yanachaga-Chemillén National Park, Región Pasco, Peru. The description is based on a freshly collected male specimen found at 2950 m a.s.l. in a cloud forest and four previously unidentifi ed museum specimens consisting of two adult males, one subadult female and a juvenile from the Yanachaga-Chemillén National Park. The new species is mainly characterized by a snout–vent length of 13.4–14.5 mm in adult males (n = 3), and 12.5 mm in the only known subadult female, and is compared morphologically and genetically with other taxonomically and biogeographically relevant species of Pristimantis. The new species is characterized by its small size, disproportionally large head with short snout, absence of a tympanic annulus and membrane, and reddish-copper iris. Phylogenetically it belongs to a speciose clade, an as yet unnamed species group, comprising both montane (Andes, Guiana Shield) and lowland (Amazon) taxa from the northern part of South America. The new species is genetically close to the sympatric P. cruciocularis. Species of Pristimantis occurring in the Cordillera Yanachaga region in the Andes of central Peru are members of six divergent phylogenetic lineages.


Introduction
In the area of the Selva Central of Peru (Regions of Pasco and Junín, central Peru), there are twelve natural areas with different levels of legal protection such as national parks, national sanctuaries, national reserves and others of lower protection categories (SERNANP 2010). In 2012 we started surveying the herpetofauna of the Yanachaga-Chemillén National Park (hereafter YCNP; Región Pasco, Fig. 1), with the aim to obtain a better knowledge of the anuran species richness of the YCNP and to provide data needed for an effective protection of the park. The expeditions in the YCNP led to the discovery of several new species of amphibians (e.g., Phrynopus badius Lehr, Moravec & Cusi, 2012, Phrynopus curator Lehr, Moravec & Cusi, 2012 and Rhinella yunga Moravec, Lehr, Cusi, Córdova & Gvoždík, 2014). Among the new fi ndings was an undescribed species of Pristimantis Jiménez de la Espada, 1870 readily distinguishable from its local congeners by its small size with a disproportionally large head, short snout, and reddish-copper iris. However, because only a single male specimen was available at that time, we refrained from formally describing it. Revision of specimens from the YCNP housed in the herpetological collection of the Museo de Historia Natural Universidad Nacional Mayor de San Marcos (Lima, Peru) in 2015 led to the discovery of four additional specimens (labeled Pristimantis sp.) of the same taxon. Close examination of the fi ve specimens revealed that they bear several morphological characteristics not shared by any other described species of Pristimantis in Peru. Therefore, we describe the new species and compare it morphologically and genetically with other taxonomically and biogeographically relevant species of Pristimantis from Bolivia, Ecuador, Peru, the Guyanas, and Venezuela. A phylogenetic analysis provided additional insights into the phylogenetic diversity of Pristimantis from the area of the Cordillera Yanachaga.

Morphological characters
The format for the description follows Lynch & Duellman (1997), and diagnostic characters are those of Duellman & Lehr (2009). Taxonomic classifi cation follows Hedges et al. (2008) and Heinicke et al. (2009), except that we followed Pyron & Wiens (2011) for family placement and Padial et al. (2014) for names of Pristimantis species groups. We consider minute species of amphibians as having an adult snout-vent length (SVL) below 20 mm, as suggested by Clarke (1996). The holotype was fi xed in 96% ethanol and stored in 70% ethanol. Liver tissue of the holotype was taken for genetic analyses. Sex and maturity of specimens were identifi ed by observing secondary sexual characters (vocal slits), and gonads through dissections. Specimens with a SVL ≤ 10 mm were considered juveniles when gonads were too small to distinguish between sexes. We used maximum known SVL for males within a species to recognize smallest body size as recommended by Lehr & Coloma (2008). We measured the following variables to the nearest 0.1 mm with digital calipers under a stereo microscope: snout-vent length (SVL, straight length distance from tip of snout to vent), 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). Fingers and toes are numbered preaxially to postaxially as 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 the fi ngers against each other. To avoid refl ection, the preserved holotype was photographed immersed in ethanol. All drawings were made by JM using a stereo microscope and a camera lucida. Photographs taken by JM and EL were used for descriptions of coloration in life. Comparisons of congeners focused on species with similar SVL from Ecuador and Peru and genetically close species as recovered in our trees. Information on species for comparative diagnoses was obtained from Duellman & Lehr (2009)

