Four new species of Pristimantis Jiménez de la Espada, 1870 (Anura: Craugastoridae) in the eastern Amazon

The Pristimantis genus (Anura: Craugastoridae) is the most diverse among all vertebrates with 531 described species. The highest diversity occurs in Ecuador (215 species), followed by Colombia (202), Peru (139), Venezuela (60), Brazil (30), Bolivia (17), Guyana (6) Suriname and French Guiana (5). The genus is divided into 11 species groups. Of these, the P. conspicillatus group (containing 34 species), distributed in extreme southeastern Costa Rica, Isla Taboga (Panama), northern South America (from Colombia to eastern Guyana), south Bolivia, and is the best represented in Brazil (16 species). The main characteristics of this group are the tympanic membrane and tympanic annulus distinct (except in P. johannesdei); dorsum smooth or shagreen; dorsal lateral fold present or absent; usually smooth belly, but may be weakly granular in some species; toe V slightly larger than the toe III. Most of the taxonomic inconsistencies in species of Pristimantis could be due to its much conserved morphology and the lack of comprehensive taxonomic evaluations. Thus, an ongoing challenge for taxonomists dealing with the Pristimantis genus is the ubiquitous abundance of cryptic species. In this context, accurate species delimitation should integrate evidences of morphological, molecular, bioacoustics and ecological data, among others. Based on an integrative taxonomy perspective, we utilize morphological, molecular (mtDNA) and bioacoustic evidence to describe four new species of the Pristimantis conspicillatus group from the eastern Amazon basin. Pristimantis giorgii sp. nov. is known from the Xingu/Tocantins interfluve and can be distinguished from the other Pristimantis species of the region by presenting discoidal fold, dorsolateral fold absent, vocalization composed of three to four notes and genetic distance of 7.7% (16S) and 14.8% (COI) from P. latro, the sister and sympatric species with respect P. giorgii sp. nov.. Pristimantis pictus sp. nov. is known to the northern Mato Grosso state, Brazil, and can be distinguished from the other species of Pristimantis by presenting the posterior surface of the thigh with light yellow patches on a brown background, also extending to the inguinal region, vocalization consisting of four to five notes and a genetic distance of 11.6% (16S) and 19.7% (COI) from P. pluvian sp. nov., which occurs in sympatry. Pristimantis pluvian sp. nov. is known to the northern Mato Grosso state, Brazil, and may be distinguished from the other Pristimantis species by having a posterior surface of the thigh reddish and vocalization composed of two notes. Pristimantis moa sp. nov. is known to the northern Tocantins state and southwestern Maranhão state. This species can be distinguished from the other Pristimantis species by possessing slightly perceptible canthal stripe, external thigh surface with dark yellow spots on brown background, vocalization consisting of three to five notes and genetic distance of 2.3–11.7 (16S) and 10.5–23.1 (COI) for the new Pristimantis species of this study.


Introduction
The genus Pristimantis Jiménez de la Espada 1870, is the most diverse among vertebrates [1], currently comprised of 531 valid species, 155 of which have only been described in the last decade [2]. Most species of this genus occur in the western part of the Amazon [3]. The topographic heterogeneity of the Andes, accompanied by the life history of Pristimantis (direct development, high endemism and the ability to colonize a wide variety of habitat types, including highlands) could be partially responsible for this great diversity [4]. In the eastern Amazon, few studies have focused on taxonomic aspects of Pristimantis populations, and most of the information about this genus comes from faunal inventories [5][6][7][8], of which many do not present specific identification of existing lineages [9].
The species of the P. conspicillatus group mentioned above, only P. gutturalis, P. chiastonotus, P. zeuctotylus, P. latro and P. dundeei occur in the eastern Amazon rainforest. Adding P. inguinalis, P. marmoratus (Boulenger, 1900), and P. zimmermanae (Heyer & Hardy, 1991) are recorded eight species of Pristimantis to the east of the Brazilian Amazon. This region has been suffering from strong deforestation pressures in recent years, causing habitat loss, which is one of the main threats to biodiversity [12], [13].
Most of the taxonomic inconsistencies within Pristimantis can be attributed to its conserved morphology and its high diversity [1], [9], [14]. The conserved morphology of the species is seen in the high cryptic diversity within the genus, where distinct lineages are usually associated with the same nominal species [15], [16]. Given this context, correctly delimiting a species often requires an integrative approach, associating morphological, molecular, and bioacoustic data [14], [17], [18]. After several expeditions in different regions of the eastern Amazon and north Cerrado, we recorded Pristimantis lineages that we could not associate with any nominal species. Based on an integrative taxonomy framework, herein we describe four new Pristimantis species belonging to the P. conspicillatus group.

