A new species of Odontophrynus (Anura, Odontophrynidae) from the southern portion of the Mantiqueira mountains

. Using an integrative approach (morphology of the adult and larvae, bioacoustics, osteology, karyotype, and molecular data), we described a new tetraploid species of Odontophrynus to the Mantiqueira mountain range, in southeastern Brazil. The data suggest that Odontophrynus toledoi sp. nov., O. juquinha and Odontophrynus sp. (aff. juquinha ) comprise a clade with specimens distributed along three distinct mountain ranges in Brazil: Mantiqueira ( O. toledoi sp. nov.) and Espinhaço ( O. juquinha ) mountains, both in southeastern Brazil, and Diamantina Plateau ( O. aff. juquinha ), in northeastern Brazil. The new species is morphologically similar and closely related to O. juquinha , but is distinguished in morphology (both adult and larval), karyotype ( O. toledoi sp. nov. is tetraploid and O. juquinha is diploid), and corroborated by phylogenetic inferences. We also show that these species do not exchange haplotypes in the 16s gene. Furthermore, although the raw acoustic parameters of Odontophrynus toledoi sp. nov. and O. juquinha overlap at the limits of their ranges, we found a clear difference in the acoustic space structure.


Introduction
The genus Odontophrynus Reinhardt & Lütken, 1862 is composed by ground-burrowing mediumsized toads with nocturnal activity, distributed throughout eastern South America, from northeastern Brazil to southwestern Argentina, and mainly associated with open formations (Caramaschi & Napoli 2012;Frost 2021).The phylogenetic position of this genus and its species has been discussed for over a decade (Pyron & Wiens 2011;Martino et al. 2019;Magalhães et al. 2020).However, in spite of previous discussions, the genus could be phenotypically diagnosed by the putative synapomorphy of foot musculature (see Blotto et al. 2017).Currently, the genus includes ten species arranged in two species groups: the O. americanus and the O. cultripes groups, plus O. occidentalis (Berg, 1896), a species not associated to any group (Martino et al. 2019;Rosset et al. 2021).
One of the major taxonomic issues in this group is the identity of O. americanus throughout its geographic range.Several studies have pointed out the presence of more than one species under this name (Juncá et al. 2005;Martino et al. 2019;Rosset et al. 2021).In addition, there are populations with different ploidies referred to as O. americanus or Odontophrynus sp.(aff.americanus), and there are major gaps in the acoustic and genetic data, mainly from northern populations (see Rosset et al. 2006;Rocha et al. 2017;Martino et al. 2019).Thus, it is necessary to better evaluate these putative new cryptic taxa under the current name of O. americanus.
Integrative taxonomy is an important approach to understanding the identity and distribution pattern of species with the intersection of evidence from two or more independent lines of investigation (Padial et al. 2010).In anurans, polymorphic traits such as color patterns that are highly variable (Hoffman & Blouin 2000) may complicate the taxonomic identifi cation of species.Herein lays the importance of combining multiple evidences of diagnostic traits.This is especially true for the genus Odontophrynus, for which there are several cryptic species (e.g., Rosset et al. 2006Rosset et al. , 2021;;Cianciarullo et al. 2019), and lack of genetic, acoustic, and larval data for several populations (see Rocha et al. 2017;Martino et al. 2019;Rosset et al. 2021).
The Brazilian Atlantic Forest comprises several types of phytophysiognomy from the Northeast to the Southern regions of the country; less than 10% of the original cover remains (Colombo & Joly 2010).The montane Atlantic Forest species diversity observed in the state of São Paulo has been attributed to factors such as the high structural complexity of the environments (Rossa-Feres et al. 2010).The highest lands in this region are in the Mantiqueira mountain range, which runs for about 900 km along the borders of the states of São Paulo, Rio de Janeiro, Espírito Santo, and Minas Gerais, in southeastern Brazil (Peixoto et al. 2020).The Mantiqueira mountain range harbors one of the most diverse anuran fauna in the Atlantic Forest and is considered a hotspot of anuran endemism (Silva et al. 2018;Muscat et al. 2020).
In the course of a survey of anurans in the highlands of the southern Mantiqueira mountains in the state of São Paulo, southeastern Brazil, we found a frog belonging to the O. americanus species group for which we found enough information (genetics, acoustics, osteology, larval and adult morphology) to consider it as a distinct species.During the course of our surveys in the district of São Francisco Xavier, municipality of São José dos Campos, state of São Paulo, we also gathered in-depth data of its natural history.Here is the description of the new species.
Specimens used for comparisons are deposited in the following Brazilian collections:
For dorsal fi n height (DFH); ventral fi n height (VFH); and for oral disc width (ODW) we followed Grosjean (2005).In the labial tooth row formula (LTRF): A-1 (fi rst anterior labial tooth row) and A-2 (second anterior labial tooth row) refers to anterior portion of labial tooth; P-1(fi rst posterior labial tooth row), P-2(second posterior labial tooth row), and P-3 refer (third posterior labial tooth row) to posterior portion of labial tooth.We followed Pinheiro et al. (2012) for spiracle length (SL).Spiracle width (SW) measurements were also taken.We took all linear measures to the nearest 5 mm using software TPSDig2 (Rohlf 2015(Rohlf , 2017)).ND, SL and SW were taken using stereo microscope Zeiss Discovery V. 20 to the nearest 0.5 mm.Lateral line system was described based on Lannoo (1987).
To compare morphology between tadpoles of the genus Odontophrynus, we performed principal component analysis (PCA) by building a comparative table of the species of the genus Odontophrynus with the same 21 measurements (Supp.fi le 1: Table S2) used in description of the tadpole O. toledoi sp.nov.The comparative table was built using information present in literature and we inferred measurements from the pictures and illustrations using TPSDig2 (Rohlf 2017).We used means of the morphological measurements provided by authors or maximum range when measurements were inferred from the illustrations.We have three main measurements present for all descriptions of tadpoles: total length (TL), body length (BL), and tail length (TAL).However, some measurements were not present in all tadpole descriptions.We had 30.5% missing data concentrated in additional measurements (e.g., ESD, NSD, END, SSD, SL, SW), but 69.5% of the most common measurements were present.For this, we used an imputation method by cross-validation, which led to the smallest mean square error of prediction to estimate the number of dimensions for the PCA using the estim_ncpPCA function of the missMDA package (Josse & Husson 2016).Also, we used imputePCA of the missMDA package (Josse & Husson 2016) to estimate the missing values based on the number of components estimated previously.All analyses were performed in R software ver.4.1.3(R Core Team 2022).
Currently, multivariate analyses that explore acoustic-space have become increasingly common for the diagnosis of cryptic species (e.g., MacLeod et al. 2013;Andrade et al. 2020).In this approach, spectrograms are compared by using a variant of eigensurface analysis to sample and compare these surfaces (MacLeod et al. 2013).The SoundShape package (Rocha & Romano 2021) in R software implements the geometric morphometric approach to compare the acoustic-space of species.SoundShape fi rst applies a 3D sampling grid to the spectrogram, which allows the extraction of coordinates (semilandmarks) that correspond to the acoustic attributes of the species and how they are distributed in the acoustic-space.These positions are later used in a principal coordinate analysis (PCA) that summarizes the acoustic information, allowing for the comparison of different species.For this reason, and since the acoustic parameters of closely related species can phylogenetically overlap, we used this approach to explore the acoustic-space of Odontophrynus toledoi sp.nov.and Odontophrynus juquinha.These species possibly have similar calls and parameters that overlap in range.Although PCA is not a hypothesis test per se, it allows visualization of the variation of acoustic attributes along fewer axes than when looking at the original dataset.

