A review of the elusive bicolored iris Snouted Treefrogs (Anura: Hylidae:Scinax uruguayus group)

The genus Scinax currently includes more than 120 species, recovered in two major clades, the S. catharinae and the S. ruber clades. The latter comprises 75 species, most of which remain unassigned to any species groups, while 12 are included in the S. rostratus and S. uruguayus groups. In this paper we present a taxonomic review of the two species currently included in the S. uruguayus group, discussing some putative phenotypic synapomorphies of this group. Although S. pinima and S. uruguayus have been considered as distinct species, this has been based on scant evidence, and several authors doubted of their distinctiveness. Our study of available specimens of S. pinima and S. uruguayus corroborates that both are valid and diagnosable species based on phenotypic evidence. Furthermore, our results show that S. pinima previously known only from its type locality, has a much widespread distribution than previously thought (including the Brazilian states of Paraná, Santa Catarina, and Rio Grande do Sul), which, added to the biological information presented here allows to suggest the removal of this species from the “Data Deficient” IUCN Red List category to “Least Concern”. Also, we describe a new species formerly reported as S. aff. pinima and S. uruguayus from NE Argentina and some localities from the Brazilian State of Rio Grande do Sul. All species are diagnosed and characterized using adult and larval morphology, osteology, vocalizations, cytogenetics, and natural history.


Introduction
The Neotropical genus Scinax Wagler, 1830 is the most species-rich in the family Hylidae with 124 species widely distributed from southern Mexico to central-eastern Argentina, Trinidad a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and Tobago, and St. Lucia [1]. The genus comprises two large clades, the S. catharinae and the S. ruber clades [2,3]. The latter includes 75 species, of which 12 are included in the two monophyletic species groups currently recognized, the S. rostratus and the S. uruguayus groups [3,4].
The Scinax uruguayus group was first recognized as the Hyla uruguaya group by Faivovich et al. [3] based on three putative morphological synapomorphies, the presence of bicolored iris in adults, enlargement of the posterior oral disc marginal papillae of larvae with respect to the lateral papillae (this was mistakenly inverted on pg, 40 and then corrected on pg, 97 in [3]), and the presence of two keratinized and dark colored plates on the sides of the lower jaw sheath [5,6]. In their phylogenetic analysis of Hylidae, Faivovich et al. [3] recovered their single exemplar of the group, H. uruguaya Schmidt, 1944 as the sister taxon of the Scinax ruber clade. These authors, consequently, transferred the H. uruguaya group to the genus Scinax in order to remedy its paraphyly. This result corroborated the proposal by Kolenc et al. [6] regarding a possible relationship of the former Hyla pinima Bokermann and Sazima, 1973 and H. uruguaya with the Scinax ruber clade (sensu [2]) as the vent tube in tadpoles of these taxa does not reach the free margin of the lower fin, which is to date the single morphological synapomorphy known for the referred clade [2].
Since the results of Faivovich et al. [3], the Scinax uruguayus group was repeatedly recovered as the sister taxon of the remaining species of the S. ruber clade in subsequent phylogenetic analyses, being S. pinima (as S. uruguayus CFBH 5788; see S1 Appendix) the only exemplar species of the group included in these analyses [3,[7][8][9][10]. Two morphological character states are considered putative synapomorphies of the S. uruguayus group: (i) bicolored iris in adults and (ii) presence of two keratinized and dark colored plates on the sides of the lower jaw sheath in larvae. The reduction of toe webbing and marginal papillae on the posterior margin of the oral disc larger than those on the lateral margins in larvae could be other synapomorphies of this group [3,6].
Scinax uruguayus occurs in Uruguay, south Brazil, and one locality in northeastern Argentina, whereas S. pinima is known only from its type locality and nearby farther north in central-eastern Brazil (e.g., [5,6,[11][12][13][14]). Scinax uruguayus was described as Hyla uruguaya by Schmidt [15], from Quebrada de los Cuervos, Departamento de Treinta y Tres, Uruguay. It was subsequently considered a junior synonym of Hyla minuta (Barrio 1967), until Langone [16] resurrected it as a valid species. Kolenc et al. [6] provided a natural history account on Uruguayan populations. Scinax pinima was described as Hyla pinima by Bokermann and Sazima [5], from Serra do Cipó, Jaboticatubas, State of Minas Gerais, and no relevant information about its biology was published since then.
Several authors raised doubts about the validity of Scinax pinima and suggested that it could be a junior synonym of S. uruguayus, given their morphological resemblance [6,11,13,17]. Two reasons that have been considered to recognize them as distinct species are (i) the geographic distance of~1,200 km between the only known population of S. pinima in Serra do Cipó, and the northernmost known record of S. uruguayus in the Municipality of Palma, State of Paraná, Brazil [5,18]; and (ii) the coloration of the head: presence of a white Vshaped dorsal blotch in S. pinima, yellow cream in S. uruguayus [5,6,16,17].
The goal of this study is to review the taxonomy of the Scinax uruguayus group on the basis of new and existing information about external morphology, osteology, bioacoustics, cytogenetics, geographical distribution, and natural history. We also describe a new species of this group from northeastern Argentina and southeastern Brazil, and discuss the taxonomic distribution of the putative morphological synapomorphies that have been proposed for the group.

Materials and methods
Examined adults, juveniles, and tadpoles, including types of Scinax pinima and S. uruguayus, are listed in the S1 Appendix. Specimens collected for this study were euthanized with lidocaine, fixed in 10% formalin, and stored in 70% ethanol (adults and juveniles) or 10% formalin (tadpoles). Institutional abbreviations follow Sabaj [19].

Adult external morphology
Dorsal and profile outline standards of snout shape follow Heyer [20]. Ten measurements (in millimeters) were taken with digital calipers (± 0.01 mm), after Duellman [21]: snout-vent length (SVL), head length (HL), head width (HW), internarial distance (IND), interocular distance (IOD), eye diameter (ED), eye-nostril distance (END), tympanum diameter (TD), tibia length (TL), and foot length (FL). We also considered additional measurements: third finger disk diameter (3FD) and fourth toe disk diameter (4TD) employed by Napoli and Caramaschi [22]. Webbing formula follows Savage and Heyer [23] as modified by Myers and Duellman [24]. Fingers were numbered II to V following Fabrezi and Alberch [25]. Terminology of nuptial pad morphology is that proposed by Luna et al. [26]. Sex was determined by visual inspection of external secondary sexual characters (nuptial pads, vocal slits, and expansion of the vocal sac) or the gonads by dissection.
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: C0C1C6B7-0FAB-4EF6-A3BE-D61B9FBA0FEB. 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 and LOCKSS.

Results
The study of the available specimens of Scinax pinima and S. uruguayus, including types and topotypic material (S1 Appendix), corroborates that S. pinima and S. uruguayus are valid, clearly diagnosable species. In addition, populations from Argentina and some localities from the Brazilian State of Rio Grande do Sul, previously reported as S. aff. pinima and S. uruguayus [14,[51][52][53][54][55][56] actually belong to a new species that is described below. hidden surfaces of thighs and tibia orange in life; (9) frontoparietals juxtaposed or slightly separated, almost completely concealing fontanelle; (10) laminar dentigerous process of the vomers without teeth; (11) palatines reduced to thin slivers; (12) intercalary elements between ultimate and penultimate phalanges partially mineralized; (13) larynx with oval arytenoids, which have a slight medial constriction in dorsal view; (14) advertisement call composed of a single, short (49-66 ms), and pulsed note (25-31 pulses/note), emitted at a rate of 3.9-4.9 notes/s; (15) pulse rate of 490-540 pulses/s; (16) notes with pulses that are increasingly modulated for the first quarter of the note, remaining with relatively constant amplitude in the second quarter, and then decrease up to the end; (17) highly pitched advertisement call, with harmonic structure; and (18) dominant frequency between 5513-6159 Hz.