Ethics statement
The permits for field samples collections were provided by the Brazilian Government (SISBIO licence N 30034-1, 32401, 54493-5, and 54493-11). Although euthanized methods were not evaluated by an institutional animal care and use committee or similar regional ethics committee, voucher collections strictly complied with the ethical conditions as dictated by the Sistema of Autorização and Informação in Biodiversidade-SISBIO, environmental authorities of Brazil (see scientific license numbers above). The collected frogs were euthanized by topical application of a 2% liquid solution of lidocaine hydrochloride, fixed in 10% formalin and transferred to permanent collections in alcohol 70%. We collected tissue samples (muscle and liver) before individuals were fixated and stored.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:A1A7A538-68C8-401B-B706-6C7C16D87296. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Sampling
We conducted active searches in 27 localities, including areas typical to the Amazonian domain (Pará state and northern Mato Grosso state), transition areas between the Amazon and Cerrado (Araguaína, in Tocantins state and Balsas, in Maranhão state) and Cerrado areas (Riachão in Maranhão state and Palmas in Tocantins state) (Fig 1)

Molecular analysis
We extracted the total genomic DNA using the 2% CTAB protocol [19]. We amplified a fragment of 16S mtDNA by polymerase chain reaction (PCR) using 16Saf primers and 16Sbr [20], and the Cytochrome Oxidase sub unit I (COI) with the primers dgLCO 1490 forward and dgHCO 2198 reverse [21]. The sequences were obtained through the di-desoxiterminal Sanger method using ABI PRISM 1 Big Dye™ Terminator V.3 Cycle Sequencing kit (Applied Biosys-tems™), with 16Saf and COI (forward and reverse). PCR details and sequencing reaction are available in Supporting information (S1 File).
GenBank sequences (https://www.ncbi.nlm.nih.gov/genbank/) of other Pristimantis species and of the P. conspicillatus group were used in the database for phylogenetic analyses (access numbers are listed in S2 File). The sequences were aligned using the ClustalW Algorithm [22] implemented in the software Geneious v. 9.1.2. The alignment of the 16S mtDNA gene presented gaps, which were removed using GBLOCKS v.0.91b [23], [24], available online (http:// molevol.cmima.csic.es/castresana/Gblocks_server.html). A gene tree was built using the MrBayes software [25], with the concatenated genes (16s + COI) using the evolutionary model HKY + G, estimated in the JModelTest software [26] under the Bayesian information criterion (BIC). We followed the standard values for two runs and four chains, running 50 7 generations, with a tree sampled every 10.000 generations. The burn in value was selected by viewing the log likelihoods associated with the distribution of the tree after the software Tracer v 1.5 [27], discarding 20% of the trees with the TreeAnnotator software v1.8 [28]. We accessed convergence by examining the average standard deviation of the split frequency between runs (< 0.01). The number of independent samples was considered sufficient when the stationary was reached and the Effective Sample Sizes (ESS) were higher than 200. As additional evidence for species delimitation, the uncorrected pairwise distance (p-distances for the 16S rRNA and Kimura 2 Parameters-K2P for the COI) among the species of Pristimantis of this study and others in the P. conspicillatus group were calculated using MEGA 6.0 [29]. Hedges and colleagues [11] suggest Oreobates genus as sister group of Pristimantis, so we used it as the outgroup (O. quixensiss of Amazonas State) in our analysis. We built a network of haplotypes among Pristimantis species from the 16S mtRNA and COI mtDNA genes in the POPART software [30] using the medianjoining network method in order to check for possible haplotype sharing.
Measurements were made using a 0.01 mm digital caliper and rounded to 0.1 mm as in [31][32][33][34]. The measures obtained were: snout-vent length (from tip of snout to posterior margin of vent), SVL; head length (from posterior margin of lower jaw to tip of snout), HL; head width, (at level of angle of jaw ), HW; snout length (from anterior corner of eye to tip of snout), SL; distance from eye to nostril (from anterior corner of eye to posterior margin of naris), DEN; internarial distance (taken between the median margins of the nares), ID; eye length (measured horizontally), EL; interorbital distance (taken between the inner margins of the orbits), IoD; eyelid width (the largest transverse width of the upper eyelid.), EW; tympanum length (the largest length of the tympanum from the anterior margin to the posterior margin of the tympanum), TL; arm length (from the tip of the elbow to the proximal edge of the palmar tubercle), AL; hand length (from the proximal edge of the palmar tubercle to the tip of finger III), HaL; thigh length (from vent to knee), ThL; tibia length (from outer edge of flexed knee to heel), TiL; tarsus length (from the heel to the proximal edge of the inner metatarsal tubercle.), TaL; foot length (from proximal border of inner metatarsal tubercle to tip of fourth toe), FL; leg length (from the knee joint to the tip of toe IV), LL (all morphometric measurements are listed in S3 File). Sex was determined by direct visualization of secondary sexual characters in adult individuals, such as the presence or absence of vocal slits, vocal sac and nuptial callus in males. Comparisons of characters states were performed with specimens from the P. conspicillatus group available in specialized literature [31], [32], [34][35][36][37].