Phylogenetic inference
We sequenced fragments of the 16S ribosomal RNA mitochondrial gene of 19 individuals from the new species and 18 from other species of Odontophrynus; we also included sequences of nine species of Odontophrynus available in GenBank (Supp.fi le 1: Table S1).All localities are listed in Supp.fi le 1: Table S1.
We extracted DNA from ethanol-preserved tissues from either liver or muscle, using an ammonium acetate precipitation method (adapted by Lyra et al. 2017) and DNeasy Blood & Tissue kit (QIAGEN Inc.) following the manufacturer's guidelines.We used the An16SF and An16SR primer pair designed by Lyra et al. (2017) following polymerase chain reaction (PCR) conditions described by the same authors.We purifi ed PCR products using a mix of 0.5 unit of thermosensitive alkaline phosphatase (FastAP) and 1 unit of Exonuclease I (Thermo Fisher Scientifi c Inc.) diluted in 2.45 lL of ultrapure water per PCR, incubated for 30 min at 37°C followed by 5 min at 95°C.We sequenced the purifi ed PCR products with BigDye Terminator Cycle Sequencing Kit (ver.3.0, Applied Biosystems) in an ABI 3730 automated DNA sequencer (Applied Biosystems) at Centro de Biologia Molecular e Engenharia Genética, Unicamp, Campinas, SP, Brazil.
We edited and aligned the resulting sequences in Geneious ver.9.1.2with the MUSCLE algorithm using default parameters (Edgar 2004).We found gaps in 16S and to avoid possible bias we removed it using Gblocks (Talavera & Castresana 2007).The fi nal dataset was 347 base pairs (bp; 92% of the original 375 positions).We deposited all sequences in GenBank (Supp.fi le 1: Table S1).
We used BEAST ver.2.6.5 (Bouckaert et al. 2019) to estimate a gene tree using Bayesian inference with the same 75 sequences used in IQ-TREE analysis.We employed a relaxed clock exponential and bModelTest for model selection (Bouckaert & Drummond 2017) and a constant size coalescent tree prior.We ran the analysis for 50 million generations, sampling every 2000 steps.We checked for stationarity by visually inspecting trace plots and ensuring that all values for effective sample size (ESS) were above 200 in Tracer ver. 5 (Drummond & Rambaut 2007).The fi rst 10% of sampled genealogies were discarded as burn-in, and we calculated the maximum clade credibility tree with median node ages with TreeAnnotator ver.1.8 (Drummond et al. 2012).
Based on our single loci (16S) data, to retrieve the evolutionary mitochondrial lineages, we implemented tree-based methods used for species delimitation.We used the BEAST generated tree in a Bayesian implementation of the Generalized Coalescent Yule (bGMYC) model with the bGMYC package (Reid & Carstens 2012), in R software (R Core Team 2022).The GMYC method performs a modelbased analysis to locate threshold points (or nodes) in the genealogy where there are transitions in branching rates, refl ecting inter-or intra-specifi c evolutionary processes (Pons et al. 2006).We also employed a Bayesian Phylogenetics and Phylogeography (BPP) analysis as a complementary method.BPP is a Monte Carlo Markov Chain (MCMC) program that analyzes DNA sequence alignments under the multispecies coalescent model (MSC) (Rannala & Yang 2003).
In order to explore the relationship among haplotypes, we estimated haplotype networks among Odontophrynus spp.for the recovered clade in the Bayesian topology, in POPART software (Leigh & Bryant 2015) using median-joining network method.