Comparisons with Scinax pinima and S. uruguayus. Scinax fontanarrosai sp. n. differs from S. pinima (character states in parentheses) as females are smaller, SVL 24.1-24.2 mm (29.0 mm in the only known female; [5]); sub-elliptical head in dorsal view (broadly rounded); two or three poorly distinguishable interorbital grooves (three grooves fairly evident in all topotype specimens and poorly marked in specimens of southern populations); bicolored iris with a golden upper half and dark brown to black lower half (golden upper half and dark brown lower half with scattered small, round, and golden chromatophores); discs of fingers and toes gray to dark brown in life (bicolored discs of fingers with a gray proximal half and a light brown to orange distal half); and hidden surfaces of thighs and tibia orange in life (light purple).
The new species also differs from Scinax pinima by having frontoparietals juxtaposed or slightly separated resulting in an almost completely concealed fontanelle (frontoparietals as slender strips at the level of the fontanelle resulting in an almost completely exposed fontanelle); laminar dentigerous process of the vomers without teeth (pointed dentigerous process without teeth); and larynx with oval arytenoids, which have a slight medial constriction in dorsal view (arytenoids oval, without medial constriction in dorsal view).
The advertisement call of Scinax fontanarrosai sp. n. differs from that of S. pinima as notes are longer ranging between 49-66 ms (30-43 ms); and composed by 25-31 pulses (11-14 pulses) that are increasingly modulated for the first quarter of the note, being relatively constant in amplitude in the second quarter, and then decreasing towards the end (notes with the first pulse distinctly lower than the second, and decreasing amplitude modulation from the second pulse to the last one); notes emitted at a rate of 3.9-4.9 notes/s (3.0-3.3 notes/s); pulse rate of 490-540 pulses/s (275-355 pulses/s); notes having harmonic structure (harmonic structure absent); and dominant frequency ranging between 5513-6159 Hz (3919-4479 Hz).
Scinax fontanarrosai sp. n. can be differentiated from S. uruguayus (character states in parentheses) by the sub-elliptical head in dorsal view (broadly rounded); presence of two or three poorly distinguishable interorbital grooves (grooves absent); anterior portion of the choanae not concealed by the palatal shelf of the maxillary arch when roof of mouth is viewed from below (anterior portion of the choanae concealed by the palatal shelf of the maxillary arch when roof of mouth is viewed from below); V-shaped cephalic blotch (subtriangular cephalic blotch); bicolored iris with a golden upper half and dark brown to black lower half (iridescent golden upper half and golden lower half with brown reticulations); discs of fingers and toes gray to dark brown in life (golden yellow to orange); and hidden surfaces of thighs and anterior surface of tibia orange in life (light purple).
Furthermore, the new species differs from Scinax uruguayus by the occurrence of juxtaposed or slightly separated frontoparietals, resulting in an almost completely concealed fontanelle (frontoparietals as slender strips at the level of the fontanelle resulting in an almost completely exposed fontanelle); laminar dentigerous process of the vomers without teeth (thick, rectangular dentigerous process with 3-4 teeth); intercalary elements between ultimate and penultimate phalanges partially mineralized (completely mineralized); and larynx with oval arytenoids, which have a slight medial constriction in dorsal view (arytenoids oval, without medial constriction in dorsal view).
The advertisement call of Scinax fontanarrosai sp. n. differs from that of S. uruguayus as notes are also longer, ranging from 49-66 ms (17-28 ms), with higher number of pulses, 25-31 (7-10 pulses), and distinct modulation (notes with pulses of decreasing amplitude from the first to the last one); pulse rate of 490-540 pulses/s (286-476 pulses/s); notes having harmonic structure (harmonic structure absent); and dominant frequency ranging between 5513-6159 Hz (3833-4651 Hz).
Description of holotype. Body slightly robust (Fig 1A). Head length 29.2% SVL, slightly longer than wide (HW/HL = 0.97). Snout nearly rounded in dorsal view and protruding in lateral view (Fig 2A). Head sub-elliptical in dorsal view (Fig 3A). Nostril tear-shaped, slightly protruded, located nearer the tip of the snout than to the eye, and laterally oriented; distance between nostrils 82.6% of IOD. Canthus rostralis rounded. Loreal region concave, slightly constricted in dorsal view. Eye protuberant (ED 9.7% SVL), ED 40% larger than END, almost equal to IOD. Tympanum oval, with distinct tympanic annulus and tympanic membrane, separated from eye by a distance almost equal to TD. TD 57.1% of ED. Supratympanic fold poorly developed. Vocal sac single, median, subgular, well-developed, externally evident by the loose skin; longitudinal folds evident in deflated position. Vocal slits present, located diagonally to the longitudinal body axis, originating laterally to the tongue and running towards the corner of the mouth. Tongue ovoid, free laterally and posteriorly not notched. Choanae rounded, widely separated medially, not concealed by the palatal shelf of the maxillary arch in ventral view. Vomerine teeth absent; premaxillary and maxillary teeth present.
Coloration of the holotype in life. Colors and pattern descriptions of the holotype in life are based on field notes. Head with a light brown V-shaped blotch, extending from the tip of the snout to the middle of upper eyelids. Dorsum dark brown-black with a light gray or white reticulated pattern and scattered, small, white spots. The dark brown-black background is defined as several ovoid to round dark blotches and a big rhomboidal black blotch behind the V-shaped cephalic blotch. Dorsal surface of arms dark brown with gray transverse bars. Hind limbs dark brown with small white spots. Hidden surfaces of thighs and tibia orange. Pectoral and abdominal regions whitish, with scattered dark spots; dark brown colored vocal sac. Ventral region of arms dark brown, bearing a few small white spots. Black horizontal elliptical pupil; bicolored iris, with upper half golden and the lower half dark brown.
Coloration of the holotype in preservative. Dorsum brown with gray blotches and bars; white spots maintained. Whitish coloration on the ventral regions maintained. Spots on pectoral and gular regions became dark gray. Orange coloration on the hidden surfaces of thighs and tibia maintained.

Etymology.
The new species is named in honor to the writer and cartoonist Roberto "El Negro" Fontanarrosa (1944−2007, in recognition of his vast contribution to the Argentinean culture. His work always included elements of nature, like the amphibians. Variation in type series. Measurements are presented in Table 1. Dorsal coloration varies in that the V-shaped cephalic blotch can be whitish or light brown, and the gray to white reticulation could extend to different areas, even covering some or most of the dorsal surface ( Fig  4). Seven metamorphs have a similar coloration to that of adults, but with the gray to white reticulation somewhat lighter. Sexual dimorphism is evident: males bear vocal slits, quite expanded, dark brown colored vocal sacs, and light-colored nuptial pads without macroscopically evident epidermal projections. The two available females (SVL 24.1-24.2 mm) are slightly larger than males (SVL 19.1-23.3 mm, n = 78).
Adult skeletal morphology. Descriptions based on skeletons of six adult male specimens (LGE 2040, 4486-90). No traces of skeletal hyperossification. Skull slightly wider than long (Fig 5A and 5B). Nasals L-shaped; anterior portion covering the tectum nasi only; inner margins at level of longitudinal axes that runs through superior prenasal cartilages; posterior margins barely overlapping the sphenethmoid posteriorly. Maxillary process terminating between postnasal wall and planum antorbitale, not articulating with the maxilla. Sphenethmoids dorsally smooth; anterior portion incorporating the proximal half of the septum nasi; laterally extending to the level of (but not including) the anterior process of the postnasal wall; and posteriorly delimiting the anterior margin of the frontoparietal fontanelle at the level of the orbitonasal foramen. Crista supraorbitalis short. The medial margins of the frontoparietals vary from being juxtaposed (LGE 4487) to slightly separated (LGE 2040; Figs 5A and 6A) at the level of the fontanelle; the fontanelle is almost completely concealed within this limited variation. The medial margins of frontoparietals can be irregularly indented (LGE 2040, 4486, 4488) or straight (LGE 4487-4489). Lamina perpendicularis poorly developed, reaching (LGE 2040) or almost reaching (LGE 4486, 4488-90) the level of the orbitonasal foramen. Frontoparietals not articulating medially over the tectum synoticum, with widely separated and roughly straight inner margins (Figs 5A and 6A). Tectum synoticum entirely (LGE 2040, 4487) or partially ossified (LGE 4488). Prootics narrow, medially separated by a strip of cartilage (LGE 4487-8) or fused (LGE 2040); crista parotica poorly ossified laterad and separated from the otic plate of squamosal by a narrow gap. Crista parotica with a cartilaginous, slender posterolateral process that extends posteriorly from its posterolateral margin.