Bioacoustic analysis
We used two types of audio recorders to capture vocalizations: Sony Digital Recorder ICD-PX240 and Tascam DR-40 digital recorder. We obtained records of advertisement calls (vocalizations) from seven males between 18h -20h, at Uberlândia Farm, Portel municipality, Pará state; four males from Jacareacanga municipality, Pará state; two males from Paranaíta municipality, Mato Grosso state; three males from Taquaruçu district, Palmas municipality, Tocantins state; and five males from Balsas municipality, Maranhão state. The species of the P. conspicillatus group selected for acoustic comparisons were P. fenestratus, P. zeuctotylus, P. chiastonotus, P. samaipatae [38], P. vilarsi, P. gutturalis and P. latro, as they present similar morphological and geographic distribution (east of the Amazon) as the species described in this study, except for P. vilarsi (western Amazon) and P. samaipatae (Bolivia).
We analyzed vocalizations at a sampling rate of 44.1 kHz and 16-bit resolution using the Raven Pro v 1.5 software for Windows (Bioacoustics Research Program 2014). Information about frequency was obtained through Fast Fourier Transformations (FFT; width of 1024 points), Frame = 100, Overlap = 75 and flat top filter. Spectrograms and oscillograms were generated using the SeeWave package [39] in the R software (R Core Team, 2018) following the same parameters as the Raven software. The following variables were measured according to specialized literature [34], [40]: call length (ms), number of notes per call, length of the note (ms), presence of pulses, fundamental frequency (frequency band in which the first sound is visualized through a spectral slice output, in Hz) and dominant frequency (measured from a spectral slice taken from the highest amplitude portion of the note, in Hz).

Species delimitation
To delimit the Candidate Species (CS), we followed the model proposed by Padial and colleagues (2010) [41]: where a given CS can be classified as an Unconfirmed Candidate Species (UCS), Confirmed Candidate Species (CCS) and Deeply Conspecific Lineages (DCL). UCS is considered any CS that presents genetic differences above the limit (> 3%) proposed for the 16S rRNA gene [18], [42], [43], but with no complementary characters verified, such as morphology, bioacoustics, ecology or distribution. A CCS is considered a CS that presents genetic differentiation in relation to other species and also has support of parallel evidence. Finally, a DCL is considered a CS that presents genetic divergence above the proposed limit (> 3%), but cannot be differentiated by parallel evidence (morphology and acoustics).

Phylogenetic relationships and genetic distances
Our tree topology, based only on 16S and COI genes (82 sequences of two concatenated mitochondrial genes, 866bp), recovered posterior probabilities support values among three main divergent lineages. (Fig 2): 1) Andean, 2) Amazon Basin, and 3) Tocantins Basin. The Andean lineages were represented by P. peruvianus, P. achatinus, P. vilarsi and P. bipunctatus [44]. The Amazon Basin lineages consists in Pristimantis zeuctotylus, P. fenestratus complex (with three cryptic lineages, one for Manaus municipality and two for Borba municipality in Amazonas state), Pristimantis sp1 (Altamira municipality), Pristimantis sp2 (Juruti municipality), P. latro, and two undescribed lineages for Mato Grosso state. Two lineages were found for the Tocantins Basin, one of which occurs in the eastern Pará state and another in Tocantins and Maranhão states.
The two undescribed Pristimantis lineages from the Amazon Basin occur in northern Mato Grosso (Cristalino, Novo Mundo, Paranaíta, Sinop, Tabaporã and Cotriguaçu municipalities), showing genetic distances (Table 1) between each other of 11.6% (gene 16S rRNA) and 25.9% (gene COI mtDNA) and distant phylogenetic positioning, even though being sympatric in some localities (e.g. Cotriguaçu and Novo Mundo). The lineage identified as Pristimantis sp1, with individuals from the left bank of the upper Xingu River, in Altamira municipality, represents the sister group of the clade formed by P. latro and three undescribed Pristimantis lineages from the Amazon Basin and Tocantins (HPD = 1), with genetic distances of 9.1-9.9% (16s rRNA gene) and 19.5-22% (COI mtDNA gene). Pristimantis sp2, represented by individuals from the municipality of Juruti, Pará state, were found as a sister group of P. fenestratus (lineage from Borba, Amazonas state, support HPD = 1), with genetic distances of 4% (16S rRNA gene) and 14.6% (COI mtDNA gene).
In the Tocantins Basin, the two lineages found showed genetic distances of 2.3% (16S rRNA gene) and 10.5% (COI mtDNA gene) among themselves, with one found only in Pará state and the other in Tocantins and Maranhão states. Regarding phylogenetic positioning, the two lineages are sisters with high support (HPD = 1). Pristimantis latro (occurring in the Amazon Basin and Tocantins Basin), is sympatric with the Pará lineage, presenting genetic distances of  7.7% (16S rRNA gene) and 14.8% (COI mtDNA gene) and is sister to the lineages from northern Mato Grosso state (HPD = 0.95). The haplotype networks for the 16S (422 bp) and COI (444 bp) genes showed seven distinct mitochondrial haplogroups, corroborating the lineages found in phylogenetic reconstruction, with no haplotype sharing among the new species of Pristimantis described in this study ( Fig  3). Based on the large genetic distance found with the 16S and COI mitochondrial markers, phylogenetic positioning, and morphological and acoustic (Fig 4) divergences (mentioned in the "Diagnosis" and "Comparison with other species" sections), herein we describe four new species occurring throughout the east of Amazon and north Cerrado.

Systematics
Allocation to species group. The new taxon described in this article belong to the Pristimantis conspicillatus group based on (1) molecular phylogenetic relationship (Fig 2); and (2) morphological characteristics such as: tympanic membrane and tympanic annulus distinct (except in P. johannesdei); dorsum smooth or shagreen; lateral dorsal fold present or absent; usually smooth belly, but may be weakly granular in some species; toe V slightly larger than toe III (see Hedges and colleagues [11]).

