A new species of Proceratophrys Miranda-Ribeiro, 1920 (Anura, Odontophrynidae) from Southern Amazonia, Brazil

Based on concordant differences in morphology, male advertisement call, and 16S mtDNA barcode distance, we describe a new species of Proceratophrys from southern Amazonia, in the states of Mato Grosso and Pará, Brazil. The new species is most similar to P. concavitympanum and P. ararype but differs from these species by its proportionally larger eyes and features of the advertisement call. Additionally, genetic distance between the new species and its congeners is 3.0–10.4% based on a fragment of the 16S rRNA gene, which is greater than the threshold typically characterizing distinct species of anurans. Using an integrative approach (molecular, bioacoustics, and adult morphology), we were able to distinguish the new species from other congeneric species. The new species is known only from the type locality where it is threatened by illegal logging and gold mining as well as hydroelectric dams.


INTRODUCTION
Integrative taxonomic approaches are considered paramount to describe new species (Padial et al., 2010). This is especially true for taxa in megadiverse regions, where new species are usually found (Toledo & Batista, 2012;Moura et al., 2018), and within speciose genera, where species are often misidentified (Fouquet et al., 2007). Such a diversity scenario occurs in Amazonia, the largest tropical forest in the world, where many anuran species are still unnamed (Funk, Caminer & Ron, 2011;Rojas et al., 2018). Many Amazonian species are considered widespread, but studies using integrative approaches have unveiled new species and lineages covering much smaller areas than previously known (Fouquet et al., 2007;Funk, Caminer & Ron, 2011;Ferrão et al., 2016). Focusing taxonomic efforts to reveal this hidden diversity is important for properly documenting Earth's biodiversity (Brito, 2010) and for illuminating diversification patterns. Undescribed species occurring in megadiverse regions may also face threats that lead them to become extinct before they can be formally described (Lees & Pimm, 2015),

Morphology
Specimens used in the description and examined for comparison are housed in 11 herpetological collections in Brazil (see Appendix 1). We followed the terminology for morphological characters of Prado & Pombal (2008) and Brandão et al. (2013). We followed Prado & Pombal (2008) for the 13 morphometric variables: snout-vent length (SVL), head length (HL), head width (HW), distance from the interocular crest to the tip of snout (DICS), internarial distance (IND), eye-nostril distance (END), eye diameter (ED), upper eyelid width (UEW), interorbital distance (IOD), thigh length (THL), tibia length (TL), foot plus tarsus length (FL), and forearm and hand length (FHL). All measurements were made by DJS using a digital caliper (0.01 mm precision, which we rounded to one decimal in order to avoid imprecision). We determined the sex of each individual by the presence of vocal slits in males and their absence in females. Finally, for the eyelid pattern tubercles formulae, we followed Brandão et al. (2013), which takes into account the number of anterior and posterior tubercles in each eyelid (right and left), the extension of the medial tubercle, expressed by the side (L or R), the number of anterior tubercles, the extension of the elongated medial tubercle (expressed in proportion), and the number of posterior tubercles.

Bioacoustics
We recorded the advertisement call of the paratype ZUFMS-AMP13680 at Jacareacanga (the type locality), and analyzed a total of eight advertisement calls. We used a Tascam DR-44 digital recorder to record the calls around 20:00 h (air temperature 24.3 C; humidity 86%) recorded at 44.1 kHz with 16-bit resolution in .wav format. We analyzed calls in Raven Pro v1.5 for Mac (Bioacoustics Research Program, 2014) and constructed audio spectrograms in R using the package 'seewave' (Sueur, Aubin & Simonis, 2008) with the following parameters: FFT window width = 256, frame = 100, overlap = 75, and flat top filter. We analyzed the acoustic parameters: call duration, pulse number per call, pulse rate, which was measured as the ratio of the absolute number of pulses and the absolute duration in which these pulses were emitted, and dominant frequency. Terminology of call descriptions follows Köhler et al. (2017), and values are reported as average ± SD (minimum-maximum). We deposited the sound recording in the acoustic collection of the Fonoteca Mapinguari da Universidade Federal de Mato Grosso do Sul (MAP-V 329).