Diagnosis
Odontophrynus toledoi sp.nov. is a medium-sized species belonging to the genus Odontophrynus based on the phylogenetic position and a combination of morphological characters: granular skin on the dorsum and venter, head wider than long, snout truncate in profi le, tympanum hidden, fi rst subarticular tubercle on toe I enlarged, inner metatarsal tubercle large, tarsal fold short (Savage & Cei 1965;Caramaschi & Napoli 2012).The new species belongs to the Odontophrynus americanus species group based on phylogenetic affi nities and the combination of the following characters: absence of large dorsal, tibia and forearm glandular warts, with postorbital, temporal, and parotoid glandular warts not distinctly developed but with a series of small glandular warts of irregular size and shape, forming glandular ridges longitudinally oriented, on postorbital-parotoid regions (Caramaschi & Napoli 2012).Odontophrynus toledoi is distinguished from the remaining species belonging to the O. americanus group by the following combination of characters: (1) medium sized (SVL = 40.4-51.8mm in males and 45.0-54.5 mm in females of O. toledoi; Table 1); (2) head wider than long (HW/HL = 1.31); (3) dorsal surface of head, arms, body and limbs dark brown with arms and limbs with light brown stripes; (4) light mid-dorsal stripe present or interrupted in most of the specimens; (5) yellowish stripe between the eyes, resembling a ')' shape; (6) increased number of longitudinally oriented dorsal glandular ridges; (7) karyotype with 2n = 4X = 44, with fundamental number = 88; (8) advertisement call with dominant frequency of 775-1033 Hz; (9) pulse rate of 89-132 pulses/s; (10) large tadpoles (mean TL = 42.91-56.18mm); (11) one-two submarginal papillae on the posterior labium of each side of the oral disc near the posterior emargination; and (12) spiracle sinistral, short, inner wall fused to the body with small distal portion free.

Etymology
The specifi c epithet honors Professor Luís Felipe Toledo for his contribution in solving the mysteries of the natural history of Neotropical amphibians, especially those from southeastern Brazil and mostly within the Atlantic Forest, where the new species resides.tips; dermal fringes absent; interdigital webbing absent.Length of fi ngers: IV>II>V≥III.Subarticular tubercles large, nearly bilobated, proximal more developed than distal.Supernumerary tubercles rounded to oval, covering the palmar surface.Nuptial pads present on thumbs and posterior surface of inner metacarpal tubercles.Inner and outer metacarpal tubercles large, inner oval and outer longitudinally divided, internal part oval, external part elongated.Nuptial pads present on thumbs and posterior surface of inner metacarpal tubercles.Hind limbs stout and relatively short.Tarsal fold present, slightly curved, as long as inner metatarsal tubercle and approximately same length as tarsus.Toes slender with rounded tips; dermal fringes slightly developed; interdigital webbing present.Lengths of toes: IV>III≥V>II>I.Webbing formula: I 1-2 II 1-3 III 2-4 IV 4-2 V. Inner metatarsal tubercle large (IMT 19.3% of FL), shovel-like, with external border keratinized; outer metatarsal tubercle slightly distinct; subarticular tubercles rounded, subarticular tubercle of toe I and II, enlarged, greater than others.Supernumerary tubercles small and rounded, aligned, and covering the plantar surface.

Coloration of the holotype in life
Dorsal surface of head, arms, body, and limbs dark brown (Fig. 2).Arms and limbs with light brown blotches resembling stripes over a dark brown background.A horizontal yellowish stripe between the eyes, resembling a ')' shape.Yellowish dorsolateral stripes, starting behind the head towards the hind limbs.A yellow interrupted mid-dorsal stripe.Irregularly sized light and dark brown blotches distributed below the dorsolateral yellowish stripe.Upper lip yellowish interrupted by dark brown blotches.Vocal sac moss green to black.Venter whitish scattered with grey blotches gradually increasing on the side of the body.Nuptial pads light brown.Foot with outer metatarsal, subarticular, and supernumerary tubercles gray over light brown background.Tarsal fold grayish.Inner metatarsal tubercle light brown with keratinized dark brown portion.Iris with three marbled colors: golden in the dorsal region, black in the middle and whitish in ventral region; pupil horizontally elliptical with golden margins.

Coloration of the holotype in preservative
Dorsal background color predominantly dark brown.Light brown blotches on the arms and limbs.The yellowish stripes became light brown; the whitish or grayish coloration of the antebrachial glandular wart, tubercles of the hand and foot, and tarsal fold became cream-colored.Venter yellowish-colored, with scattered dark brown blotches gradually increasing on the sides of the body.Gular area with dark grey pigmentation.be poorly distinguished in some individuals and one individual had a distortion, interrupting the stripe.Mid-dorsal stripe is discontinuous and can begin on the head or not.