Diagnosis
Pristimantis giorgii sp. nov. distinguishes from other species in the P. conspicillatus group by the following combination of characters (summarized in Table 2): (1) shagreen dorsal skin, with dorsal tubercles in the scapular region, supra ocular and laterally; lateral dorsal fold absent; discoidal fold present; smooth and granular belly skin texture laterally; (2) males with slit and vocal sac; (3) thigh surface with dark yellow spots on brown background; (4) four supernumerary tubercles present on palms of the hands and almost the same size as the subarticular tubercles projected; (5) side fringe along the fingers; (6) supernumerary tubercles present in the base of the foot's IV, conical and small format; (7) basal webbing and lateral fringe present between toes; (8) vocalization with three to four notes pulsed in length 0.017-0.074 s (N = 7);

Comparison with other species
Pristimantis giorgii sp. nov. distinguishes from P. latro by the absence of lateral dorsal fold (present in P. latro) and vocalization composed of three to four notes (seven notes, see  Table 3); from P. vilarsi by presenting smooth belly texture in the center and granular laterally (smooth throughout the belly), presence of basal webbing in the feet (absent), vocalization of three to four notes (eight notes) [45], [46]; from P. fenestratus by presenting smooth belly texture in the center and granular laterally (smooth throughout the belly in P. fenestratus), fringe on toes prominent (weak) [31], [34], [47]; from P. koehleri by having a lateral fringe on the fingers (absent in P. koehleri), presence of basal webbing in the feet (absent), vocalization composed of three to four notes (four to eight notes) [14]; from P. dundeei by presenting a flat belly in the center and granular laterally (areolated in P. dundeei), presence of fringes on fingers (absent) [14], [48]; from P. zeuctotylus by presenting a divided palmar tubercle (undivided in P. zeuctotylus), white belly coloring with scattered black dots (black belly) [37]; from P. chiastonotus by presenting basal webbing and fringe on toes (absent in P. chiastonotus), tarsal fold present (absent); rostrum subacuminated in dorsal view (acuminate), dorsal tubercles present (absent), vocalization composed of three to four notes (one note) [37].

Description of the holotype
Adult male 34.2 mm SVL. Dorsal skin shagreen, dorsal tubercles present, dorsolateral fold absent; smooth belly texture and granular laterally, granular posterior surface of thighs; head longer (39.5% of the SVL) than wide; snout subacuminated in dorsal view and curved anteroventrally in lateral view; canthus rostralis weakly concave in dorsal view and in cross section, flat loreal region; ovoid tongue, wider than long; dentigerous process of vomer oblique and posterior to choanae, eight visible teeth; eye 93% of distance from the eye to the nose; elliptical pupil; supraocular tubercles and inter-supraocular bar present; cranial crests absent; prominent supra tympanic fold, not contacting the eyelid; tympanic membrane 47.5% of EL, rounded and translucent, tympanic annulus prominent; relatively small hands, 26% of the SVL; relative length of fingers: II < IV < I < III; discs of fingers III and IV are wider than fingers I and II; prominent, semi divided, heart-shaped external metacarpal tubercle; large internal palmar tubercle; one subarticular tubercle prominent on fingers I, II, III and IV; one supernumerary tubercles present at the base of fingers I, II, III and IV; long legs, tibia 56.1% of the SVL; relative length of toes: I <V <II <III <IV; well developed and oval inner metatarsal tubercle; external metatarsal tubercle much smaller than the internal; one subarticular tubercle on toes I and II; two subarticular tubercles on toes III and V; and three subarticular tubercles on toe IV; basal webbing and lateral fringes (weak) present on toes; inner tarsal fold present; outer tarsal tubercles absent.

Color in life
The dorsum is predominantly light brown with scattered dark brown bars. Posterior and anterior limbs with dark brown bars. Black labial bars. Black canthal stripe. White belly with tiny scattered black dots. Iris presents a golden coloration at the top and bottom, while in the anterior and posterior region is predominantly red. White groin area with few yellow spots. Posterior thigh surface predominantly light brown.

Etymology
The specific epithet is a patronymic to the Professor José Adriano Giorgi † of the Universidade Federal do Pará, due to his great contributions to knowledge about entomofauna of the Xingu region and since he accompanied us during the first collection of the species.

Distribution, ecology and habitat
Pristimantis giorgii sp. nov. has been recorded in the interfluve Xingu/lower Tocantins in Pará state, Brazil throughout the municipalities of Altamira (Assurini, right bank of the middle Xingu river), Portel (left bank of the lower Tocantins River), Marabá and Melgaço (Flona de Caxiuanã) (Fig 1). This species can be found in preserved forest areas (Flona Caxiuanã and

PLOS ONE
Portel) or in some disturbed environments, e.g. forest fragments surrounded by pastures (agrovila do Assurini, Altamira municipality, Pará state). During the rainy season, this species becomes reproductively active, and males move up in the vegetation to call at a height of two meters; during the dry season, they can be found in the leaf litter. Pristimantis pictus sp. nov. (Fig 5E-5H) urn:lsid:zoobank.org:act:AEE3D30E-203C-4215-BFA8-5EBFBEFD78FC Holotype. (Fig 6E-6H

Diagnosis
Pristimantis pictus sp. nov. differs from the other species in the P. conspicillatus group by the following character combinations (summarized in Table 2): (1) dorsal skin shagreen, dorsal tubercles present distributed mainly in the lateral dorsal region, supra-ocular tubercles absent, smooth belly texture in the central part and granular laterally; (2) males with vocal slit and vocal sac present; (3) four supernumerary tubercles on the palm of the hands almost the same size as the subarticular, conical shaped; (4) lateral fringe along the fingers of the hands; (5) basal webbing and lateral fringe between toes; (6) vocalization with four to five notes with length ranging from 0.017-0.033 s (N = 4) and pulsed; and (7) posterior surface of the thighs with light yellow patches on brown background, white groin with yellow spots.