Phylogenetic inference and genetic distances
We sequenced fragments of the 16S ribosomal RNA mitochondrial gene from three individuals of the new species and four individuals of P. strussmannae from its type locality (Document S1). We extracted genomic DNA from liver samples using the phenolchloroform protocol of Sambrook & Russell (2001). We used the 16Sa/16Sb primer pair of Palumbi et al. (1991), following PCR conditions described by Costa et al. (2016). PCR reactions consisted of 1× buffer, dNTPs at 0.2 mM, each primer at 0.2 mM, MgCl 2 at two mM, one U Taq polymerase, and two ml of template DNA, in a total reaction volume of 25 ml. We used the following PCR cycling program: 94 C for 2 min, followed by 35 cycles of 94 C for 30 s, 59 C for 1 min, and 72 C for 1 min, and a final 5 min extension at 72 C. We purified PCR products with Ethanol/Sodium Acetate and sequenced them on an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Resulting sequences were edited and aligned using Geneious v9.1.2 with the MUSCLE algorithm using default parameters (Edgar, 2004). We aligned our 16S sequences with 16S sequences of other species of Proceratophrys and with the outgroups Odontophrynus spp., Macrogenioglottus alipioi, Cycloramphus acangatan and Thoropa miliaris, which are available in GenBank (Document S1). The final aligned dataset used in all analyses comprised 421 base pairs (bp) of 16S. We used the Bayesian Information Criterion in jModelTest (Darriba et al., 2012) to determine that HKY+I+G was the best model of nucleotide substitution for our 16S data set.
We performed a Bayesian phylogenetic analysis of 16S using BEAST v.2.6.3 (Bouckaert et al., 2019) for 50 million generations, sampling every 5,000 steps using a Yule Process tree prior. We checked for stationarity by visually inspecting trace plots and ensuring that all values for effective sample size were above 200 in Tracer v1.7.1 (Rambaut et al., 2018). The first 10% of sampled genealogies were discarded as burn-in, and the maximum clade credibility tree with median node ages was calculated with TreeAnnotator v2.6.3 (Bouckaert et al., 2019). We also calculated sequence divergence (uncorrected p-distance) among species/individuals using MEGA v10.1.1 (Kumar et al., 2018). In order to explore the relationship among haplotypes, we estimated haplotype networks among species closely related to the new species (P. concavitympanum clade) for the 16S mtDNA gene in POPART (Leigh & Bryant, 2015) using the median-joining network method. We identified each species using different colors in the haplotype network.