Tadpoles
Larvae external morphology Tadpoles of Odontophrynus toledoi sp.nov.have a total length of 42.91-56.18mm (47.71 ± 5.34 mm) in Gosner stages 37-40 (Lot ZUFMS-AMP 15276, Fig. 3, Table 2).Body is depressed (BH/BW = 0.85-0.86;Fig. 3A-C), rounded in lateral view (BL/BH = 1.81-1.86),elliptical in dorsal view (BL/ BW = 1.55-1.58),and slightly longer than one third of the total length (BL/TL = 0.36-0.37).Snout is rounded in lateral view and ovoid in dorsal view.Eyes dorsal, dorsolaterally oriented.Nostrils dorsal with oval shape, anterolaterally positioned with an elevated marginal rim, undeveloped inner margin projection, and closer to the tip of the snout than the eye (NSD/END = 0.77-0.79;Fig. 3G).Spiracle sinistral, short, inner wall fused to the body with small distal portion free, lateroventrally positioned, posterodorsally directed, and placed at half of the body length (SSD/BL = 0.57-0.60;Fig. 3F).Ventral tube median, dextral opening, ventrally directed, fused to the ventral fi n with posterior portion free and positioned slightly below ventral margin (Fig. 3D-E).The dorsal membrane of the ventral tube is slightly shorter than the ventral membrane (Fig. 3D).Intestinal mass is circular, located at the center and slightly displaced to the left from the abdomen (Fig. 3C).Tail has acute tip, comprising 62.3% of the total length and higher than the body (MTH/BH = 1.02-1.06).Tail musculature is slightly developed (TMH/BH = 0.46-0.51),myotomes visible and more developed on the anterior third and mid portion, and reach the tip of the tail.Dorsal fi n has slightly more convex margin than the ventral fi n.Dorsal fi n high (DFH/TMH = 0.83-1.00)and ventral fi n high (VFH/TMH = 0.52-0.60)with maximum height at the mid of the tail.Dorsal fi n emerges at the posterior third of body, and the origin of ventral fi n is at the inferior margin of ventral tube.Lateral line system has four neuromasts, almost indistinguishable, accumulated near the ventral tube, and others that we could delimit as: supraorbital around the eyes (SO); infraorbital around the snout and nares (IO); posterior supraorbital (PSO); posterior infraorbital near the musculature insertion (PIO); dorsal near the middle of tail musculature (D); and middle (M) on the half of body side (Fig. 3H-I).Oral disc small (ODW/BW = 0.29-0.30)anteroventrally positioned, laterally and posteriorly emarginated (Fig. 3A, J-K).Anterior labium has one single row of laterally long marginal papillae aligned on the top and alternated near the emargination with a wide gap anteriorly equivalent to P-2.Papillae aligned, reduced and spaced in lateral emargination.Posterior labium has one single row of long marginal papillae alternated without pigmentation and some bifi d aligned papillae, about 14 papillae/mm with four bifi d papillae (estimated on the posterior labium; Fig. 3J).There are one-two submarginal papillae on the posterior labium of each side of the oral disc near the posterior emargination.Submarginal papillae may be absent or not visible during initial stages (e.g., 30-37;Gosner 1960).Labial tooth row formula (LTRF) is 2(2)/3(1) with a short gap in A-2 and P-1; A-1 slightly shorter than A-2; P-1 and P-2 have the same size, both longer than P-3 (Fig. 3J-K).Labial teeth dark-colored arranged in a single row, one per tooth ridge with variable size.There are about 52 labial teeth/mm (estimated on P-3).Jaw sheaths serrated, heavily keratinized (with about 37 serrations/mm), and dark with lower sheath base ranging from brown to dark yellow.Upper jaw is arc-shaped with long lateral projection and lower sheath V-shaped, with lower jaw sheath slightly wider than the upper one (Fig. 3K).Coloration of tadpoles in life Body greenish in life with scattered dark blotches distributed all over it and concentrated on the lateral view of the body.Ventral region cream.Tail light cream with dark blotches, with concentration on last third of the tail.Musculature of the caudal portion easily seen due its reddish coloration (Fig. 4A).Mid dorsal cream stripe present in imagoes dorsum (Fig. 4B).

Coloration of tadpoles in preservative
Body coloration in preservative 10% formalin is light yellow or cream on the lateral view and dark yellow on dorsal view with melanophores located mainly near the eyes, nares, and on the anterior insertion of tail musculature on dorsal view.There are some angular patches on tail musculature and dorsal and ventral fi ns on the lateral view.Fins translucent with iridophores, mainly on the dorsal fi n.The intestinal region is translucent with small melanophores near to oral disk.

Osteology
Odontophrynus toledoi sp.nov.has a semicircular shaped skull (Figs 5-6), wider than long (SKW/ SKL = 1.41 ± 0.19; Table 3) and more elevated posteriorly.Frontoparietal relatively large, broad (width of frontoparietal 5.04 mm ± 0.59 mm), anterior margin rounded, posterior margin crescent-shaped, in contact medially without exposing frontoparietal fontanelle.Nasals triangular, wider than long, keeled, slightly separated from one another medially and well separated from the frontoparietal posteromedially; maxillary process of the nasals in contact with the pars facialis of the maxilla, and with the nasal process of the pterygoids.Maxillary arch complete.Short and wide alary process of the premaxilla.Pars facialis of maxillae bearing around 32 teeth, pars palatina broad, pterygoid process well developed, reaching the level of quadratojugal, wide anterior portion oriented to nasal and posterior portion that contacts maxillary process of nasals.Quadratojugals well developed, contacting maxilla.Vomers moderately sized, separated medially, prechoanal, postchoanal and anterior processes of the vomer well developed, anterior process contacts maxillary arch, dentigerous process of vomer bearing 4-5 teeth.Parasphenoid triradiate, cultriforme process pointed with four peaks, alae perpendicular to cultriform process.Zigomatic ramus of the squamosal moderately sized, anteroventrally directed, separated from the maxilla, enlarged posteriorly and rounded at the anterior end; otic ramus longer than zygomatic ramus.Sphenethmoid well developed, extending anteriorly to the anterior margin of the nasals.Palatines large, strong, narrowly separated medially, expanded laterally.Pterygoides triradiate, relatively large; anterior ramus long connected with maxilla, extending anteriorly to palatine, dorsally curved reaching maxillary process of nasals; medial ramus in contact with otic capsule.Prootic and exoccipital well developed.Occipital condyles large and broadly separated.Crista parotica broad and quadrangular.Posteromedial hyoid process long and well ossifi ed.Vertebral column with 8 presacral vertebrae, I and II imbricate;