Comparison with other species
Pristimantis pictus sp. nov. distinguishes from P. giorgii sp. nov. by presenting yellow spots on a brown background on the posterior thigh surface (light yellow spots absent in P. giorgii sp. nov.), occurs in northern Mato Grosso state (eastern Pará state), vocalization composed of four to five notes (three to four notes); from P. latro by vocalization composed of four to five notes (consisting of seven notes in P. latro), occurs in northern Mato Grosso state (Tapajós/ lower Tocantins interfluve); from P. vilarsi by presenting smooth belly texture in the central and granular side laterally (smooth in P. vilarsi), presence of basal webbing in toes (absent), vocalization composed of four to five notes (composed of eight) [45], [46]; from P. fenestratus by presenting smooth belly texture in the central and granular side laterally (smooth in P. fenestratus), presence of light yellow spots on brown background on the posterior thigh surface (light yellow spots absent) [14], [31], [47]; from P. koehleri by the smooth belly texture in the central and granular side laterally (finely granular in P. koehleri), vocalization consisting of four and five notes (from four to eight notes) [14]; from P. samaipatae by possessing white belly with tiny black dots scattered randomly (immaculate in P. samaipatae), semi-divided external palmar tubercle (divided) [14], [49]; from P. dundeei by possessing smooth belly texture in the center and granular laterally (areolate in P. dundeei), presence of fringe on the fingers (absent) [14], [48]; from P. zeuctotylus by presenting semi-divided palmar tubercle (whole in P. zeuctotylus), white belly with tiny black dots scattered randomly (black belly) [37]; from P. chiastonotus by presenting basal webbing and fringe on toes (absent in P. chiastonotus), inner tarsal fold present (absent), dorsal tubercle present (absent), vocalization composed of four to five notes (one note) [37].

Description of the holotype
Adult male 30.6 mm SVL. Dorsal skin strongly shagreen, dorsal tubercles present, mainly in the lateral region of the dorsum, dorsolateral fold present or absent; smooth belly texture and granular laterally, granular posterior surface of thighs with yellow spots on brown background; head longer (38.2% of the SVL) than wide; long snout (49.5% do HL), subacuminate in dorsal view and protruding in lateral view; canthus rostralis slightly concave in dorsal view and in cross section, flat loreal region; ovoid tongue, wider than long; dentigerous process of vomer oblique and posterior to choanae; the distance from the eye to the nose is the same horizontal diameter of the eye; elliptical pupil; supraocular tubercles present; cranial crests absent; prominent supra tympanic fold, not contacting the eyelid; tympanic membrane 41% of EL, rounded and translucent tympanic annulus prominent; relatively small hands, 28.7% of the SVL; relative length of fingers: II < IV < I < III; discs of fingers III and IV are wider than fingers I and II; prominent, semi divided, heart-shaped external metacarpal tubercle; large internal palmar tubercle; one subarticular tubercle prominent on fingers I and II, two prominent subarticular tubercles on fingers III and IV; one supernumerary tubercles present at the base of fingers I, II, III and IV almost the same size as subarticular tubercles; long legs, tibia 59.1% of the SVL; relative length of toes: I <II <V <III <IV; well developed and oval inner metatarsal tubercle; external metatarsal tubercle much smaller than the internal one; one subarticular tubercle on toes I and II; two subarticular tubercles on toes III and V; and three subarticular tubercles on toe IV; basal webbing and lateral fringes present on toes, absent supernumerary tubercle; long inner tarsal fold present.

Color in life
Reddish dorsum with yellow dots on the flanks forming a division between the lateral and the whitish venter. Posterior and anterior limbs present dark brown bars. Evident labial bar. Canthal stripe weak,. White belly with tiny black dots scattered randomly. Iris features a golden coloration at the top and bottom, whereas in the anterior and posterior region it is predominantly red. The posterior surface of the thigh presents bright yellow spots on a brown background. Groin area with yellow spots, extending across the lateral half of the body.

Coloration in preservative
Coloration is predominantly brown in dorsal view, both in females and males. The belly can be white with tiny black dots or black spots randomly distributed. The rostral band present in some individuals appears more erased, hindering its visualization. The yellow spots on the posterior surface of the thigh are whitish.

Etymology
The specific epithet "pictus" comes from the Latin (pictus = painted, stained) and refers to the yellow dots that are characteristic to individuals of this species, facilitating its identification in relation to other Pristimantis species in its occurrence region.

Distribution, ecology and habitat
Pristimantis pictus sp. nov. has been registered in the municipalities of Cotriguaçu, Novo Mundo, Tabaporã, Paranaíta and Sinop in Mato Grosso state and Jacareacanga in Pará state, Brazil (Fig 1). It can be found at the edges of preserved forests, close to pastures.