Premaxilla anteriorly inclined; pars palatine shallow, barely noticeable, with a developed palatine process; pars dentalis with 12-17 pedicellate teeth continuously distributed along its entire extension. Alary processes narrow, rectangular shaped, anteriorly inclined, the base slightly wider than the tip, not articulating with the nasals, and with its longitudinal axes laterally directed. Maxilla weakly overlapping the lateral portion of the pars dentalis of the premaxilla, and posteriorly reaching (LGE 4488, 4490) or surpassing (LGE 2040, 4486, 4489) the anterior margin of the tympanic annulus. Pars facialis of maxilla in one plane only; two times higher than the pars dentalis, reaching approximately half of the distance between maxilla and processus lingularis of the nasal cartilage, and expanding posteriorly into a preorbital process. Pars dentalis with 25-34 pedicellate teeth that extend posteriorly to the anterior end of the pterygoid fossa. Maxilla overlaps approximately half of the quadratojugal length, which extends to the anterior end of the pterygoid fossa. The posterior part of the quadratojugal contacts the ventral ramus of squamosal and palatoquadrate cartilage; basal process rounded, non-bicapitate.
Squamosal composed of ventral, zygomatic, and otic rami. Ventral ramus posteriorly arched and directed posteroventrally forming an angle of 70˚with the horizontal axis of the skull (Fig 5C). The distal end of the ventral ramus invests the palatoquadrate cartilage and articulates with the quadratojugal. Zygomatic ramus short, flat, pointed, directed anteroventrally, and not articulating with maxilla. Otic ramus flat, pointed in lateral view, slightly longer than the zygomatic ramus, expanded into an otic plate that does not overlap the ossified portion of crista parotica. Pterygoid triradiate; medial ramus short, not in contact with the otic capsule and concealing less than half of the anterior face of the basal process; posterior ramus elongate, reaching the distal portion of palatoquadrate; anterior ramus elongated, investing the maxilla laterally (Figs 5B and 6C).
Tympanic annulus incomplete dorsally, without contact with the crista parotica. Pars externa plectri elongated, mediodistally expanded; pars media plectri slightly expanded distally and with an ossified stapedial footplate that fills the rostral portion of the fenestra ovalis and contacts the operculum; a cartilaginous, round, and flat pars interna plectri that lies in the rostral half of the fenestra ovalis and overlaps the dorsal portion of the operculum.
Septum nasi narrow, vertical, bearing an anterior, median prenasal process that extends anteroventrally surpassing the alary process of the premaxillar: triangular (LGE 4488) or rounded (LGE 2040, 4487) in dorsal view. Alary cartilage cup-shaped, squared, and synchondrotically fused to the superior prenasal cartilage. Superior prenasal cartilage short, rectangular, and extending anteroventrally to abut the dorsomedial margin of the posterior surface of the alary process of the premaxilla. Inferior prenasal cartilage extending anteroventrally from the solum nasi to the posterior side of the alary process of the maxilla; it contacts the alary process on the basal portion of its posterior surface. The portion of the inferior prenasal cartilage that articulates with the solum nasi is oriented mediolaterally. The oblique cartilage is a semicircular cartilaginous sheet that lies lateral to each side of the septum nasi; it extends from the ventrolateral to the dorsomedial aspect of the nasal capsule. The ventrolateral portion of the oblique cartilage, the planum terminale, is a vertical plate of cartilage, with a fenestra. The inferior edge of the planum terminale has a caudal, rod-shaped, short, lingular process. The crista subnasalis is elongated, with a posteriorly opened V-shaped channel. The septomaxilla is located within the nasal capsules, and posteroventral to the corresponding alary cartilage.
Dentary lacking odontoids; angulosplenial bearing a poorly developed, laminar coronoid process in the prearticular region. Meckel's cartilage lies between the dentary and the angulosplenial, extending posteriorly from the anterior end of the mandible, where it articulates with the mentomeckelian bone ( Fig 5D). The hyoid plate is cartilaginous, slightly mineralized posteromedially in some individuals (LGE 2040, 4487, 4490), and 1.5-2 times wider than long ( Fig 5E). Hyoglossal sinus U-shaped, deep, wide; the posterior margin at the level of the anterior margin of the posterolateral process. Anterior process present, laminar, slightly expanded distally, and medially curved. Anterolateral process slender, expanded distally. Posterolateral process slender, expanded distally, about three times shorter than the total length of posteromedial process. Posteromedial process abuts directly on hyoid plate (no cartilaginous stalks), and with a long cartilaginous epiphysis, one third of the posteromedial process length. Hyales slightly divergent anteriorly, and attached to the otic capsule on the skull.
Larynx oriented slightly ventral in relation to the posteromedial processes. Arytenoids oval with a slight medial constriction in dorsal view. Dorsomedial prominence of the arytenoids well developed, right triangle shaped, with major axis parallel to arytenoids ( Fig 5G). Arytenoid concavity with one medial internal buttress supporting the frenulum, bounded at each end by a process for attachment of the vocal cords. Cartilaginous support rods on each distal end of the vocal cords ( Fig 5F). Fibrous masses evident but not chondrified (absence of alcian blue and red). Cricoid ring with the bronchial process slender, elongated, with the distal end bifid; esophageal process absent ( Fig 5H); cardiac process laminar, width similar or slightly thinner than adjacent parts of the ring, and not curved ventrally ( Fig 5F). Posterior portion of the cricoid ring not cardiacly elongated.
The pectoral girdle has a typical arciferal arrangement ( Fig 5I). The epicoracoids are synchondrotically fused to one another anteriorly to the interclavicle region; posteriorly to the clavicles they are free and overlapping; the right epicoracoid overlaps the ventromedial margin of the left epicoracoid and may slide over it. Omosternum and sternum cartilaginous. The former is distally expanded; the latter distally simple (LGE 2040, 4487) or bilobed (LGE 4488, 4490). Clavicle slightly longer than coracoid and scapula, with the anterior margin concave and posterior margin convex. Scapula 3/4 of clavicle length, with the anterior and posterior margins concave. Pars acromialis subspherical and slightly more expanded than the pars glenoidalis, which is concave. Anterior process of suprascapula present. Cleithrum consists of a narrow, strip of bone that encompasses the anterior margin of the suprascapula. Scapula and clavicle, as well as clavicle and coracoid separated by cartilage. Coracoids are slightly smaller than clavicles; the medial head is slightly more expanded that the lateral head; coracoid ridge present.
Vertebral column with eight procoelus, nonimbrincate, presacral vertebrae (Figs 5L and 6D). Transverse process inclined anteriorly in presacral vertebrae II, III, VII, and VIII; inclined posteriorly in presacral vertebrae IV and V, and perpendicular (LGE 4487-8, 4490) or inclined posteriorly (LGE 2040, 4486) to body axis in Presacral Vertebra VI. Sacral diapophysis rounded, index maximum/minimum length of diapophysis < 3.5. Posteromedial process on the posterior margin of the sacral diapophysis absent (LGE 2040; Fig 5L), poorly developed, reduced to a small bump (LGE 4487), or developed (LGE 4488). Distal ends of the sacral diapophysis without contact with the anterior shaft of ilium; the articulation between these elements is through a partially ossified sacral sesamoid element that lies laterally to the distal terminus of each diapophysis. Sacral sesamoids are slightly expanded medially with the transversal axis at least two times longer than their longitudinal axis (Figs 5L and 6D). Pelvic girdle V-shaped in dorsal view, and composed of three pairs of elements, the ilia, ischia, and pubes, which unite in a medial symphysis ( Fig 5L). Ilium about 55% of the vertebral column length; dorsally, it bears a longitudinal crest that extends for 65% of their length. A small ilial protuberance is located anterior to the level of the anterior border of the acetabulum. Ischium with a well developed and dorsally flattened interischiadic crest. Pubis markedly mineralized.