Karyotype
Odontophrynus toledoi sp.nov. is a tetraploid species, with 2n = 4X = 44, with all chromosomes biarmed (FN = 88).The karyotype is composed of fi ve metacentric chromosome groups (1, 5-7, and 11) and six submetacentric chromosome groups (2-4 and 8-10; Fig. 8A).In cells in meiosis I, tetravalents were commonly seen, although in variable numbers (Fig. 8B).In Giemsa-stained metaphases, secondary constrictions were observed in the long arm of chromosomes 9, which corresponded to silver-stained NORs revealed by the Ag-NOR method.A notable variation was observed in NOR size in specimen ZUEC-AMP 24764.While three of its chromosomes 9 had large silver-stained NORs, which were easily seen as secondary constrictions in Giemsa-stained metaphases, one chromosome 9 had a small NOR (Fig. 8C), hardly seen in some metaphases.Such variation in NOR size justifi es the differences observed in chromosome 9 length (Fig. 8A, C).Constitutive heterochromatin was detected in the centromeric region of all chromosomes (Fig. 8D).

Phylogenetic inferences and mitochondrial DNA divergences
Our ).The two delimitation analyses, bGMYC and BPP, were almost entirely congruent with each other, supporting the existence of nine and ten species, respectively, in the clade of the Odontophrynus spp.(Fig. 9), which mostly agrees with the current taxonomy of the group.Furthermore, the mitochondrial haplotype network shows that O. toledoi does not share haplotypes with other species of the O. americanus species group (Fig. 10).

Geographical distribution
The distribution of Odontophrynus toledoi sp.nov.comprises the southern portion of the Serra da Mantiqueira and adjacent areas that share species with the mountain range, such as Jundiaí and Atibaia in the state of São Paulo.The occurrence area covers two states in Brazil (São Paulo and Minas Gerais; Fig. 10).The type locality is São Francisco Xavier (SFX; district of the municipality of São José dos Campos, São Paulo) in the 'Rio Manso' area.The species can also be found in the Projeto Dacnis private reserve and in other fl ooded areas within the district.In the state of São Paulo, the municipalities of Campos do Jordão (Parque Estadual de Campos do Jordão (Horto Florestal), Santo Antônio do Pinhal (RPPN Fazenda Renópolis) São Bento do Sapucaí, Caçapava, Pindamonhangaba, Piquete and Queluz (Martins, I.A. pers. com.) are also in its range.In the state of Minas Gerais, the species can be found in the municipalities of Airuoca, Itamonte, Cristina (Mata da Prefeitura; Fazenda Pouso Frio), Maria da Fé (Fazenda Pomária), Poços de Caldas, Rio Preto, Inconfi dentes and Marmelópolis.

Natural history
Odontophrynus toledoi sp.nov. is a terrestrial, nocturnal species commonly found in open and fl ooded areas.In the type locality (SFX), it was found mainly in valleys.In these locations, ephemeral wetlands receive more water from rivers and precipitation, especially during the rainy season (October-March).
Although the frogs are predominantly nocturnal, they can also be heard during daylight on rainy days.They spend most of their time buried in aestivation and generally only come out after heavy rains.
We also observed that females leave the aestivation period before males.Females emerge from holes approximately 7cm deep at the beginning of the rains, while males are only observed when there is a greater accumulation of precipitation.When noticing our presence, the species exhibited the defensive behaviors of burrowing (Fig. 11A-B), puffi ng up the body, stiff leg (Fig. 11C).Thanatosis occurred when they were handled.The species' coloration also provides camoufl age in its environment (Fig. 11D).It is an explosive breeder and males cluster around temporary pools.Adult males can be seen calling with the body partially submerged in fl ooded areas, hidden in vegetation.The amplexus is axillary.Tadpoles are exotrophic, benthonic and inhabit mainly lentic environments, such as temporary ponds (ecomorphological guild II: A: 1; sensu Altig & McDiarmid 1999).