Diagnosis
Pristimantis pluvian sp. nov. differs from the other species in the group by the following combination of characters (summarized in Table 2): (1) dorsal skin smooth or shagreen, lateral dorsal fold present, dorsal tubercles present mainly in the lateral part of the body and few in the scapular region, smooth and granular belly texture laterally; (2) males with slits and vocal sac present; (3) light venter with tiny black dots scattered; (4) four or five supernumerary tubercles in the palm of the hands almost the same size as the subarticular and conical; (5) basal webbing and fringe between toes; (6) one to three small supernumerary tubers present in the foot; (7) vocalization consisting of two notes with length ranging from 0.047-0.073 s (N = 3) and pulsed; (8) posterior surface of the thighs reddish and without spots.

Comparison with other species
Pristimantis pluvian sp. nov. distinguishes from P. giorgii sp. nov. by presenting vocalization consisting of two notes (from three to four notes in P. giorgii sp. nov.), lateral dorsal fold present (absent); from P. pictus sp. nov. by the presence of the posterior thigh surface reddish (light yellow patches in P. pictus sp. nov.), vocalization consisting of two notes (from four to five notes); from P. latro by vocalization composed of two notes (seven notes), northern Mato Grosso state (Tapajós/Xingu interfluve); from P. vilarsi by presenting smooth belly texture in the center and granular laterally (smooth along the whole belly in P. vilarsi), presence of basal webbing in the feet (absent), vocalization consisting of two notes (eight notes) [45], [46]; from P. fenestratus by presenting smooth belly texture in the center and granular laterally (smooth in P. fenestratus), vocalization consisting of two notes (from three to five notes), northern Mato Grosso state (western Amazonia) [31], [47]; from P. koehleri by the texture of the flat belly in the center and granular laterally (finely granular along the belly in P. koehleri), vocalization consisting of two notes (from four to eight notes) [14]; from P. samaipatae by possessing white belly with black dots scattered randomly (immaculate in P. samaipatae), external palmar tubercle semi-divided (divided) [14]; [48]; from P. dundeei by possessing smooth belly texture in the center and granular laterally (areolate along the belly in P. dundeei), lateral dorsal fold present (absent) [14], [48]; from P. zeuctotylus by a semi-divided palmar tubercle (undivided in P. zeuctotylus), white belly with black dots scattered randomly (dark belly) [37]; from P. chiastonotus for presenting basal webbing and fringe on toes (absent in P. chiastonotus), tarsal fold present (absent), dorsal tubercles present (absent), vocalization consisting of two notes (one note) [37].

Description of the holotype
Adult male 28.7 mm SVL. Dorsal skin shagreen, dorsal tubercles present, mainly on the side of the body and dorsal lateral fold present; texture of the belly smooth and granular laterally, granular posterior surface of thighs; head longer (34.1% of the SVL) than wide; long snout (54.1% do HL), truncate in dorsal view and long and protruding in lateral view; canthus rostralis slightly concave in dorsal view and in cross section,, flat loreal region, obvious facial mask, weak labial bars; ovoid tongue, wider than long; dentigerous process of vomer oblique and posterior to choanae; distance from the eye to the nose (3.7 mm) is greater than the horizontal diameter of the eye (3.4 mm); elliptical pupil; absent supraocular tubercles; absent cranial crests; prominent supra tympanic fold, contacting the eye; tympanic membrane 41.2% of EL, rounded and translucent, tympanic annulus prominent; relatively small hands, 25.4% of the SVL; relative length of fingers: II < IV < I < III; discs of fingers III and IV are wider than fingers I and II; prominent, semi divided, heart-shaped external metacarpal tubercle; large internal palmar tubercle in contact with nuptial pad; one subarticular tubercle prominent on fingers I and II, two prominent subarticular tubercles on fingers III and IV; supernumerary tubercles present at the base of fingers I, II, III and IV and above the inner palmar tubercle; long legs, tibia 56.4% of the SVL; relative length of toes: I <V <II <III <IV; well developed and oval inner metatarsal tubercle; external metatarsal tubercle much smaller than the internal; one subarticular tubercle on toes I and II; two subarticular tubercles on toes III and V; and three subarticular tubercles on toe IV; one to three plantar supernumerary tubercle; basal webbing and lateral fringes present on toes; inner tarsal fold present and long.

Coloration in preservative
The coloration is predominantly brown in the dorsal region, both in males and females. The belly can be white with tiny black or immaculate dots. The transversal bands present in some individuals appear more erased and in others may be difficult to observe.

Etymology
The specific epithet "pluvian" comes from Latin and means rain. The frogs of this genus are known as Amazon Rain Frogs.

Distribution, ecology and habitat
Pristimantis pluvian sp. nov. is registered in the municipalities of Cotriguaçu, Ipiranga do Norte and Paranaíta in Mato Grosso state, Brazil (Fig 1). It can be found in conserved areas of forests or areas with some environmental disturbances, e.g., forest fragments surrounded by pastures. During the reproductive period, males rise in vegetation and vocalization. During the dry period they are predominantly on the ground.