Taxon sampling
We included samples of various Pristimantis species collected by us in the YCNP during a survey in 2012, and from the nearby Bosque de Shollet Protected Area (BS) and Pui Pui Protected Forest (PPPF) collected between 2012 and 2014. The aim was to detect phylogenetic diversity of Pristimantis species distributed in these and nearby montane regions of the Cordillera Yanachaga. Most of the specimens were identifi ed to species level, with the exception of the single 'bigheaded' male (mentioned above) and a few specimens from the Pui Pui forest, which are tentatively named Pristimantis sp. Pui Pui. A list of the newly genetically investigated material and its GenBank accession numbers is in Table 1. For the fi nal dataset, we retrieved additional sequences conspecifi c with our samples from GenBank to show phylogenetic positions of our new material in relation to DNA sequences published earlier (most importantly in the review by Hedges et al. 2008). We also retrieved and included species known to occur in the Cordillera Yanachaga region (Duellman & Hedges 2005, 2007 but not recorded by us (P. albertus Duellman & Hedges, 2007, P. minutulus Duellman & Hedges, 2007 (Duellman & Hedges, 2005), P. sagittulus (Lehr, Aguilar & Duellman, 2004)). In addition, we specifi cally focused on the 'bigheaded' specimen and applied a BLAST search (Altschul et al. 1990) using the BLASTN v. 2.3.1 program (Zhang et al. 2000;Morgulis et al. 2008) for the most similar DNA sequences available in GenBank based on the 16S rRNA gene fragment (see below). We selected and included into our dataset sequences of species or main evolutionary lineages of species complexes (e.g., the P. platydactylus complex) with nucleotide sequence identity in relation to our 'bigheaded' specimen ≥ 90%, and which formed a clade in the BLAST minimum-evolution tree. We also included species from the sister clade, which also contained one species known from the YCNP, P. minutulus. As outgroups, we used the craugastorid genera Oreobates Jiménez de la Espada, 1872 (O. cruralis (Boulenger, 1902)) and Phrynopus Peters, 1873 (P. bracki Hedges, 1990), retrieved from GenBank. The fi nal dataset was composed of 87 samples of 37 nominal taxa, including the new species and outgroups. All sequences acquired from GenBank can be identifi ed by the GenBank accession numbers as given in Fig. 2.

DNA extraction, PCR, sequencing and sequence alignment
Genomic DNA was extracted from tissues stored in 96% ethanol. A fragment of the mitochondrial gene for 16S rRNA (16S), which is commonly used in amphibian DNA barcoding (Vences et al. 2012), was targeted using the primers 16SL1 and 16SH1 adapted or directly taken from Palumbi et al. (1991). For primer sequences and PCR conditions see Moravec et al. (2009). Sequencing was performed by using the PCR primers. Additionally, a newly designed internal primer (prist16SinR1: 5'-ATGTTTTAAGCTCCA-3') had to be used for the 'bigheaded' Pristimantis to overcome a problem with a G-C homopolymer region in the middle of the fragment. New sequences have been deposited in GenBank (KY006082-006112). The multiple sequence alignment was performed using MAFFT v. 7.1 (Katoh & Standley 2013), producing a 561 bp long alignment. Ambiguously aligned positions were eliminated by Gblocks v. 0.91b under options for a less stringent selection (Castresana 2000), producing a fi nal alignment of 510 bp.