Tadpole description. Measurements taken from 12 tadpoles at stages 35-37 (Lot LGE 8529) are presented in Table 2. Description is based on 12 tadpoles at stages 31-37 (Lot LGE 8529; Fig 7A). Body slightly higher than wide (BMH/BMW = 1.07 ± 0.09, 0.88-1.18); body length about a third of the total length (BL/TL = 0.33 ± 0.02, 0.30-0.36); body shape oval in https://doi.org/10.1371/journal.pone.0222131.t002 Submarginal commissural papillae present. Papillae simple, longer than wide, sub-conical; those on the posterior margin of the oral disc longer and not so tightly arranged as the others; submarginal papillae shorter and blunt. Upper jaw sheath U-shaped, lower jaw sheath with V-shaped free margin. Jaw sheaths massive, well developed, serrated, and heavily pigmented on the distal two thirds. Labial tooth row formula 2(2)/3(1). Row A1 typically bent with an angle directed to the front; P3 slightly shorter than P2; one specimen with a short ridge with labial teeth between A1 and A2. Thin, keratinized, and dark colored sheets placed between the lower jaw sheath and P1, on each side of the midline.
Tadpole coloration in preservative. The general appearance of tadpoles is that of scarcely pigmented anuran larvae, with distinct black spotted fins and a fine longitudinal black bands, and dark abdomen. All flecks, lines, and blotches are brown in preservative. The body is mainly translucent, with a lateral longitudinal narrow line visible from almost the tip of the snout that surrounds the eyes ventrally, and continues over the upper edge of the abdominal region and extends on the caudal myomers for about 2/3 of the tail (approximately occupying their middle third of height). Some scattered small flecks occur in the posterior half of the tail. In dorsal view, two narrow lines extend over the dorsal surface of body and tail, on both sides of the dorsal fin and reaching the tail end. Anteriorly, they are roughly continuous with a large blotch of the interocular region in correspondence with the chondrocranium. Two small semilunar blotches are present in the internarial region along with some tiny stains. In ventral view, a narrow line is evident on both sides of the ventral fin, all over the tail. The body wall is completely translucent in the ventral and most of the lateral region of the abdomen, and in the buccopharyngeal area. The oral disc is almost devoid of pigment. Although scarcely pigmented, the abdomen is dark in lateral and ventral views given the presence of some blotches in the skin of the upper and posterior portions, but mainly due to the viscera evident through the translucent body wall. The spiracle and vent tube are also unpigmented. Hind limbs are scarcely pigmented, mostly on the dorsal surface. Both fins are conspicuously colored with scattered blotches surrounded by tiny flecks that concentrate posterior to the body-tail junction, particularly over the free margins and on its last third. The anterior portion of the ventral fin concealing the vent tube is unpigmented. Both fins are transparent at their distal ends.
Buccopharyngeal morphology of tadpoles. Buccopharyngeal morphology and musculoskeletal system were described by Alcalde et al. [53] as Scinax aff. pinima. Advertisement call. We analyzed calls (LGE-B 50-4) of five males: LGE 4451 (holotype), one topotype specimen (unvouchered), and three (LGE 4387 (paratype) and two unvouchered specimens) from Ruta Nacional 12, 2.6 km NE San Borjita (27.477167˚S, 56.073861˚W; 142 m a.s.l.), Departamento Ituzaingó, Corrientes, Argentina. Air temperature was 21.7-23.1˚C. The advertisement call consists of a single, pulsed, and very highly pitched note, emitted at a rate of 3.9-4.9 notes/s ( Fig 8A; Table 3). The note duration is 49-66 ms, with intervals between notes of 130-241 ms. Each note is composed of 25-31 pulses that are increasingly modulated for the first quarter, remaining with a relatively constant amplitude in the second quarter and then start decreasing up to the end of the note. Pulses are released at a rate of 490-540 pulses/s. The notes have harmonic structure (fundamental frequency 432-582 Hz). The dominant frequency is 5513-6159 Hz.
Diagnosis. Scinax pinima can be differentiated from S. fontanarrosai sp. n. and S. uruguayus by the combination of (1) large size in females (SVL 29.0 mm in the only female available; [5]); (2) broadly rounded head in dorsal view ( Fig 3A); (3) presence of two or three well or poorly marked interorbital grooves ( Fig 3A); (4) anterior portion of the choanae not Table 4. Morphometric analysis of chromosomes. Scinax fontanarrosai sp. n. (above) and S. uruguayus (below; data taken from Cardozo et al. [103]. Abbreviations: AR = arm relation, CI = centromeric index, SD = standard deviation, CT = chromosome, type: m = metacentric, and Sm = submetacentric. concealed by the palatal shelf of the maxillary arch when roof of mouth is viewed from below; (5) V-shaped cephalic blotch; (6) bicolored iris, with a golden upper half and a dark brown lower half, with small round, scattered, and golden chromatophores ( Fig 3B); (7) bicolored discs of fingers and toes with a gray proximal half and a light brown to orange distal half in life ( Fig 3C); (8) hidden surfaces of thighs and tibia light purple in life ( Fig 3D); (9) frontoparietals as slender strips at the level of a completely exposed fontanelle; (10) pointed dentigerous process of the vomers without teeth; (11) palatines reduced to thin slivers (palatines overlapping Comparison with Scinax uruguayus. Scinax pinima differs from S. uruguayus (character states in parentheses) by the following character states: large size in females (SVL 23.8-27.6 mm in females; n = 6); two or three well or poorly marked interorbital grooves (interorbital grooves absent; Fig 3A); anterior portion of the choanae not concealed by the palatal shelf of the maxillary arch (choanae concealed by the palatal shelf of the maxillary arch when roof of mouth is viewed from below); V-shaped cephalic blotch (subtriangular cephalic blotch); bicolored iris with a golden upper half and a dark brown lower half with scattered, small, round, and golden spots (iridescent golden upper half and a golden lower half with brown reticulations; Fig 3B); and bicolored discs with a gray proximal half and a light brown to orange distal half in life (discs of fingers and toes golden yellow to orange; Fig 3C).
Scinax pinima also differs from S. uruguayus by having a pointed dentigerous process of the vomers without teeth (thick, rectangular dentigerous process with teeth), and intercalary elements between ultimate and penultimate partially mineralized (completely mineralized). Furthermore, the advertisement call of S. pinima differs from that of S. uruguayus in the following character states: note duration ranging from 30-43 ms (17-28 ms); notes composed of 11-14 pulses (7-10 pulses), released at the rate of 3.0-3.3 notes/s (3.7-5.0 notes/s); and notes with the first pulse distinctly lower than the second, and decreasing amplitude modulation from the second pulse to the last one (notes with pulses of decreasing amplitude modulation from the first to the last one).
Description of holotype. See Bokermann and Sazima [5] for a description of the holotype.
Coloration in life of the holotype. See Bokermann and Sazima [5] for a description of the coloration in life of the holotype.
Coloration in preservative of the holotype. The holotype of Scinax pinima is well preserved. Its dorsum is dark brown colored with a light brown reticulated pattern. A brownish V-shaped cephalic blotch and dark brown colored vocal sac are visible.
Variation and morphological observations on the adult specimens examined. Measurements of some adult specimens are presented in Table 1. We observed variation in the development of the interorbital grooves and in the degree of pigment concentration on the dorsum (Fig 12). All specimens from Serra do Cipó, Minas Gerais, Brazil have two or three well marked dermal interorbital grooves; specimens from southern populations (states of Paraná, Santa Catarina, and Rio Grande do Sul) have poorly marked grooves. Also, the degree of dorsal dark pigmentation is quite variable with some specimens lighter than others.