Diagnosis
Pristimantis moa sp. nov. differs from other species in the P. conspicillatus group by the following combination of characters (summarized in the Table 2): (1) shagreened dorsal skin, dorsal tubercles present mainly in the scapular region, discoidal fold present, smooth belly skin texture in the center and granular laterally; (2) ventral coloration with tiny dots or black spots on white background; (3) a subarticular tubercle in fingers I and II, and two in fingers III and IV, all are large and conical; (3) posterior surface of the thigh strongly stained dark yellow on brown background;(4) supernumerary tubercles presented at the base of the finger I, II, III and IV, almost the same size as the subarticular tubercles; (5) side fringe along the fingers of the hand; (6) supernumerary tubercle present at the base of toes III and IV, smaller than subarticulars; (7) basal webbing well developed and lateral fringe present between toes; (8) vocalization consisting of three to five notes, notes are on average 45 ms long (minimum 23.8 and maximum 64.6, ± 9.1); (9) occurs in the right margin of the Araguaia River and left and right margins of the Tocantins River.

Comparison with other species
In parentheses, the character state of the nominal species compared to the new species. Pristimantis moa sp. nov. distinguishes from P. giorgii sp. nov. by presenting vocalization composed of three to five notes (three to four in P. giorgii sp. nov.), posterior surface of the thigh strongly stained yellow on a dark background (weakly stained yellow on a dark background), it occurs on the right bank of the Araguaia river and left and right banks of the Tocantins river (left bank of the lower Tocantins river); from P. pictus sp. nov. by presenting vocalization composed of three to five notes (four to five in P. pictus sp. nov.), posterior surface of the thigh strongly stained yellow on a dark background (yellow spots on brown background); from P. pluvian sp. nov. by posterior surface of the thigh strongly stained yellow on a dark background (reddish in P. pluvian sp. nov.), vocalization composed of three to five notes (two notes); from P. latro by vocalization composed of three to five notes (seven notes in P. latro), posterior surface of the thigh strongly stained yellow on a dark background (almost spotless); from P. vilarsi by presenting the smooth belly texture in the center and granular laterally (smooth in P. vilarsi), presence of basal webbing in toes (absent), vocalization with three to five notes (eight notes) [45], [46]; from P. fenestratus by presenting the smooth belly texture in the center and granular laterally (smooth in P. fenestratus), vocalization consisting of three to five notes (from two to three notes), occurs in the eastern Brazilian Amazon, Tocantins state (occurs in the western Amazon) [14], [31], [47]; from P. koehleri by presenting the smooth belly texture in the center and granular laterally (finely granular in P. koehleri), vocalization composed of three to five notes (four to eight notes), shorter vocalization 0.294s (0.421s) [14]; from P. samaipatae by having a white belly with randomly scattered black dots (immaculate in P. samaipatae), semi-divided external palmar tubercle (split) [14], [48]; from P. dundeei by presenting the smooth belly texture in the center and granular laterally (areolate in P. dundeei) [14], [48]; from P. zeuctotylus by a divided palmar tubercle (entire in P. zeuctotylus), a white belly with randomly scattered black dots (black belly) [37]; from P. chiastonotus by presenting basal webbing and fringe on the toes (absent in P. chiastonotus), tarsal fold present (absent), dorsal tubercles present (absent), vocalization composed of three to five notes (one note) [37].

Description of the holotype
Adult male 32.9 mm SVL. Dorsal skin shagreened, presence of dorsal tubercles and dorsal lateral fold, five tubercle in the scapular region, with the dark base forming a "W", interorbital bar; texture of the belly smooth and granular laterally, granular anterior surface of thighs; head longer (38.6% of the SVL) than wide; long snout, truncate in dorsal view and long and protruding in lateral view; convex canthus rostralis, flat loreal region; dentigerous process of vomer oblique and posterior to choanae, longer than wide tongue; eye diameter (4.4 mm) greater than distance from eye to nostril (4.0 mm); elliptical pupil; supraocular tubercles absent; cranial crests absent; prominent supra tympanic fold with tubercle present in the posterior region, not contacting the eye; tympanic membrane 47.7% of EL, rounded and translucent, tympanic annulus prominent; relatively small hands, 26.7% of the SVL; relative length of fingers: II < IV < I < III; discs of fingers III and IV are wider than fingers I and II; prominent, semi divided, heart-shaped external metacarpal tubercle; large internal palmar tubercle; one subarticular tubercle prominent on fingers I and II, two prominent subarticular tubercles on fingers III and IV; supernumerary tubercles present at the base of fingers I, II and III; long legs, tibia 57.4% of the SVL; relative length of toes: I <II <V <III <IV; well developed and oval inner metatarsal tubercle; external metatarsal tubercle much smaller than the internal one; one subarticular tubercle on toes I and II; two subarticular tubercles on toes III and V; and three subarticular tubercles on toe IV, all large and conical; plantar supernumerary tubercle present at the base of the toe IV and smaller than the subarticular; basal webbing well developed and lateral fringes present on toes (weak); inner tarsal fold present and long.

Color in life
Brown dorsum with darker transversal bars. Posterior and anterior limbs present dark brown bars. Black lip bars. Canthal stripe weak, almost absent in some individuals. White belly with tiny dots or dark spots. The iris has golden coloration on top and bottom, while in the anterior and posterior region it is predominantly red.

Coloration in preservative
The coloration is predominantly brown on the back, in both males and females. The belly can present tiny black dots or black spots scattered randomly.

