A new species of Proceratophrys Miranda-Ribeiro, 1920 (Anura, Odontophrynidae) of the P. bigibbosa species group from Southern Brazil

The monophyletic Proceratophrys bigibbosa species group has a stable taxonomic history. Despite the increasing number of Proceratophrys described in the last decades, the P. bigibbosa group, for a long time, has consisted of four species distributed in south of South America; except for P. palustris that occurs in southeastern Brazil. Herein, based on concordant evidence of morphology and mtDNA barcoding, we describe a new species of Proceratophrys for specimens assigned to the P. bigibbosa group collected in Tibagi municipality, Paraná state, Brazil. The new species is diagnosed by its small size, snout rounded in dorsal view, palpebral ridge with small and rounded tubercles, small postocular swellings, presence of a line of small and round tubercles on dorso-lateral region of body, and dorsal region covered by small, sparse, and rounded tubercles. We also highlight the potential occurrence of the new species in other areas along the Campos Gerais of Paraná, given the strong association of the species with this vegetational formation in South Brazil.


Introduction
The knowledge on the systematics and phylogenetic relationship among species in the genus Proceratophrys have rapidly increased in the last decades (Dias et al. 2013;Mângia et al. 2018Mângia et al. , 2020Magalhães et al. 2020). Nevertheless, the traditional morphological groups of Proceratophrys still lacks phylogenetic support. Despite that most of the groups recovered are paraphyletic, three clades were recurrently consistent with available phylogenetic analysis: a clade of restricted Caatinga species (P. cristiceps, P. minuta, and P. redacta), the P. concavitympanum clade (P. ararype, P. concavitympanum, P. moratoi, P. salvatori, and P. strussmannae) distributed in Amazonia, Cerrado, and Brejos de Altitude within the Caatinga, and the P. bigibbosa species group, composed of P. avelinoi, P. bigibbosa, P. brauni, and P. palustris (Teixeira-Jr et al. 2012;Mângia et al. 2018Mângia et al. , 2020Magalhães et al. 2020). Although the P. bigibbosa group has been recovered as monophyletic with high support, P. palustris still lacks DNA information and is placed in this group based on putative morphology basis.
The number of species of Proceratophrys has increased by 55% in the last two decades. However, the P. bigibbosa species group (revised 20 years ago by Kwet and Faivovich 2001) has a stable taxonomic history. Species within this group are mainly diagnosed by the presence of post ocular swellings and being distributed in southern of South America, except for P. palustris which is only known from its type locality in Poços de Caldas Plateau, south of Minas Gerais state, southeastern Brazil (Giaretta and Sazima 1993;Kwet and Faivovich 2001;Caldart et al. 2010).
During field expeditions in the Tibagi municipality, Paraná state, South Brazil, we collected specimens of Proceratophrys belonging to the P. bigibbosa species group, presenting one of the most northern distribution (except for P. palustris) that aroused questions about their taxonomy. We combined morphological and mtDNA barcoding evidence to elucidate its status and concluded that no available name could be applied to this population. Herein, we describe the population from Tibagi as a new taxon.

Sampling
We conducted visual surveys at Tibagi municipality, in the Guartelá Canyon region from Campos Gerais of Paraná state, Brazil, in February 2016. All specimens were captured manually and killed using 5% lidocaine, fixed in 10% formalin, and transferred to 70% ethanol for permanent storage (following Conselho Federal de Biologia-CFBio No 148/2012. Voucher specimens are housed in the Coleção de Anfíbios do Laboratório de Anfíbios e Répteis da Universidade Federal de Santa Maria (acronym ZUFSM), Santa Maria, Brazil, and Coleção Zoológica da Universidade Federal de Mato Grosso do Sul (acronym ZUFMS-AMP), Campo Grande, Brazil (Appendix I). We state here that appropriate protocols for the collection and handling of the individuals were followed for the present research according to Brazilian federal law. Collect permit was issue by ICMBio (SISBIO #49876-1) and IAP (#03.15).