Males have expanded, dark brown colored vocal sacs with longitudinal folds evident in deflated position (Fig 12F), and light-colored nuptial pads without macroscopically evident epidermal projections. Pectoral fold with only a preaxillar element. The discs in hand and toes are rounded. Hand webbing is basal. Toe webbing formula I 2 + -2 + II 2 --3 + III 3 + -2 -IV 3 + -2 -V. The only available female (SVL 29.0 mm; [5]) is larger than males (SVL 22.1-26.5 mm). (2) Sphenethmoid reduced; anterior portion incorporates only the base of the septum nasi, not laterally extended, and posteriorly covering slightly the anterior margin of the frontoparietal fontanelle (anterior portion incorporating at least half of the septum nasi length; laterally extending to the level of the anterior process of the postnasal wall; and posteriorly covering the anterior margin of the frontoparietal fontanelle, at the level of the orbitonasal foramen). (3) Frontoparietals as slender strips at the level of the fontanelle resulting in an almost completely exposed fontanelle (inner margins of the frontoparietals juxtaposed or slightly separated resulting in an almost completely concealed fontanelle in S. fontanarrosai sp. n.; Fig 6A). (4) Tectum synoticum entirely cartilaginous (tectum synoticum entirely or partially ossified). (5) Prootics with a non-ossified crista parotica laterad (crista parotica poorly ossified laterad). (6) Maxilla posteriorly overlapping approximately one third of the quadratojugal length (overlapping approximately half of the quadratojugal length). (7) Anterior process of the vomers absent, and prechoanal and postchoanal processes poorly developed, embracing a small portion of the medial margin of the choanas (anterior process present; prechoanal and postchoanal processes limiting the medial margin of the choanas). (8) Dentigerous process of the vomers pointed without teeth (laminar, without teeth in S. fontanarrosai sp. n. and thick, rectangular, with teeth in S. uruguayus; Fig 6B). (9) Palatines reduced to thin slivers (palatines overlapping about 3/4 of planum antorbitale in S. uruguayus). (10) Posteromedial process of the parasphenoid absent (present, but extremely reduced in S. fontanarrosai sp. n.). (11) Arytenoids oval, without medial constriction in dorsal view (arytenoids oval, with a slight medial constriction in dorsal view in S. fontanarrosai sp. n.). (12) Intercalary elements between ultimate and penultimate partially mineralized (completely mineralized in S. uruguayus).
Additionally, we observed some characteristics in the only available skeleton of Scinax pinima that vary intraspecifically in S. fontanarrosai sp. n. and/or S. uruguayus (character states in parentheses). These are as follows. (1) Prootics separated by a strip of cartilage medially (prootics medially fused in some specimens of S. fontanarrosai sp. n.). (2) Pars dentalis of premaxilla and maxilla with 12-13 and 30-32 pedicellate teeth, respectively (8-17 and 25-36 pedicellate teeth in the premaxilla and maxilla, respectively). (3) Maxilla posteriorly surpassing the anterior margin of the tympanum (maxilla posteriorly reaching the anterior margin of the tympanum in some specimens of S. fontanarrosai sp. n.), and contiguous to the main body of the vomer by a medial, constricted portion (continuous to the main body of vomer in some specimens of S. fontanarrosai sp. n. and S. uruguayus; and separated from the main body of vomer in some specimens of S. fontanarrosai sp. n.; Fig 6B). (4) Parasphenoid with a pointed cultriform process, whose anterior end is posterior to the orbitonasal foramen (rounded cultriform process with the anterior end extending to the level of the orbitonasal foramen in some specimens of S. fontanarrosai sp. n.). (5) Median prenasal process of the septum nasi at the level of the alary process of the premaxilla anteriorly (surpassing the alary process of the premaxillar in S. fontanarrosai sp. n. and some specimens of S. uruguayus), and rounded shaped in dorsal view (triangular in some specimens S. fontanarrosai sp. n.). (6) Hyoid plate slightly mineralized posteromedially, and two times wider than longer (entirely cartilaginous and one and a half times wider than longer in some specimens of S. fontanarrosai sp. n.). (7) Sternum distally simple (bilobed in some specimens of S. fontanarrosai sp. n. and S. uruguayus). (8) Transverse process perpendicular to body axis in the Presacral Vertebra VI (inclined posteriorly in S. uruguayus and in some specimens of S. fontanarrosai sp. n.). (9) Posteromedial process on the posterior margin of the sacral diapophysis absent (absent or poorly developed in some specimens of S. fontanarrosai sp. n. and S. uruguayus; and developed in some specimens of S. fontanarrosai sp. n.).
Buccopharyngeal morphology of tadpoles. The buccopharyngeal cavity has keratinized, colored, and multicuspidate spurs, behind the posterior edge of the lower jaw sheath. Multifid papillae project from the medial region of the cartilago meckeli, overlapping at the midline. The lingual anlage presents no papillae, and prepocket papillation is absent. Buccal pockets are narrow, transverse, and medially curved slits. The buccal floor arena is delimited by 3-5 papillae. The ventral velum is sinuous, with conspicuous ondulations on each side and a noticeable median notch. The glottis is exposed. The buccal roof presents prenarial arena without pustules, and choanae-like oblique slits in the posterior margin form narial valves. Postnarial arena has two long papillae and any few pustules, and a high and triangular median ridge with a pustulate margin. Lateral ridge papillae are wide, flat and pustulate. The buccal roof arena is not clearly defined, presenting numerous (~20) and scattered small pustules, and a few small lateral papillae. The dorsal velum is short, smooth, and with large secretory pits.
The advertisement call of Scinax pinima was first reported by Bokermann and Sazima [5]. Our analyses on calls (CBUFMG 991, 993-7) of six topotype specimens showed that it is composed of a single pulsed note, emitted at a rate of 3.0-3.3 notes/s (Fig 8B; Table 3). Note duration ranges from 30-43 ms, and intervals between notes from 260-330 ms. Each note is composed of 11-14 pulses that are decreasingly modulated within each one, except for the first pulse, which is consistently lower than the second. Pulses rate of 308-355 pulses/s. The notes lack harmonic structure. Dominant frequency is 3919-4479 Hz. Spectral and temporal parameters of the advertisement call of the single specimen from the State of Santa Catarina are very similar to those of topotype specimens (see Table 3).
Cytogenetics. No samples were available for this study.
Geographic distribution. Scinax pinima shows an interesting disjunct geographic distribution (Fig 10). The northernmost known population inhabits the mountain system of Serra do Cipó, Minas Gerais, Brazil, which belongs to the Campos Rupestres Montane Savanna Ecoregion (1100-1400 m a.s.l.). Other populations are known nearly 900 km southwards, in altitude grasslands of the Araucaria Moist Forest Ecoregion of the Brazilian states of Paraná, Santa Catarina, and northern of Rio Grande do Sul (787-1027 m a.s.l.).
Natural history. Some notes on the natural history of Scinax pinima, including comments on breeding sites, clutch structure and egg size, were provided by Bokermann and Sazima [5]. Scinax pinima is an explosive breeder (sensu [59]), detectable mainly after the heavy rains of the rainy season. A female kept in captivity laid a clutch with~300 eggs (single eggs sensu [70]) in water, attached to the glass at the bottom of the aquarium [5]). The ovum has a diameter of 1.5 mm, and 1.7 mm when the viteline membrane is considered, and the egg jelly diameter is 4.5 mm [5].
Additional adult and juvenile specimens examined. One hundred and eight specimens from several localities in the Brazilian State of Rio Grande do Sul, and 44 specimens from many localities in the Uruguayan departments of Cerro Largo, Maldonado, Rivera, Rocha, Tacuarembó, and Treinta y Tres. See S1 Appendix for account.
Description of holotype. See Schmidt [15] for a morphological description of the holotype.
Coloration in life of the holotype. See Schmidt [15] for a brief description of the coloration in life of the holotype.
Coloration in preservative of the holotype. The holotype of Scinax uruguayus is well preserved but quite discolored, with faded dorsal pattern. A whitish subtriangular cephalic blotch and light rounded spots are visible in its light brown dorsum; and the vocal sac is brown colored.