Phylogenetic analysis
The Bayesian inference (BI) was applied to construct a phylogenetic tree. First, the software jModelTest v. 2.1.7 (Darriba et al. 2012) using the PhyML algorithm (Guindon & Gascuel 2003) was used to fi nd the best-fi tting model of nucleotide evolution, which was the GTR+I+G model based on both the Akaike and Bayesian information criteria. The BI analysis was run in MrBayes v. 3.2.5 (Ronquist et al. 2012), with two runs and four chains in each run for 6 × 10 6 generations, sampling every 100 th generation. Appropriate sampling was controlled by examining the stationarity of log-likelihood scores against the generation time using Tracer v. 1.6 (Rambaut et al. 2013; all parameters had effective sample size > 1000), and convergence between the two simultaneous runs was confi rmed by the convergence diagnostics of the average standard deviation of split frequencies and the potential scale reduction factor values. From the sampled trees, 25% were discarded as a burn-in and a 50% majority-rule consensus tree was produced from the remaining post burn-in trees. The posterior probabilities (pp) were calculated as the frequency of samples recovering any particular clade. Clades supported with pp values ≥ 0.95 were considered highly supported (Huelsenbeck & Rannala 2004). Genetic uncorrected p-distances were calculated in PAUP* (Swofford 2003).

Molecular analysis
Our morphology-based species identifi cations of taxa recorded in the Cordillera Yanachaga were supported by 16S DNA barcodes as they were identical or closely related to haplotypes available in GenBank (Pristimantis aniptopalmatus (Duellman & Hedges, 2005), P. bipunctatus (Duellman & Hedges, 2005), P. cruciocularis , P. cf. mendax (Duellman, 1978), P. rhabdocnemus (Duellman & Hedges, 2005)). The inferred phylogenetic tree ( Fig. 2) showed that species of Pristimantis occurring in the Cordillera Yanachaga and nearby regions belonged to at least six deeply divergent lineages (marked as species groups in Fig. 2). Five species (including samples retrieved from GenBank) clustered together forming a well-supported lineage: P. albertus, P. aniptopalmatus, P. ornatus ) (from a site near the Cordillera Yanachaga), P. sagittulus, and P. stictogaster. We also uncovered one distinct lineage within 'P. aniptopalmatus' (3.8% average genetic uncorrected p-distance to other 'conspecifi cs'), which we tentatively name P. cf. aniptopalmatus. Pristimantis bipunctatus, P. cf. mendax, P. minutulus (taken from GenBank), and P. rhabdocnemus are members of four other divergent lineages. The 'bigheaded' Pristimantis from the YCNP is, together with P. sp. Pui Pui from the geographically close PPPF and P. cruciocularis from both the YCNP and PPPF, a member of another lineage. This lineage represents a speciose clade, which comprises montane species from the Andes and the Guiana Shield and also lowland species from the intervening Amazon lowlands. The topology within this clade is not resolved, but the clade is highly supported (pp = 0.98). Among the sympatric species, the genetically closest of the 'bigheaded' Pristimantis is P. cruciocularis, with a genetic uncorrected p-distance of 5.8%. The genetic uncorrected p-distances among the main lineages range between 13 and 17%.  Nodes were collapsed if they appeared in less than 50% of the post burn-in tree samples. Support values of posterior probabilities (pp) given only for discussed lineages (stars indicate full support, pp = 1.00). Taxa in red occur in the Cordillera Yanachaga region and represent six divergent lineages/species groups (uncorrected p-distance 13-17%). The new species, P. boucephalus sp. nov., is a member of a speciose clade, an as yet unnamed species group, comprising both montane taxa from the Andes and Guiana Shield, and lowland taxa from the intervening Amazon Basin. Species-group names follow Padial et al. (2014). DNA sequences of taxa retrieved from GenBank are labelled by standard GenBank accession numbers, all other codes stand for our new material.

Etymology
The species epithet boucephalus is derived from the Greek prefi x "bou-" meaning large, huge, or great, and the Greek noun "cephalo" meaning head. The name refers to the disproportionally large head of the new species.

Generic placement
We assign this species to Pristimantis based on its general morphological similarity to other members of the genus and our molecular data (Fig. 2).