Morphology
Specimens used in the description of the new species, as well as specimens examined for comparisons, are housed in 11 herpetological collections in Brazil (Appendix 1).
Terminology for morphological characters follows Kwet and Faivovich (2001), Prado and Pombal Jr. (2008), and Brandão et al. (2013). We follow Kwet and Faivovich (2001) for the ten morphometric variables: snout-vent length (SVL); head length, defined as the diagonal distance from the tip of the snout to the right angle of the jaw (HL); head width, defined as the distance between the angles of the jaw (HW); horizontal eye diameter (ED); eye-nostril distance (EN); nostril-snout distance (NS); internarial distance (IN); tibia length (TL); foot length (FL); inner metatarsal tubercle length (ML). All measurements were taken by DJS using a digital caliper (0.01 mm precision). We determined the sex of each individual by the presence of vocal slits in males and their absence in females.

Phylogenetic inference and genetic distances
We extracted genomic DNA from liver samples using the phenol-chloroform protocol (Sambrook and Russell 2001) and sequenced fragments of the 16S ribosomal RNA mitochondrial gene from three individuals of the new species (genbank numbers provided in Appendix II). We used the 16Sa/16Sb primer pair (Palumbi et al. 1991), following the polymerase chain reaction (PCR) conditions described by Costa et al. (2016). PCR reactions consisted of 1× buffer, dNTPs at 0.2 mM, each primer at 0.2 μM, MgCl2 at 2 mM, 1 U Taq polymerase, and 2 μl of template DNA, in a total reaction volume of 25 μl. We used the following PCR cycling program: 94ºC for 2 min, followed by 35 cycles of 94ºC for 30 s, 59ºC for 1 min, and 72ºC for 1 min, and a final 5 min extension at 72ºC. We purified PCR products with Ethanol/Sodium Acetate and sequenced them on an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, California). 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 along with 16S sequences of 28 species of Proceratophrys and with the outgroups Odontophrynus spp., Macrogenioglottus alipioi, Cycloramphus acangatan and Thoropa miliaris, which were available in Gen-Bank (Appendix II). The final aligned dataset used in all analyses comprised 498 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 20 million generations, sampling every 2,000 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 v.2.6.3 (Bouckaert et al. 2019). We also calculated sequence divergence (uncorrected p-distance, Appendix II) among species/individuals using MEGA v10.1.1 (Kumar et al. 2018).
Description of the Holotype. Head wider than long (HL/ HW = 0.70), head length 32% of SVL, snout rounded in dorsal and ventral views, obtuse in profile; nares elliptical and prominent, canthal crests well marked, prominent, and covered by small tubercles; no preocular crests; eyes directed anterolaterally, eye diameter 38% of head length; eyelid with distinct, rounded tubercles, with the contact point between the ocular-dorsal ridge of warts and the external eyelid margin tubercles in a tubercle posterior to the post-ocular swellings, six warts on the border of the left eyelid and five on the right; sparse tubercles on the eyelid; distinct tympanum; vomerine teeth in two short rows between and below the choanae; frontoparietal crests well developed; region between frontoparietal crests shallow; interocular ridge of warts not organized in a row, with sparse small rounded tubercles; ocular-dorsal ridge of warts incomplete, 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 rounded; single outer metacarpal small, both internal and external are rounded; scarce small, rounded supernumerary tubercles; subarticular tubercles large, rounded, but grooved anteriorly and posteriorly. Toe lengths I > II > V > III > IV; inner metatarsal tubercle  Color Pattern in Life (Fig 3). Dorsal coloration overall in variable shades of brown, with regular patterns of dark brown blotches in the dorsum. Presence of longitudinal irregular stripes of light brown in dorsolateral region. Gu- lar region cream colored with mottling dark brown. Belly dark brown to black, irregularly spotted with yellow. Ventral surface of limbs dark brown to black, spotted with yellowish marks. Palm, fingers, soles of foot and toes are black, with two to three transverse dark-brown bars on fingers and toes.