Discussion
We described a new species of Odontophrynus within the O. americanus group based on multiple lines of evidence, such as external morphology (adults and tadpole), osteology, molecular data, karyotype, and acoustic attributes.On the basis of the 16S tree, this new species is sister to the clade (O.juquinha + Odontophrynus sp.[aff.juquinha]).The use of this gene in the present work suggests possible phylogenetic relationships and genetic distance that comprise a reliable line of evidence with the corroboration of other data for an integrative approach (see Padial et al. 2010).Morphology is maintained across the lineages, as in other species of the Odontophrynidae family, which can lead to subtle differences between closely related species (Rocha et al. 2017;Rosset et al. 2021;Santana et al. 2021).On the other hand, differences in karyotypes can result in reproductive isolation, being generally associated with distinctions in geographic distribution and acoustic parameters (Wasserman 1970;Stöck 1998;Martino & Sinsch 2002;Holloway et al. 2006;Rocha et al. 2017).In addition, we found that the clade species of O. toledoi sp.nov.occur in different biogeographic units.Odontophrynus toledoi is restricted to the Atlantic Forest of Serra da Mantiqueira, while O. juquinha is restricted to the Serra do Espinhaço savanna.Both mountain ranges are notable for their high number of endemic amphibian species (Cruz & Feio 2007;Silva et al. 2018).Another evidence is that, although the raw acoustic  S2.
parameters of Odontophrynus toledoi and O. juquinha overlap at the limits of the ranges, we found a clear difference in the acoustic space structure.These reasons, taken together, reinforce the reproductive isolation between the two lineages and add another recognized species to a genus that is restricted to southeastern Brazil.
We also added the osteological description of another species of Odontophrynus to the body of knowledge about the genus.In the O. americanus species group, only O. maisuma and O. reigi have their osteological structures described, and now O. toledoi sp.nov.(Rosset 2008;Rosset et al. 2021).We couldn't compare O. toledoi with O. juquinha because no osteology analysis was made of the latter.However, we reinforce the need for osteological characters in taxonomic and systematic studies, since some osteological features can be extremely well preserved, particularly in the skull (Trueb 1977;Duellman & Trueb 1994).This is especially true in the genus Odontophrynus, which was fi rst defi ned using skull osteological features alongside external morphology (Lynch 1971).The use of CT scans for osteological description provides the advantage of a non-invasive method, which can be applied in rare or even extinct species and also allows deeper reexamination of type specimens.However, this method is resolution-limited by the tomograph, which can determine the possibility of seeing or not small structures and forms, especially in the skull.
Two putative species within Odontophrynus had already been suggested by Martino et al. (2019) Beçak et al. (1966) for specimens from Campos do Jordão, São Paulo.Specimens from Campos do Jordão were also included in our molecular analyses and clustered together with topotypes O. toledoi, supporting the hypothesis that those sequences previously karyotyped by Beçak and collaborators belong to O. toledoi.The only difference between the karyotype we describe here and that described by Beçak et al. (1966) refers to the numbering of the smaller chromosomes.Probably, Beçak et al. (1966) disregarded the secondary constriction in the size analysis of the NOR-bearing chromosomes and considered that it was present in the short arm of chromosomes 11 (and not in the long arm of chromosomes 9, as we did).Although our cytogenetic study was based on males, Beçak and collaborators (1966) included males and females in their analyses, and heteromorphic sex chromosomes were not evidenced.Our study reinforces the importance of karyotypes for the diagnosis of species of Odontophrynus.
We found that Odontophrynus toledoi sp.nov., O. juquinha and Odontophrynus sp.(aff.juquinha) constitute a clade that is distributed along three different elevation areas in Brazil: Mantiqueira (O.toledoi) and Espinhaço (O.juquinha) ranges, and Chapada Diamantina (Odontophrynus sp.[aff.juquinha]).These three lineages corresponded to distinct haplogroups in the genetic analyses based on 16S rRNA partial gene and were recovered as independent evolutionary entities in the species delimitation tests.It is known that mountains constitute geographic barriers for several taxa, including frogs (Roberts et al. 2006;Vijayakumar et al. 2016).New studies should investigate how past climate may have infl uenced the speciation process of these three species.As they are associated with swampy areas and naturally exposed environments, they could be good models for understanding past connections of such different domains.
We observed that females leave the aestivation period before males.We suggest that this is likely due to greater investment in female reproduction, but this hypothesis still needs to be tested.Although the new species was found in at least 17 localities, wetlands are still constantly modifi ed in the type locality by urbanization and water drainage for human use.These actions could be of conservation concern for this species, since forested areas are more protected than naturally open environments.However, following the International Union for Conservation of Nature (IUCN) criteria, O. toledoi sp.nov.should be classifi ed as Least Concern (LC) in spite of ongoing and potential habitat loss.

Fig. 5 .
Fig. 5. Odontophrynus toledoi sp.nov., holotype, ♂ (ZUEC-AMP 24833), skeleton, generated by microtomography.A. Dorsal view of the individual.B. Ventral view of the individual.C. Lateral view of the head and arms.D. Ventral view of the skull.E. Dorsal view of the skull.F. Ventral view of the hand.G. Ventral view of the foot.
Fig. 7. A. Advertisement call of Odontophrynus toledoi sp.nov.B. Acoustic-space of Odontophrynus toledoi sp.nov.and Odontophrynus juquinha Rocha, Sena, Pezzuti, Leite, Svartman, Rosset, Baldo & Garcia, 2017, and the respective grid with the position of each semilandmark.The positions of the semilandmarks were obtained in the 3D spectrogram and summarized in a Principal Coordinate Analysis (PCA).With this, the ordering graphic recovered 73.8% of all the acoustic-space variance in the fi rst two PCA axes, resulting in a non-overlapping of the acoustic-space of the two species.