Defi nition
A new species of Pristimantis, not assigned to any species group, having the following combination of characters: (1) Skin on dorsum smooth with few low scattered tubercles, skin on venter areolate with low scattered tubercles; discoidal and thoracic folds absent; dorsolateral folds absent; (2) tympanic membrane and tympanic annulus absent; (3) snout short, rounded in dorsal and lateral views; (4) upper eyelid with one enlarged conical tubercle at its center and one enlarged conical tubercle at its posterior end; EW slightly shorter than IOD; cranial crests absent; (5) dentigerous processes of vomers absent; (6) males with vocal slits, nuptial pads absent; (7) Finger I shorter than Finger II; discs of digits expanded, rounded; (8) fi ngers with lateral fringes; (9) small conical ulnar and tarsal tubercles present; (10) heel with a conical tubercle; inner tarsal fold absent; (11) inner metatarsal tubercle ovoid, 2.5 times as large as outer; outer metatarsal tubercle small, rounded; numerous low, supernumerary plantar tubercles; (12) toes with lateral fringes; basal toe webbing present; Toe V longer than Toe III; toe discs slightly smaller than those on fi ngers; (13) in life, dorsal ground coloration greenish gray with reddishbrown blotches and scattered brown fl ecks surrounded by black; canthal and supratympanic stripes absent; groin and anterior surfaces of thighs greenish yellow with black blotches; venter gray with pale reddish and greenish brown mottling and scattered dark gray fl ecks; iris reddish copper with fi ne black vermiculation and black narrow vertical streak from pupil across lower half of iris; (14) SVL in adult males 13.4-14.5 mm (n = 3), in adult females unknown (12.5 mm in single subadult female).

Differential diagnosis
Pristimantis boucephalus sp. nov. is readily distinguished from its congeners in Ecuador and Peru by its minute SVL, short snout, absence of a tympanum, large head, groin and anterior surfaces of thighs greenish yellow with black blotches, and reddish-copper iris. It is currently one of the smallest of the 130 species of Pristimantis from Peru (AmphibiaWeb 2017) and the 199 species of Pristimantis from Ecuador (Ron et al. 2017, see Table 2).