Variation and morphological observations on the adult specimens examined. Measurements of some adult specimens are presented in Table 1. The dorsal coloration pattern in life is somewhat variable in specimens of Scinax uruguayus (Fig 13). A light brown to gold subtriangular cephalic blotch is present in the region from the nostrils to the middle of the upper eyelids. The subtriangular blotch is laterally bordered by a thin dark brown line that extends from the nostrils to the eyes; this line can be posteriorly and anteriorly enlarged in some specimens. A fine light cream line along the posterior border of the cephalic blotch also can be present.
The dorsum is gray with scattered irregular, small, dark brown blotches. Irregular brown blotches are present at the border of the maxilla. An irregular, small, dark blotch in the posterior portion of the nostril can be present in some specimens. The dorsal surface of arms is light golden, with transversal dark blotches; dorsal surface of hindlimbs is grayish to dark brown, with irregular light golden blotches. The dorsal blotches can merge forming longitudinal irregular stripes and a transversal dark band adjacent to the subtriangular cephalic blotch in some individuals. Blotches might also form a fine reticulated pattern on the flanks.
Hidden surfaces of thighs and tibia are light purple. Pectoral and abdominal regions white. Ventral region of arms grayish. Black horizontal elliptical pupil; bicolored iris, with an iridescent golden upper half and a golden lower half with brown reticulations.
Adult skeletal morphology. The skeletal morphology of Scinax uruguayus (LGE 4569, 4578-81) shares several characteristics with those of S. fontanarrosai sp. n. (LGE 2040, 4486-90) and S. pinima (UFMG 20185). We only point here the differences observed in S. uruguayus with respect to these species. (1) Nasals L-shaped (nasals narrow in S. pinima), with inner margins weakly overlapping the sphenethmoid posteriorly (non-overlapping in S. pinima). (2) Sphenethmoid with the anterior portion incorporating at least half of the septum nasi length, laterally extending to the level of, but not including the anterior process of the postnasal wall; and posteriorly covering the anterior margin of the frontoparietal fontanelle, at the level of the orbitonasal foramen (sphenethmoid with the anterior portion incorporates only the base of the septum nasi, not laterally extended, and posteriorly covering slightly the anterior margin of the frontoparietal fontanelle in S. pinima). (3) Frontoparietals as slender strips at the level of the fontanelle resulting in a fontanelle almost completely exposed (inner margins of the frontoparietals juxtaposed or slightly separated; the fontanelle is almost completely concealed in S. fontanarrosai sp. n. Fig 6A). (4) Prootics with a crista parotica poorly ossified laterad (with non-ossified laterad in S. pinima). process of the parasphenoid absent (present, but extremely reduced in S. fontanarrosai sp. n.). (10) Arytenoids oval, without medial constriction in dorsal view (arytenoids oval, with a slight medial constriction in dorsal view in S. fontanarrosai sp. n.). (11) Intercalary elements between ultimate and penultimate phalanges completely mineralized (partially mineralized).
We also observed that some characters in Scinax uruguayus vary intraspecifically in S. fontanarrosai sp. n. (character states in parentheses). These are as follows. (1) Parasphenoid with a pointed cultriform process; anterior end is posterior to the orbitonasal foramen (rounded cultriform process; anterior end extending to the level of the orbitonasal foramen in some specimens of S. fontanarrosai sp. n.). (2) Prootics separated by a strip of cartilage medially (prootics medially fused in some specimens of S. fontanarrosai sp. n.). (3) Maxilla posteriorly surpassing the anterior margin of the tympanum (maxilla posteriorly reaching the anterior margin of the tympanum in some specimens of S. fontanarrosai sp. n.). (4) Hyoid plate slightly mineralized posteromedially, and two times wider than longer (entirely cartilaginous and one and a half times wider than longer in some specimens of S. fontanarrosai sp. n.). (5) Transverse process inclined posteriorly to body axis in the Presacral Vertebra VI (perpendicular in some specimens of S. fontanarrosai sp. n.).
Finally, there are some characters that vary intraspecifically in Scinax uruguayus but showed no variation in the skeletons of S. fontanarrosai sp. n. and/or S. pinima. These are as follows (character states of S. fontanarrosai sp. n. and S. pinima in parentheses). Tadpoles. See Kolenc et al. [6] for a detailed description of the external morphology of the tadpole of Scinax uruguayus (Fig 7C). Measurements from 15 tadpoles at stages 31-33 (Lots ZVCB 10235, 10237, and 10240) are presented in Table 2.
Buccopharyngeal morphology of tadpoles. See Alcalde et al. [53] for a description of the buccopharyngeal morphology and musculoskeletal system of the tadpole of Scinax uruguayus. Advertisement call. We reanalyzed calls (LGE-B 55) previously published for one topotype specimen (ZVCB 8224) of S. uruguayus [6]. In addition, we analyzed advertisement calls (LGE-B 56-67) of 12 specimens from three Uruguayan populations: five topotypic specimens (MNHN 9870-4), six specimens (MNHN 9875-80) from Valentines, Departamento de Treinta y Tres, and one specimen (MNHN 9881) from Laguna de Rocha, Departamento de Rocha. The advertisementcall of this species was described for specimens from Brazil [17] and Uruguay [6]. Our analyses of the advertisement calls of 13 specimens from Uruguayan populations showed that the advertisement call of S. uruguayus consists of a single, pulsed note, emitted at a rate of 3.7-5.0 notes/s (Fig 8C; Table 3). The note lasts 17-28 ms and the interval between notes is 157-290 ms. Each note has 7-10 pulses that are decreasingly modulated from the first to the last pulse within the note. Pulse rate of 286-476 pulses/s. Dominant frequency ranges from 3833-4651 Hz (Fig 8C).
Geographic distribution. Scinax uruguayus inhabits mostly the southern and southeastern portions of the Uruguayan Savanna (5-500 m a.s.l.), associated mainly to hilly environments, although some Uruguayan populations are present in lowland grasslands of the Laguna Merin basin (Treinta y Tres) and coastal areas close to the Atlantic Ocean at Laguna de Rocha. In addition, it is known from a few localities in the southeastern limits of the altitude grasslands (700-900 m a.s.l.) in the Araucaria Moist Forest Ecoregion, in the Brazilian State of Rio Grande do Sul (Fig 10).
Natural history. Detailed notes on the natural history of Scinax uruguayus, including data on its phenology, breeding sites, and clutch structure, were provided by Kolenc et al. [6]. We observed the passive defensive "crouching down" behavior in one juvenile of S. uruguayus (unvouchered specimen; Fig 11F). The amplexus is axillary and one female kept in captivity laid clutches with 398 eggs (single eggs sensu [70]) in water, attached to the aquatic vegetation ( [6]; Fig 11G and 11H), and positioned singly or in irregular masses [6]. The ovum has a dark brown to black animal pole and a cream vegetal pole ( [6]; Fig 11G), with a diameter of 1.3 ± 0.07 mm, and 1.62 ± 0.07 mm when the viteline membrane is considered; and the egg jelly diameter is 5.2 mm [6].

Discussion
In a recent molecular phylogenetic study of Hylidae, limited to DNA sequences available in GenBank, Duellman et al. [9] erected the new genus Julianus for the species in the Scinax uruguayus group as defined by Faivovich et al. [3]. The genus was poorly defined, without any regard to synapomorphies proposed neither by earlier authors nor by themselves, based on few characteristics shared by most species included in Scinaxini. For example, the characters used to define Julianus are identical to those of Scinax [9]. Although ignored by Duellman et al. [9] and by Ferrão et al. [69], there are adult and larval morphological putative synapomorphies that diagnose the Scinax uruguayus group [2,3,6] as commented earlier in this study, and further discussed below.

Synapomorphies of the Scinax uruguayus group
The monophyly of the Scinax uruguayus group has not been formally tested, since S. pinima (as S. uruguayus) was the only species included in previous phylogenetic analyses (e.g., [3,9,10]). However, four morphological synapomorphies were proposed for this group: (i) bicolored iris in adults, (ii) reduction of toe webbing, (iii) presence of two keratinized and dark colored plates on the sides of the lower jaw sheath, and (iv) marginal papillae on the posterior margin of the oral disc larger than those on the lateral margins [3,6]. They are discussed below.