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 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 LSID (Life Science Identifier) for this publication is: LSIDurn: lsid:zoobank.org:pub:4077F2CC-A0B1-49AC-B562-2FE7CC929668. The electronic edition of this work was published in a journal with an ISSN, has been archived, and is available from the following digital repository: www.peerj.com/.
Proceratophrys korekore sp. nov. has fused small pointed warts on the upper eyelid border (short, fused, and not pointed in P. ararype; small, rounded, and not fused in P. cururu and P. rotundipalpebra; slightly fused without appendage in P. branti, P. huntingtoni, P. vielliardi, and P. moratoi; conical and pointed in P. bagnoi; enlarged, pointed, and with the largest tubercle in the middle more projected than lateral tubercles in P. minuta; small and rounded in P. redacta; multiple short and pointed expansions in P. schirchi). From P. bagnoi, P. concavitympanum, P. dibernardoi, and P. goyana, P. korekore sp. nov. differs by the presence of a single row of tubercles of different sizes bordered with some sparse tubercles on the forearm (two well-delimited rows of tubercles in P. bagnoi, P. concavitympanum, and P. dibernardoi; tubercles not organized in rows in P. goyana). From P. concavitympanum, P. korekore differs by its proportionally larger eyes ED/END 1.1-1.3 (ED/END 0.8-1.0 in P. concavitympanum). From P. salvatori, P. korekore sp. nov. differs by the presence of an ocular-dorsal ridge of warts (lack of ocular-dorsal ridge of warts in P. salvatori). Proceratophrys korekore sp. nov. also differs from P. ararype by the shorter duration and lower number of pulses/call in the advertisement call (0.162-0.332 s, 18-31 pulses/call versus 0.374-0.648 s, 38-65 pulses/call, respectively). The lower pulse rate differentiates P. korekore sp. nov. (96.4-111.1 pulses/s) from P. moratoi (69-86 pulses/s), and the lower dominant frequency (861.3 Hz) from P. ararype and P. moratoi (1,033.6-1,378.1 Hz and 1,153-1,594 Hz, respectively).
Description of the holotype. Head wider than long, head length 70% of SVL, snout semi-circular in dorsal and ventral views, obtuse and slightly vertical in profile; nares elliptical and prominent, canthal crests marked, prominent, and covered by small tubercles; no preocular crests; eyes directed anterolaterally, eye diameter 38% of head length and 95% of upper eyelid width; eyelid with short warts, with the contact point between the ocular-dorsal ridge of warts and the external eyelid margin tubercles in the posterior third region, eight warts on the left eyelid and six on the right (L 2, 2/5, 5; R 2, 2/5, 3), with one more prominent; presence of one row of tubercles on the eyelid; indistinct tympanum; vomerine teeth in two short rows between and above the choanae; frontoparietal crests well developed; region between frontoparietal crests shallow; interocular ridge of warts organized in a row, markedly curved; ocular-dorsal ridge of warts complete from the eyes to the sacral diapophysis, and discontinued to the coccyx region. Dorsal surface, including flanks, arms and legs, with various warts of different sizes and shapes, a single row of tubercles in different sizes bordered with some sparse tubercles on the forearm; ventral surfaces, except hands and feet and cloacal region, covered by numerous small, rounded, uniform warts. Finger lengths IV > II > I > III (Fig. 2B); interdigital webbing absent; inner metacarpal tubercle large and elliptical; outer metacarpal divided in two parts, both internal and external are elliptical; scarce small, rounded supernumerary tubercles; subarticular tubercles large, rounded, but grooved anteriorly and posteriorly. Thigh length longer than tibia length (THL/TL = 1.1), the sum of thigh and tibia lengths 81% of SVL; toe lengths I > II > V > III > IV; inner metatarsal tubercle long, elliptical, spatulated; outer metatarsal tubercle small, rounded; scarce small, rounded supernumerary tubercles; subarticular tubercles large, nearly rounded, grooved anteriorly and posteriorly.  (Table 1).
Color of the holotype in preservative. Dorsal background color brown. Area delimited by the ocular-dorsal ridge of warts light brown, bordered by four dark brown triangular blotches on each side. Two light brown bands from the eye to the upper lip. From two to three transverse dark-brown bars on the fingers and toes. Ventral surface cream with mottling dark brown, becoming darkish in the gular region (Fig. 1B). Variation. Color variation is related to the size and shape of blotches, and background color varies from light to dark brown in life (Fig. 3) and in preservative (Fig. 4). Some individuals do not have a complete ocular-dorsal ridge of warts, and it varies within individuals with some more complete than others (Fig. 4). Within the collected type series, females are larger than males. Measurements of the type series are provided in Table 1.
Distribution. The new species is known only from the type locality, Jacareacanga municipality, Pará state, and few sites on the other side of the Teles Pires River, Paranaíta municipality, Mato Grosso state (Fig. 8).