LEHR E. et al., New species of Pristimantis
and areolate venter, but P. minutulus has the snout moderately long (short), males without vocal slits (present), groin with large yellow spot (greenish yellow with black blotches), and iris reddish gray (reddish copper with fi ne black vermiculation). Pristimantis caeruleonotus Lehr, Aguilar, Siu-Ting & Jordán, 2007 from elevations of 2500-2900 m a.s.l. of humid montane forests in northern Peru has males of similar size (SVL 11.6-14.6 mm [n = 4] vs 13.4-14.5 mm [n = 3] in P. boucephalus sp. nov.; ). Both P. caeruleonotus and P. boucephalus sp. nov. have fi ngers and toes with lateral fringes, heel with conical tubercle, and males that have vocal slits, but lack nuptial pads. However, P. caeruleonotus has a distinct tympanum (absent), dorsolateral folds (absent), small dentigerous processes of vomers (absent), a discoidal fold (absent), prominent dorsolateral folds ending in a sacral tubercle (absent), and a long snout with terminal papillae (short snout without papillae). Furthermore, P. caeruleonotus has the groin blackish brown with yellow and pale blue blotches (greenish yellow with black blotches). Pristimantis coronatus Lehr & Duellman, 2007 is known from a single female specimen of 15.3 mm SVL from 2850 m a.s.l. in northern Peru (Lehr & Duellman 2007). It shares with P. boucephalus sp. nov. the absence of dorsolateral folds and a tympanum, and the presence of conical tubercles on the upper eyelid and fi ngers and toes with lateral fringes. However, P. coronatus has dentigerous processes of vomers (absent), groin red and black (greenish yellow with black blotches), and the iris dark brown (reddish copper). Pristimantis trachyblepharis (Boulenger, 1918) from elevations of 100-1250 m a.s.l. in southern Ecuador and northern Peru and P. boucephalus sp. nov. have smooth dorsum, areolate venter, dorsolateral folds absent, and males that lack nuptial pads. However, P. trachyblepharis tends to have larger males (SVL 12.1-15.8 mm [n = 20] vs 13.4-14.5 mm [n = 3] in P. boucephalus sp. nov.), tympanum present (absent), males without vocal slits (present), dentigerous processes of vomers , males that have vocal slits but lack nuptial pads, fi ngers and toes with lateral fringes, and supernumerary plantar tubercles (Lehr & Coloma 2008). However, Pristimantis andinognomus has short dorsolateral folds that end in a conical sacral tubercle (absent), tympanic membrane and tympanic annulus present (absent), a short snout with terminal tubercle (terminal tubercle absent), minute dentigerous processes of vomers (absent), fl anks with tubercles coalescing into short ridges (ridges absent), heel with two enlarged tubercles (one heel tubercle), and inner tarsal fold present (absent  Lynch 1980), no dorsolateral folds, and a groin with yellow coloration. However, P. carvalhoi has a long snout (short), tympanic annulus present (absent), prominent dentigerous processes of vomers (absent), vocal slits absent (present), and fi ngers and toes without lateral fringes (present). Pristimantis imitatrix (Duellman, 1978) from the Amazonian lowlands has slightly smaller males (SVL 13.0-14.0 mm [n = 9] vs 13.4-14.5 mm [n = 3] in P. boucephalus sp. nov. ;Duellman 2005) and shares with P. boucephalus sp. nov. the absence of a tympanum, dorsolateral folds, dentigerous processes of vomers, and nuptial pads, and both species have fi ngers and toes with lateral fringes. However, P. imitatrix lacks vocal slits (present), has the groin mottled black and white (greenish yellow with black blotches) and the iris is bronze (reddish copper). Pristimantis lirellus (Dwyer, 1995) from elevations of 470-1200 m a.s.l. in northern Peru and P. boucephalus sp. nov. both have males with vocal slits, and fi ngers and toes with lateral fringes, and both species lack a tympanum, dorsolateral folds, and nuptial pads, but P. lirellus has low longitudinal dermal ridges (absent), prominent dentigerous processes of vomers (absent), groin with single pale yellow to orange-yellow spot (greenish yellow with black blotches), and iris bronze to reddish brown (reddish copper). Pristimantis martiae (Lynch, 1974) from the Amazonian lowlands up to 1330 m a.s.l. and P. boucephalus sp. nov. lack dorsolateral folds, a tympanum, and nuptial pads, and both species have males with vocal slits, fi ngers and toes with lateral fringes. However, P. martiae has larger males (SVL 11.8-16.8 mm vs 13.4-14.5 mm [n = 3], Lynch 1980), small dentigerous processes of vomers (absent), a groin that is dull cream, pale orange or tan with brown to black bars or mottling (greenish yellow with black blotches), and bronze iris (reddish copper). Pristimantis croceoinguinis (Lynch, 1968) from the Amazonian lowland forests in Ecuador and Peru has larger males (13.9-18.2 mm vs 13.4-14.5 mm [n = 3], Lynch 1968) which lack vocal slits (present). Pristimantis llosintuta (Köhler & Lötters, 1999) Lehr et al. 2006) and share the absence of a tympanic annulus and membrane, but male P. cruciocularis lack vocal slits (present).
European Journal of Taxonomy 325: 1-22 (2017) Furthermore, P. cruciocularis has the iris with a cruciform mark (absent) and a groin that is orange-red (greenish yellow with black blotches). Pristimantis sp. Pui Pui and P. boucephalus sp. nov. share the absence of a tympanic annulus and membrane, but P. sp. Pui Pui is much larger (SVL up to 31.3 mm vs 13.4-14.5 mm [n = 3]), and has a gray groin (greenish yellow with black blotches).