(i) Adults of Scinax fontanarrosai sp. n., S. pinima, and S. uruguayus share a bicolored iris with a golden upper half and a dark brown to black or reticulated lower half (with or without golden chromatophores or brown reticulations in the lower half; Fig 3B), a somewhat unique character state in Scinax. A bicolored iris with a reddish upper half and a grey lower half was reported for S. ruberoculatus Ferrão, Fraga, Moravec, Kaefer, and Lima, 2018 so far (S. ruber clade; [111]). In Hylinae, eyes with bicolored iris-with different coloration pattern-have also been observed for adults of Osteocephalus leoniae Jungfer and Lehr, 2001 (Lophyohylini; [112]) and all species of Aplastodiscus (Cophomantini; [113]).
(ii) The toe webbing is reduced between toes II and V in the species of the Scinax uruguayus group with respect to remaining species of Scinax. However, the interspecific variation in the degree of webbing is relatively continuous and difficult to partition into discrete states; exceptions are the webbing between toes II and III, and the preaxial and postaxial webbing of Toe IV (Figs 2D and 3C). The extreme reduction of webbing between toes II and III-the webbing reaches the distal margin of the subarticular tubercle of Toe II-occurs in species of the S. uruguayus and S. perpusillus groups and was considered a synapomorphy of the latter [2,114]. While the webbing reaches the midlength of the first phalanx of Toe II (e.g., S. alter (Lutz, 1973), S. canastrensis (Cardoso and Haddad, 1982), S. haddadorum Araujo-Vieira, Valdujo, and Faivovich, 2016, and S. humilis (Lutz and Lutz, 1954); [115][116][117][118]) or the base of the disc in the remaining species of Scinax (e.g., S. eurydice (Bokermann, 1968), S. hayii (Barbour, 1909), and S. perereca Pombal, Haddad, and Kasahara, 1995;[20,119,120]) and related genera (e.g., Dendropsophus, Pseudis, Sphaenorhynchus, and Scarthyla; [121][122][123][124]). Therefore, under the recent phylogenetic hypotheses proposed for Hylidae (e.g., [9,10]) this character state could be a putative synapomorphy of both species groups.
(iii) Tadpoles of Scinax fontanarrosai sp. n., S. pinima, and S. uruguayus share the presence of two keratinized and dark colored plates on the sides of the lower jaw sheath ( [3,6,53]; see also Fig 7). These keratinized plates also occur in tadpoles of S. camposseabrai (Bokermann, 1968) [139]. The phylogenetic position of S. camposseabrai within Scinax remains unstudied. This information is essential to understand the optimization of this character state in Scinax.
(iv) Enlarged marginal papillae on the posterior margin of the oral disc are present in larvae of Scinax fontanarrosai sp. n., S. pinima, and S. uruguayus ([6,53]; see also Fig 7). The presence of these papillae has also been reported in larvae of S. camposseabrai [139] and some species of Sphaenorhynchus [123,137]. We consider the presence of marginal papillae on the posterior margin of the oral disc larger than those on the lateral margins as a putative synapomorphy of the S. uruguayus group, with at least two instances of homoplasy in Hylinae-in S. camposseabrai and in an internal clade of Sphaenorhynchus (see [137]).
Apart from the six character states mentioned above (characters i, iii, and iv plus three independent characters from the former character ii), there are some morphological characters that could be considered synapomorphies of the Scinax uruguayus group or of a less inclusive clade. The presence of a subtriangular or V-shaped cephalic blotch in the S. uruguayus group (subtriangular in S. uruguayus; and V-shaped in S. fontanarrosai sp. n. and S. pinima; Figs 3A, 4, 12 and 13) could be another possible synapomorphy of this group. No other species of Scinax or related genera (Dendropsophus, Lysapsus, Pseudis, Scarthyla, Sphaenorhynchus, and Xenohyla; [136,143,144]) have a cephalic blotch alike (subtriangular or V-shaped). Some species in the S. catharinae clade have a light triangular blotch in the anterior portion of the head from the interorbital area to the tip of snout. However, unlike the color pattern in the S. uruguayus group, the triangle results from an interruption of the light dorsal pattern color of the body by a darker interocular triangular blotch and not a distinct coloration patch (e.g., S. catharinae, S. centralis, S. hiemalis (Haddad and Pombal, 1987), S. rizibilis (Bokermann, 1964), S. trapicheiroi (Lutz and Lutz, 1954); [115,134,145,146]). The presence of dermal interorbital grooves in S. fontanarrosai sp. n. and S. pinima (absent in S. uruguayus; Fig 14 in [5], Fig 26 in [13]; see also Figs 3A and 13) could be a putative synapomorphy of S. fontanarrosai sp. n. and S. pinima. These interorbital grooves are known so far only in these species among hylines. The histological structure of the interorbital grooves might correspond to connective tissue trabeculae between the subjacent epymisium and the hypodermis (C Taboada, personal communication), but more studies are required to corroborate this.
Studies on skeletal morphology of species of Scinax are scarce, limited to a few comparative studies (S. acuminatus (Cope, 1862), S. fuscovarius, S. nasicus (Cope, 1862), and S. aff. ruber; [147,148]) and some characters used in phylogenetic analyses [2,149]. Two novel osteological character states were observed by us in S. fontanarrosai sp. n., S. pinima, and S. uruguayus: the short medial ramus of pterygoid (concealing less than half of the anterior face of the basal process; Fig 6C) and expanded iliosacral sesamoids (at least two times longer than their longitudinal axis; Fig 6D). The short medial ramus of the pterygoid present in the S. uruguayus group might be unique within Scinax, since other species in this genus have an elongate medial ramus that covers the basal process but does not contact the prootic [2,149] (K Araujo-Vieira and J Faivovich, personal observations). An elongated medial ramus of pterygoid was reported in species of other hylid genera that had been related to Scinax (Dendropsophus, Lysapsus, Pseudis, Scarthyla, and Sphaenorhynchus; [21,136,137,149,150]). The only known exception is Xenohyla truncata (Izecksohn, 1959) (Fig 2 in [144]) that has a short medial ramus similar to that present in species of the S. uruguayus group. The short medial ramus of pterygoid could be another synapomorphy of this group.
The round or trapezoid iliosacral sesamoids occur in some species of Scinax (Fig 14 in  [147]), whereas a medially expanded iliosacral sesamoid-the transversal axis at least two times longer than the longitudinal axis-is present in S. fontanarrosai sp. n., S. pinima, and S. uruguayus. While a round or trapezoid sesamoid might be present in all species of Scinax (K Araujo-Vieira and J Faivovich, personal observations), the medially expanded sacral sesamoid seems to be unique among anurans. Other hylids have an anteroposteriorly elongated sacral sesamoid, with the longitudinal axis at least two times longer than the transversal axis (Fig 14 in [147], [151,152]). The shape and size of the sacral sesamoids is one of the elements (the other being degree of sacral expansion, the iliolumbaris muscle, and the articular ligament; [147,153]) that determine the direction of movement at the iliosacral articulation of a frog. A medially expanded sacral sesamoid may provide greater lateral and vertical rotation capability to the iliosacral joint. However, the origin and insertion of the articular ligaments are also decisive for the functional interpretation of the patterns of iliosacral articulation [147,153]. Histological studies on the iliosacral joint and functional analyses are necessary to better understand the role of the sesamoids in the iliosacral articulation in Scinax. Meanwhile, we suggest that the medially expanded iliosacral sesamoid is a putative synapomorphy of the S. uruguayus group.