Natural History. Proceratophrys korekore sp. nov. was found inhabiting only dense ombrophilous forest formations on both banks of the Teles Pires River. During nocturnal surveys between 18:00 and 21:00 h in the rainy season (from October to June), we observed males of P. korekore sp. nov. calling from the leaflitter on the banks of temporary streams. Some males were also found calling on bare soil near the edges of waterbodies. Additionally, individuals of the new species were observed during surveys (both diurnal and nocturnal) moving through the forest or captured using pitfall traps, even far from water bodies. Sympatric species include Ameerega munduruku, Ceratophrys cornuta, and Lithodytes lineatus. The main anthropogenic threats to the type locality are illegal  (2012) Note: Values are presented as mean ± SD (range).
logging and the often-subsequent deforestation for cattle grazing. Furthermore, illegal gold mining inside the forests (locally called "garimpo") was observed during fieldwork. The activity of these illegal miners consists of clearing all vegetation of a target area and then destroying entire sections of stream bank to extract gold using mercury, a heavy metal that pollutes water supplies and poisons fish stocks (Silva et al., 2018). Finally, the Teles Pires River has also been dammed by multiple hydroelectric power plants, which have caused considerable natural habitat losses and fragmentation.
Etymology. The specific epithet "korekore" is a noun in apposition that means frog in the language of the Mundurukus, an indigenous group that inhabits the southwestern parts of Pará state and the northern region of Mato Grosso state, Brazil. We suggest the following Portuguese vernacular names "sapo-korekore" or "sapo-de-chifre-dosmundurukus".
In previous studies on Proceratophrys using molecular data, only three clades were consistently recovered as monophyletic: the P. bigibbosa species group distributed in southern South America (Kwet & Faivovich, 2001), the clade formed by P. cristiceps, P. minuta, and P. redacta distributed in northeastern Brazil, and the P. concavitympanum clade composed of P. concavitympanum, P. korekore (Amazonia), P. ararype (Caatinga), P. moratoi, P. salvatori and Proceratophrys aff. ararype (Cerrado) (Amaro, Pavan & Rodrigues, 2009;Pyron & Wiens, 2011;Teixeira et al., 2012;Dias et al., 2013;Mângia et al., 2018;Magalhães et al., 2020;Cruz & Napoli, 2010). However, the relationships among the remaining sequenced (13 species) and not sequenced (15 species) species of Proceratophrys lack phylogenetic resolution and there are no morphological features that could be used to group them, highlighting the need for more robust phylogenetic analysis using multi-locus approaches and coalescent-based methods. The population from Tocantins state, which we called P. aff. ararype here, has an average 1.8% genetic divergence in 16S from P. ararype. Genetic distance between species in the 16S barcode mtDNA locus is usually low within Proceratophrys, with some morphologically distinct species being less than 1.5% divergent (e.g., P. belzebul-P. appendiculata-P. izecksohni; P. itamari-P. mantiqueira) (Dias et al., 2013, Document S2). Nevertheless, based on the distributions of P. aff. ararype and P. ararype and no shared haplotypes among populations, further investigation is needed to clarify the taxonomic status of the population from Tocantins.
The description here of P. korekore sp. nov. constitutes the third new species described from the area under the influence of the São Manoel and Teles Pires hydroelectric power plants, the others being the watersnake Helicops apiaka (Kawashita-Ribeiro, Ávila & Figure 7 Haplotype network. Median-joining haplotype network of specimens from P. concavitympanum clade based on 16S mtDNA. Each haplotype is represented by a circle whose area is proportional to its frequency. Traits indicate additional mutational steps for branches with more than one mutation. Different colors indicate species-level units. The black dots are median vectors (hypothesized sequences).
Full-size  DOI: 10.7717/peerj.12012/ fig-7 Morais, 2013) and the poison-frog Ameerega munduruku (Neves et al., 2017). These three taxa are known only from this region, which is also located in the 'arc of deforestation' (Souza et al., 2019). Habitat loss in Amazonia due to river dams and deforestation are directly causing vertebrate extinctions (Benchimol & Peres, 2015;Silva et al., 2018). This scenario underscores the need for sampling surveys and integrative taxonomic approaches to reveal Neotropical anuran diversity, which will lead to a better understanding of species distributions and evolutionary history.  Animal Ethics

APPENDIX I Additional specimens examined
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): Collection permits for this study were issued by ICMBIO (SISBio 45889-1).

Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): Collection permits for this study were issued by ICMBIO (SISBio 45889-1).

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The 16S mtDNA sequences described here are available at GenBank: MW889928 to MW889930.

Data Availability
The following information was supplied regarding data availability: Data are available at GitHub: https://github.com/Rhinella85/Proceratophrys_korekore.