Description of the holotype
Head broader than body, as long as wide; head length 41.1% of SVL; head width 41.1% of SVL; cranial crests absent; snout short, rounded in dorsal view, rounded in lateral view (Figs 3, 5A-B); eye-nostril distance 50% of eye diameter; nostrils slightly protuberant, directed dorsolaterally; canthus rostralis short, broadly rounded in lateral view, weakly concave in dorsal view; loreal region slightly concave; lips rounded; upper eyelid each with an enlarged conical tubercle at its center and one enlarged conical tubercle at its posterior end; upper eyelid width 57.1% of IOD; few small tubercles on scapular region (see photos in life Fig. 3A-B); supratympanic fold short and broad, extending from posterior margin of upper eyelid slightly curved to insertion of arm; tympanic membrane and annulus absent; small low Skin on dorsum and fl anks smooth, with low scattered tubercles (denser on fl anks than on dorsum), dorsolateral folds absent; occipital and scapular region with sinusoidal ridge; skin on throat, chest, and belly areolate, with scattered low tubercles; discoidal and thoracic folds absent; cloacal sheath short.
Outer ulnar surface with minute low tubercles; palmar tubercle partially divided distally; thenar tubercle ovoid; subarticular tubercles well defi ned, most prominent on base of fi ngers, round in ventral view, subconical in lateral view; supernumerary tubercles distinct, ovoid, approximately half the size of subarticular tubercles; fi ngers with lateral fringes; Finger I shorter than Finger II; discs on digits of fi ngers expanded, round (Fig. 5C).
Hind limbs moderately long, slender, tibia length 54.6% of SVL; foot length 48.9% of SVL; upper surfaces of hind limbs smooth, with low scattered tubercles; inner surface of thighs smooth, posterior and European Journal of Taxonomy 325: 1-22 (2017) ventral surfaces of thighs weakly areolate; heels each with a prominent conical tubercle; outer surface of tarsus with scattered minute low tubercles; inner tarsal fold absent; inner metatarsal tubercle ovoid, two and a half times the size of round outer metatarsal tubercle; subarticular tubercles well defi ned, round in ventral view, subconical in lateral view; plantar supernumerary tubercles distinct, about half the size of subarticular tubercles; toes with lateral fringes; basal webbing present; discs expanded, round, less expanded than those on fi ngers; relative length of toes: 1<2<3<5<4; disc on Toe III not reaching distal subarticular tubercle on Toe IV, disc on Toe V reaching distal subarticular tubercle on Toe IV; Fig. 5D. Coloration of the holotype in life (Fig. 3) The dorsal ground coloration is greenish gray with reddish-brown and scattered brown fl ecks surrounded by black; a reddish-brown blotch mid-dorsally behind the eyes; sinusoidal ridge on occipital and scapular region dark brown; upper lip with two irregularly shaped grayish-brown bars bordered by black on each side of head below eye, bars separated by an irregular pale tan blotch; narrow brown bar laterally in area of canthus rostralis, but not forming a canthal stripe, supratympanic stripe grayish brown, bordered by black; fl anks pale greenish and pale reddish brown with scattered dark brown fl ecks; groin and anterior surfaces of thighs greenish yellow with black blotches and black stripes that extend dorsally to posterior surfaces of thighs; throat, chest, belly and thighs gray with pale reddish and greenish brown mottling and scattered dark gray fl ecks; palmar and plantar surfaces gray, fi ngers I and II cream, fi ngers III and IV gray except for cream discs, toes I and II cream, toes III-V gray with reddish-brown lateral fringes; iris reddish copper with fi ne black vermiculation and narrow black vertical streak from pupil across lower half of iris.