Color Pattern in Preservative.
Overall coloration about the same as in life. However, the color became faded, and the light tones became darker. The longitudinal irregular stripes are brown in dorsal-lateral region. Gular region color beige with mottling dark brown. Belly dark brown irregularly spotted with beige.
Variation. The main variation within this species relies on the sexual size dimorphism, with females ( Fig. 4; 33.46-39.36 mm) bigger than males ( Fig. 5; 22.97-27.10 mm); in addition, males have a darker gular region (Fig.  5). Overall, the tubercles on the dorsum can vary in size, and some can be rounded to triangular. The variation of the dorsal coloration is more prominent in the dark brown blotches that border the dorsal row of tubercles. The ventral pattern varying slightly on shape and size of yellowish blotches (Fig. 4 and 5).
Phylogenetic Inferences and Mitochondrial DNA Divergences. Our 16S tree (Fig. 5) confidently recovered P. kaingang sp. nov. nested within the P. bigibbosa species group, and as the sister taxon of P. brauni (pp > 0.95). All nodes for species in the P. bigibbosa species group are well supported (pp > 0.95); however, some deeper nodes within Proceratophrys had low posterior probabilities, probably due to the single based gene tree. Average sequence divergence between the new species and its congeners within the P. bigibbosa species group ranges from 2.1% (P. brauni) to 5.6% (P. bigibbosa) (Appendix II).
Distribution and Natural History. Proceratophrys kaingang sp. nov. is known only from its type locality, the Guartelá Canyon region, Tibagi municipality, in the Campos Gerais of Paraná state, Brazil (Fig. 7). Grassland physiognomies (e.g., rocky vegetational refuge, hygrophilous steppe, and grassy-woody steppe) are predominant in this region (Fig. 8), consisting of relictual vegetation that include Mixed Ombrophilous Forest and Cerrado mosaics (Carmo et al. 2012;Souza et al. 2018). Calling males and a female were found in temporary puddles and slow running waters associated to flooded grasslands in the Private Reserve of Natural Heritage (RPPN Rancho   Table 1. Morphometric measurements (mm) for the type series of Proceratophrys kaingang sp. nov. Measurement acronyms: snout-vent length (SVL); head length, defined as the diagonal distance from the tip of the snout to the right angle of the jaw (HL); head width, defined as the distance between the angles of the jaw (HW); horizontal eye diameter (ED); eye-nostril distance (EN); nostril-snout distance (NS); internarial distance (IN); tibia length (TL); foot length (FL); inner metatarsal tubercle length (ML).

Measurement
Holotype ZUFSM11127 Sonho Meu) and in a wetland in agricultural landscape. Calling activity was recorded in both diurnal and nocturnal periods (from early afternoon until at least ~11:00 h pm) during a historic event of heavy rains in the Paraná state. Males called from moist soil, exposed at the muddy edges of puddles as well as partially submerged in shallow flowing water, hidden among hygrophilous vegetation. Males of at least 12 other species were calling in the Etymology. The specific epithet kaingang is a noun in apposition referring to the Kaingang (or Caingangue) ethnic group, which inhabits the plateau regions of the states of Paraná, São Paulo, Rio Grande do Sul and Santa Catarina, Brazil. We suggest the following Portuguese vernacular names "sapo-de-chifre-dos-caingangue" or "sapo-de-chifre-do-guartelá".

Discussion
Proceratophrys kaingang sp. nov. is the fifth species of the P. bigibbosa species group. Similarly to other species within this group, the new species also occurs in open formations in ecotone areas with subtropical forests (Giaretta and Sazima 1993; Kwet and Faivovich 2001;Caldart et al. 2010). The geographical distribution of P. bigibbosa species group is similar to those reported for the genus Julianus that include a disjunct gap of J. pinima, i.e., a northern relictual distribution associated to rock outcrops of the Espinhaço Mountain Range of Minas Gerais state, and a southern distribution along the highland grasslands   Our search for anuran fauna in ponds in the region, among crops and silviculture, but did not find any individual. However, we found the new species inside a conservation unit, where the population was in breeding activity. Despite the stable taxonomic history of the P. bigibbosa species group in the last two decades, the description of P. kaingang sp. nov. and the rapid increase of species discovered in the genus highlights the need to study biogeographic and evolutionary patterns to better understand the species distribution. In addition, recent integrative approaches used in Proceratophrys taxonomy (Teixeira-Jr et al. 2012;Mângia et al. 2018Mângia et al. , 2020Magalhães et al. 2020;) should be applied to populations assigned to the P. bigibbosa group recently (Santos et al. 2009;Caldart et al. 2010;Carosini et al. 2010). Although P. kaingang sp. nov. is known only from its type locality, other populations belonging to the P. biggibosa group along the Campos Gerais of Paraná may reveal the real distribution of the new species and improved our knowledge to understand its conservational status. for financial support. We thank all the landowners who granted access to the study sites, and to Sonia Z. Cechin (UFSM) for allowing us to examine specimens under her care.