Fig. 8 .
Fig. 8. Karyotype of Odontophrynus toledoi sp.nov. A. Giemsa-stained karyotype.Note the length variation among chromosomes 9 due to the NOR size variation (see text for details).B. Giemsastained chromosomes extracted from two distinct cells in meiosis I.The number of tetravalents differed between both cells.C. Chromosome group 9 from one metaphase of the specimen ZUEC-AMP 24764 sequentially subjected to Giemsa-staining (G) and the Ag-NOR method (Ag-NOR).Arrows indicate secondary constrictions.Note the NOR size variation among the homologues.D. C-banded karyotype.Scale bars = 10 μm.

Fig. 9 .
Fig. 9. Phylogenetic analysis of partial sequences of the 16S mtDNA gene of 10 species of the genus Odontophrynus Reinhardt & Lütken, 1862.Nodes are labeled with the Bayesian posterior probability.Scale bar (0.4) in million years.The black bars on the side represent the species delimitated by the bGMYC and BPP methods.The colored bars represents the haplotype groups shown and map in Fig. 10.

Fig. 10 .
Fig. 10.Geographical distribution of ten species of Odontophrynus Reinhardt & Lütken, 1862 in South America and the respective haplotype network with the colors of each cluster also indicated on the map.Median-joining haplotype network of specimens from genus Odontophrynus based on partial sequences of the 16S mtDNA gene.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).Highlighted, the altitude map shows the distribution of the genus in central, south, and southeast of Brazil.Abbreviations: BA = Bahia; ES = Espirito Santo; GO = Goiás; MS = Mato Grosso do Sul; MT = Mato Grosso; PR = Paraná; RJ = Rio de Janeiro; SC = Santa Catarina; SP = São Paulo; TO = Tocantins.

Fig. 12 .
Fig. 12. Principal component analysis (PCA) of the linear measures showing distribution of the species in the reduced space composed by the fi rst two principal components.Yellow circle = O. cultripes group, grey circle = O. americanus group, and blue circle = O. occidentalis group.Tadpole illustrations and pictures were obtained from the original species descriptions: O. juquinha image obtained from Rocha et al. 2017; O. maisuma image, from Borteiro et al. 2010; O. monachus image, from Menegucci et al. 2016; O. carvalhoi image, from Santos et al. 2017; O. americanus, O. cultripes, and O. occidentalis, from Savage & Cei 1965; and O. cordobae image, from Grenat et al. 2009).Linear measures used to perform PCA are in the Supp fi le 1: TableS2.

Table 1 .
Measurements (in mm) of the holotype and 10 paratypes of Odontophrynus toledoi sp.nov.The results are presented as the mean ± standard deviation (sd) and minimum-maximum values (range).Abbreviations of morphometric characters are defi ned in Material and methods.

Table 2 .
Tadpole measurements of Odontophrynus toledoi sp.nov. between Gosner stages 37 and 40 (n = 7).Data are in millimeters and presented as mean ± sd.*sd = standard deviation.For abbreviations see material and methods.