Measurements (in mm) of the holotype
Coloration of the holotype in preservative (Fig. 4) Dorsal coloration grayish tan with scattered brown fl ecks; brown blotch middorsally behind eyes; sinusoidal ridge on occipital and scapular region dark brown; upper lip with two irregularly shaped grayish-brown bars on each side of head below eye, bars separated by an irregular pale gray blotch; narrow brown bar laterally in area of canthus rostralis, but not forming a canthal stripe, supratympanic stripe grayish brown; fl anks tan with scattered brown fl ecks; groin and anterior surfaces of thighs tan with black blotches and black stripes that extend dorsally to posterior surfaces of thighs; throat, chest, belly and thighs grayish tan with dark gray fl ecks; palmar and plantar surfaces pale gray, fi ngers I and II tan, fi ngers III and IV gray except for tan discs, toes I and II tan, toes III-V gray; iris gray with fi ne black vermiculation and black narrow vertical streak from pupil across lower half of iris.

Variation
All paratypes are similar to the holotype regarding morphology (Tables 3-4) and coloration pattern (Fig. 6). They can easily be assigned to the species by having a small size with a disproportionally large head with short snout. The juvenile specimen (MUSM 24474) has two prominent tarsal and two prominent ulnar tubercles on each hind limb. One male specimen (MUSM 24477) has black bars dorsally, laterally and ventrally on the tibia and a brown blotch midventrally on the throat. One specimen (MUSM 24479) is considered a subadult female because of its small size, presence of ovaries, but absence of ovarian eggs.

Distribution, natural history, and threat status
Pristimantis boucephalus sp. nov. is currently only known from the YCNP. The type locality (Figs 1, 7) is located on a mountain crest at 2950 m a.s.l. covered with primary cloud forest. The holotype was found at night, sitting on a leaf at 1 m above the ground. Syntopic anurans included Pristimantis cf.

Discussion
The molecular phylogeny (Fig. 2) shows that the new species, Pristimantis boucephalus sp. nov., belongs to a well-supported speciose clade, which comprises mainly montane but also some Amazonian lowland taxa from northern South America. The clade contains species from the Andes in the west and the Guiana Shield in the east, and corresponds well to a clade uncovered in other studies (Hedges et al. 2008;Pyron & Wiens 2011;Padial et al. 2014). In addition, our results point out a surprisingly high phylogenetic diversity of Pristimantis associated with the relatively small area (1220 km 2 between 460 and 3643 m elevation, Yallico & Rose 1998) of the Cordillera Yanachaga. The six divergent lineages of Pristimantis that we identifi ed in this study correspond with lineages detected in previous phylogenetic reconstructions of the genus (Hedges et al. 2008;Pyron & Wiens 2011). Three lineages were assigned to species groups (Fig. 2), whereas the three other lineages remain unassigned to species groups (Padial et al. 2014). The high phylogenetic diversity indicates that members of Pristimantis probably colonized this region of the central Peruvian Andes through several independent events or underwent unusually high diversifi cation in the region. Today, 18 species of Pristimantis (including three unnamed species) are known to occur in the cloud forests of the YCNP and its buffer zone (see Angulo et al. 2016).
In recent years, several new species of minute Pristimantis have been described from the Andes of Ecuador (Lehr & Coloma 2008: P. andinognomus;Terán-Valdez & Guayasamin 2010: P. minimus), and Peru (Duellman & Hedges 2007: P. minutulus: : P. caeruleonotus; this paper), indicating that the diversity of small and minute species of this genus is highly underestimated due to diffi culties in discovering them. With the description of P. boucephalus sp. nov., the number of Pristimantis known from Peru rises to 131 species (updated from AmphibiaWeb 2017).
Miniaturization of frogs has been documented in many species of different families (Wells 2007;Zimkus et al. 2012), and miniaturized frogs often have lost (e.g., digits) or reduced (e.g., phalanges) elements of  (Yeh 2002). Body proportions of P. boucephalus sp. nov. indicate that the process of miniaturization affected various body structures, leaving the size of the head less affected. However, whether the head of P. boucephalus sp. nov. experienced increased growth or the postcranial body experienced increased miniaturization cannot be determined at this moment. Table 4. Measurements (in mm) and proportions of the male type series of Pristimantis boucephalus sp. nov.; ranges followed by means and one standard deviation in parentheses. For other abbreviations see Materials and methods.