Variation
The texture of the belly in ZUFMS-AMP 8575 appears almost completely granular and smooth only in the anterior portion of the belly, while the ZUFMS-AMP 8552, which is a juvenile, presents a smooth belly and may be difficult to observe granules. Individuals ZUFMS-AMP 8551, 8557, 8574, 8577 do not present dorsolateral fold, present in the rest of the type series. Individuals ZUFMS-AMP 8575 and ZUFMS-8550 present a face mask more evident than other individuals, making it difficult to visualize the canthal stripe. The side folds are present in the dorsal part of the series type (ZUFMS-AMP 8548/50, ZUFMS-AMP 8558, 8575/76) and absent in the others. Females are larger than males (Average = 31.7, N = 7; Average = 28.5, N = 9)

Etymology
The specific epithet is a patronym of the Capoeira Master Romualdo Rosário da Costa †, known as Mestre Moa do Katendê, for his struggles for the black movement of Bahia.

Distribution, ecology and habitat
Pristimantis moa sp. nov. is registered in the municipalities of Palmas, Palmeirante and Araguaína in Tocantins state and Carolina, Balsas and Riachão in Maranhão state, Brazil (Fig 1). It can be found in conserved forest areas or in areas with some environmental disturbances, e.g., forest fragments surrounded by pastures. During the reproductive period, males rise in vegetation and vocalization. During the dry period they are predominantly on the ground.

Discussion
Currently, only two species of the Pristimantis conspicillatus group are known in the eastern Brazilian Amazon, in the southern Amazon River, P. latro (Pará state) and P. dundeei (northern Mato Grosso state). With the four new species described in this work, this number increases to six, representing a 200% increase in the number of species for this region. Likewise, this represents a 12% increase in the diversity of species of the P. conspicillatus group, leading the numbers from 34 to 38 species in total, of which 20 right now are occupying the Brazilian territory. The fact of having discovered four new species of Pristimantis exploring just 11.5% of the total area of the distribution of this group, suggests the existence of a greater diversity still underestimated and highly promising in that remaining 88.5% of territory inhabited by the species of the group along the Neotropic; even when the group currently inhabits the Andes and Amazon, the worldwide regions with the principals ecological and hydro-geomorphological process causing diversification [50][51][52][53][54]. This study confirms that in the specious Pristimantis conspicillatus group, apparently a lot of efforts on integrative taxonomic research is needed for studies on new species, cryptic diversity, speciation processes and biogeography, due to their great capacity and varied mechanisms of diversification [55]. In the Neotropical region, estimates based on approaches integrating different character types (e.g. morphology, molecular and bioacoustics) showed increased frog richness between 150 and 300% [49]. Studies indicate that in the last 250 years of taxonomic classification only 14% of all terrestrial species have been described [56]. Our results are part of a broad set of evidence that indicates that the Neotropical biodiversity, particularly through the Amazon, cannot be trivialized [57][58][59][60][61][62].
In the last decade, 155 new species of Pristimantis have been described for South America, mainly from the Andean regions of Peru, Colombia, Ecuador and the Guianas Shield. The heterogeneity in the topography of the Andes, accompanied by the life history of Pristimantis (direct development, high endemism and ability to colonize various habitats, including high altitudes) could be partly responsible for this notorious biodiversity [4]. The regions with the most homogeneous topography (Amazon Basin) present a high number of species that undergo a process called morphological stasis [16], where species that suffer strong selection by the environment or physiological characters for adaptation in a specific habitat, may not present morphological changes between species [63]. This morphological conservatism in Pristimantis represents one of the main problems for accurate identification of the species, resulting in an underestimated number of species for this genus [64], [65].
The occurrence of sympatric Pristimantis species has been recorded in several studies [45], [66], [67]. In this study, P. giorgii/P. latro and P. pictus/P. pluvian can be found in sympatry, with subtle morphological differences, although with great variation in genetic distance and vocalization (see Tables 1 and 3). Many cryptic species complexes are sympatric and not necessarily sister species, providing strong indirect evidence that represent independent haplogroups and cannot hybridize [68], [69]. The current sympatric distribution may be the result of secondary contact after the expansion of past populations [70]. The great variation observed in the vocalization of sympatric species may be the result of cryptic diversification and acoustic radiation, making it potentially evident in highly diversified and widely distributed anuran groups, as the Pristimantis genus [48]. Phylogenetic reconstruction shows that P. pluvian, P. fenestratus, P. sp. (municipality of Juruti, PA) and P. sp. (municipality of Borba, AM) belong to a hard polytomy, which may be common in organisms experiencing rapid speciation but with imcomplete lineage sorting in mtDNA. On the other hand, this may be because we do not have enough molecular data to describe how these lineages are related.
The concern with the global decline of amphibian species is an old debate in the academic community [70][71][72][73]. One of the main causes of this phenomenon is the loss of habitats through deforestation and fragmentation [74][75][76]. Once many species have not yet been described, efforts to catalog and describe biodiversity need to be prioritized [16], as in the case of Pristimantis sp1, that occurs only in the area called Terra do Meio, deserving greater attention for its taxonomic resolution, incorporating morphological and molecular data, in order to help in the knowledge of the biodiversity of the eastern Brazilian Amazon and the conservation of the species.