Table 3 .
Measurements (in mm) and proportions of the holotype and fi ve paratype males of Odontophrynus toledoi sp.nov.Values presented as mean ± sd, minimum-maximum values (range).See Material and methods for abbreviation defi nitions.
Martino et al. (2019)13);Van Buskirk & McCollum 1999)omthespecies of the O. cultripes group (O.cultripes, O. carvalhoi, and O. monachus) and O. occidentalis by the absence of distinctly enlarged postorbital, temporal, and parotoid glandular warts (three or more pairs of enlarged rounded-oval postorbital, temporal, and parotoid glandular warts in O. cultripes and O. occidentalis species groups).The coloration of the species also provides crypsis by camoufl age in the microhabitat where it lives.O.cordobae, O. juquinha, O. lavillai and O. maisuma by having longitudinally oriented dorsal glandular ridges (scarce or absent in O. cordobae, O. juquinha and O. lavillai, and scarce or absent, except on postorbital and parotoid regions in O. maisuma), from O. cordobae by having the rostral glandular wart well developed (poorly developed or absent in O. cordobae), from O. reigi by the eye-nostril glandular wart poorly developed or absent (well developed in O. reigi).The skin texture of densely scattered big warts on dorsum can distinguish O. toledoi from O. lavillai (small warts scattered on dorsum; Cei 1980; Rosset 2008).The new species can be distinguished from O. americanus, O. cordobae and O. maisuma by the presence of few distinct postorbital and parotoid glands undifferentiated from other glands (glands form a longitudinal ridge in O. maisuma, rounded and curved postorbital and parotoid glands or ridges on O. americanus and O. cordobae; Savage & Cei 1965; Martino & Sinsch 2002; Rosset 2008).Odontophrynus toledoi also differs from the others by the temporal gland undistinguishable from other glands (distinctly rounded temporal gland in both O. americanus and O. cordobae, and absent in O. maisuma; Savage & Cei 1965; Martino & Sinsch 2002; Rosset 2008).The toe length can distinguish O. toledoi from O. maisuma (toe I does not reach the subarticular tubercle of the toe II in O. toledoi, and toe I reaches the subarticular tubercle of the toe II in O. maisuma; Rosset 2008).In general, external morphology of tadpoles of O. toledoi sp.nov.areverysimilar to other members of the O. americanus group, O. cultripes group, and O. occidentalis group.However, in comparison with members of the O. cultripes group (O.carvalhoi, O. monachus, O. cultripes), tadpoles of Odontophrynus toledoi differ by their larger size, TL = 42.91-56.18mmatstages37-40,againstO.monachus(TL=36.9mmatstage36;Meneguccietal.2016),O.cultripes(TL=41mmatstage37;Savage&Cei 1965; Nascimento et al. 2013), with exception of O. carvalhoi (TL = 58.59 at stage 34; Caramaschi 1979; Santos et al. 2017).By lateral and ventral emarginations on the oral disc (lateral in O. monachus and O. carvalhoi; absent in O. cultripes).By 1-2 submarginal papillae on the posterior labium of each side of the oral disc (5-9 grouped laterally in both labia in O. monachus; 2-3 on upper labium and 3 on lower labium in O. carvalhoi).Furthermore, differentiated from O. monachus by tip of tail acute (rounded in O. monachus), ventral tube positioned slightly below ventral margin (at ventral margin in O. monachus), by body rounded in lateral view (globular in O. monachus).It can also be diagnosed from O. carvalhoi by having the spiracle's inner wall with small distal portion free (fused in O. carvalhoi), In comparison with tadpoles of the O. americanus group (O.maisuma, O. juquinha, O. cordobae, O. americanus, O. lavillai), tadpoles of O. toledoi sp.nov.canbedistinguished by their smaller size at same or near stages, TL = 42.91-56.18mm,stages37-40,O. maisuma (TL = 47 mm at stages 33-36; Borteiro et al. 2010), O. cordobae (TL = 35.9-50.9mmatstage37;Grenatetal.2009), O. lavillai (TL = 55.8 mm at stage 37; see Lavilla & Scrocchi 1991; Fabrezi & Vera 1997; Nascimento et al. 2013), except by being larger than O. juquinha (TL = 24.3-35.7 mm at stages 30-38; Rocha et al. 2017) and O. americanus (TL = 42 mm, stages 38-39; see Fernández & Fernández 1921; Fabrezi & Vera 1997; Nascimento et al. 2013).By body rounded in lateral view and elliptical in dorsal view (globular and ovoid in O. maisuma, O. juquinha; ovoid in dorsal view in O. cordobae; globular in dorsal view in O. lavillai and O. americanus).By by 1-2 submarginal papillae on the posterior labium (4-8 in both sides of oral disc in O. juquinha; 1-2 submarginal papillae lower/posterior lip in O. cordobae).By lateral and ventral emarginations on the oral disc (lateral in O. juquinha, O. maisuma, and lateral and subterminal in O. cordobae).By nostrils with small projection on marginal rim (absent in O. maisuma, O. cordobae).Moreover, differentiated from O. juquinha by vent tube with posterior portion free (entirely fused in O. juquinha).It can also be diagnosed from O. maisuma by having the spiracle's inner wall with small distal portion free (fused in O. maisuma), by having one-single row of papillae (varies from 1-2 rows on anterior/posterior labium in O. maisuma), and by tip of tail acute (rounded in O. maisuma). of O. toledoi sp.nov.canbediagnosedfromO. occidentalis (O.occidentalis group) by being smaller (O.toledoiTL = 42.91-56.18mm,stages37-40,O. occidentalis TL = 74 mm at stage 37;Savage & Cei 1965;Cei 1987), by one-single row of marginal papillae (two-rows along oral disc in O. occidentalis according toNascimento et al. 2013).However, O. barrioi(González et al. 2014)synonym of the O. occidentalis (seeMartino et al. 2019)have one-single row of marginal papillae.Furthermore, O. toledoi can be distinguished from species of the O. occidentalis group by its spiracle's inner wall with small distal portion free (inner wall absent in O. occidentalis/O.barrioi);andbyitsacutetip of tail (rounded in O. barrioi / acute in O. occidentalis; seeMartino et al. 2019).These phenotypic variations may be due to plastic responses to environments and predators(Van Buskirk et al. 1997;Van Buskirk & McCollum 1999)as mentioned byNascimento et al. (2013)andMartino et al. (2019).Odontophrynus toledoi can be distinguished from O. maisuma and O. reigi by: (1) phalangeal formula of hand and the foot (O.toledoi has one additional bone in each phalange, compared to other species); (2) skull with the termination of the cultriform process ending in four cusps (two cusps in both O. maisuma and O. reigi); (3) frontoparietal fontanelle not exposed (exposed in O. maisuma); (4) nasals and frontoratietal well separated (slightly separated in O. reigi).firsttwoeigenvectors of the PCA describe 79.5 percent of the total variation present on tadpoles' morphological measures.PC1 show that tadpole of O. toledoi sp.nov. is not associated with any negative or positive axis, while in PC2 O. toledoi is associate to negative axis.However, PCA show nonoverlapping between tadpoles of the genus Odontophrynus in comparison with tadpole of O.The fi rst two eigenvectors of the PCA describe 73.8% of the total variation present on the acoustic traits of Odontophrys toledoi sp.nov.andO.juquinha, indicating a separate structuring in the advertisement call of the two species in the acoustic-space (Fig.7B).
: O. aff.americanus 2, which was described as O. reigi by Rosset et al. (2021), and O. aff.americanus 1, which we are describing now.Currently, the Odontophrynus americanus group comprises seven described species, fi ve of which are diploid: O. lavillai, O. cordobae, O. juquinha, O. maisuma, and O. reigi; and two tetraploids: O. toledoi sp.nov.and O. americanus.The karyotype of O. toledoi was similar to that described by