Tadpoles of the Scinax uruguayus group have the intestinal coiling axis subparallel to the main body axis (Character 78 in [2], [16]). This character state was previously suggested as a synapomorphy of the clade including S. acuminatus + the S. rostratus group by Faivovich [2]. This character has an ambiguous optimization in a phylogenetic framework in which S. uruguayus is recovered as the sister taxon of the clade including S. acuminatus + the S. rostratus group plus the remaining species of the S. ruber clade [3,[7][8][9][10]. The intestinal coiling axis subparallel to the main body axis would have arisen in the ancestor of the S. ruber clade with a subsequent reversion in the sister taxon of S. acuminatus + the S. rostratus group. Alternatively, it may have evolved independently in the S. uruguayus group and the clade including S. acuminatus + the S. rostratus group.
Considering our observations and the putative synapomorphies mentioned above, the Scinax uruguayus group (combined SVL, males 19.1-27.0 mm, females 23.8-29.0 mm; [5]) can be diagnosed by the following phenotypic characters: (1) presence of pectoral fold (only the preaxillar element); (2) light-colored nuptial pads without macroscopically evident epidermal projections; (3) round discs in hand and toes; (4) reduced webbing between toes II and II (the webbing reaches the distal margin of the subarticular tubercle of Toe II); (5) reduced preaxial webbing in Toe IV (webbings reach the proximal half of antepenultimate phalanx in Toe IV); (6) reduced postaxial webbing in Toe IV (webbings reach the proximal half of antepenultimate phalanx in Toe IV); (7) bicolored iris (golden upper half and dark brown to black or golden lower half) in adults ( Fig 3B); (8) subtriangular or V-shaped cephalic blotch ( Fig 3A); (9) darkcolored, externally expanded vocal sac, evident by the presence of loose skin with numerous longitudinal folds (Figs 4, 12 and 13); (10) short medial ramus of pterygoid, concealing less than half of the anterior face of the basal process ( Fig 6C); (11) presence of anterior process of the suprascapula; (12) medially expanded iliosacral sesamoids, at least two times longer than their longitudinal axis ( Fig 6D); (13) tadpoles with marginal papillae on the posterior margin of the oral disc larger than those on the lateral margins; (14) dark colored keratinized plates on the sides of the lower jaw sheath; (15) well-developed, higher than wide, conspicuously keratinized jaw sheaths (Fig 7); and (16) intestinal coiling axis subparallel to the main body axis. The character states 4-8, 10, and 12-14 are putative synapomorphies of this group.

Buccopharyngeal and musculoskeletal morphology of tadpoles
Larvae of Scinax fontanarrosai sp. n. (as S. aff. pinima; [53]), S. pinima (this work), and S. uruguayus [53] have distinctive traits in the oral cavity that differentiate them from other larvae of Scinax that have been studied (see Appendix I in [53]). They are multifid and overlapping infralabial papillae, large lateral ridge papillae, and well defined buccal floor arena and ventral velum. Some musculoskeletal features seem to be also characteristic of S. fontanarrosai sp. n. and S. uruguayus; e.g., cornua trabeculae with short and wide free portions, processus articularis short and wide, processus muscularis narrow and directed anteriorly, cartilago suprarostralis forming a single structure, lower jaw cartilages massive, ceratobranchialia II-IV continuous with the planum hypobranchiale, and m. subarcualis rectus I formed by two slips [53]. The buccopharyngeal morphology, chondrocranium, and cranium muscles remain poorly studied in tadpoles of Scinax (see Appendix I in [53]). The taxonomic distribution of these character states requires a more extensive sampling to assess their polarity.

Advertisement calls
The advertisement call of Scinax uruguayus was described by Kwet [17], based on a single individual from the north of the State of Rio Grande do Sul, Brazil. Kolenc et al. [6] further described the advertisement call of one individual from Department of Lavalleja, Uruguay with some differences (number of pulses, interval between notes they regarded them as mainly produced by the different analytical methods employed). Other differences (duration of the set of notes, note rate, dominant frequency) could be associated to variables such as temperature, size, and/or degree of excitation of the specimens [154]. Our analyses of S. uruguayus (calls of 13 individuals from three localities), including a topotype, encompass the range values of the spectral and temporal parameters provided by Kolenc et al. [6] (Fig 8C; see also Table 3). Furthermore, the number of pulses per note (7-10 pulses/note) was exactly the same, and broadly overlap with the range (6-9 pulses/note) resulting from the analyses of calls by Kwet [17].
The advertisement call of Scinax pinima was described by Bokermann and Sazima [5]. Some parameters of the original description differ from our results. For instance, our analyses showed calls with note rates of 3.0-3.3 notes/s vs.8-9 notes/s reported by these authors. We noticed that 8-9 notes/s is not congruent with note duration of 0.1 s and interval between notes of 0.2 s already reported. Bokermann and Sazima [5] also reported a harmonic structure for the call of S. pinima, not seen in our analyses of this species. The poorly differentiated harmonics described by these authors seem to be sidebands, a phenomenon resulting of high pulse rates [155].
The advertisement call of Scinax pinima seems to be fairly constant along its wide latitudinal distribution (Fig 8B; see also Table 3). Temporal and spectral parameters did not show significant differences between calls recorded at the type locality (Serra do Cipó, Minas Gerais) and Lebón Regis (Santa Catarina,~1100 km from the type locality). Moreover, the pulse amplitude modulation of the notes, with the first pulse of each note being distinctly lower than the second one, is also present in calls from both populations. Although with similar pulse amplitude modulation, call notes of S. uruguayus and S. pinima can be promptly distinguished by the presence of a lower first pulse in the latter.
Scinax fontanarrosai sp. n. has the most distinctive advertisement call when compared to those of S. pinima and S. uruguayus. It has higher values of note duration, number of pulses per note, pulse rate, and dominant frequency than those of S. pinima and S. uruguayus (Fig 8; see also Table 3). Values of pulse rate and dominant frequency overlap between S. pinima and S. uruguayus but not between these and the new species. The advertisement call of S. fontanarrosai sp. n. is unique in the S. uruguayus group for having notes with pulses forming an elliptical shape on the oscillograms. This is a result of pulses that are increasingly modulated for the first quarter of the note, remaining with relatively constant amplitude in the second quarter and then decreasing up to the end of the note. Additionally, the occurrence of harmonics in the advertisement call of S. fontanarrosai sp. n. distinguishes it from S. pinima and S. uruguayus (absent in these species). The presence of harmonic structure in the advertisement calls of Scinax was previously suggested as being restricted to the S. catharinae group [120,156]. However, this is well known for many species of the S. ruber clade (e.g., [157,158]).
Cardozo et al. [103] pointed also that the differences in size and morphology of the chromosome Pair 1 between species of the Scinax catharinae and S. ruber clades could be attributed to pericentric inversions, addition/loss of repetitive sequences, or still unidentified structural chromosome alterations. Gruber et al. [176], based on replication bands, attributed these differences to the loss of repetitive sequences in the chromosomes Pair 1 in two species of the S. catharinae clade (S. littoralis (Pombal and Gordo, 1991) and S. hiemalis). However, their results were not conclusive and new approaches to the molecular cytogenetic of Scinax would help to elucidate the underlying mechanisms.
Brazilian herpetologists [181]. A similar disjunct distribution pattern is observed for species of the Proceratophrys bigibbosa group, with P. palustris Giaretta and Sazima, 1993 known only from Minas Gerais and the remaining species (i.e., P. avelinoi Mercadal de Barrio and Barrio, 1993, P. bigibbosa (Peters, 1872), and P. brauni Kwet and Faivovich, 2001) occurring in the Brazilian states of Paraná, Santa Catarina, Rio Grande do Sul; northeast Argentina; and eastern Paraguay [182]. Extensive molecular studies (i.e., phylogenetic and phylogeographic) would allow understanding these intriguing convergent biogeographic patterns in South American frogs.
Populations of Scinax fontanarrosai sp. n. are dense, and seem to be tolerant to anthropogenic disturbances. For instance, we observed several specimens near national highways and in areas used for agriculture and extensive livestock production in Corrientes and Misiones, Argentina. Nevertheless, S. pinima was previously considered a microendemic species, whose range was restricted to the type locality and nearby areas in Serra do Cipó, Minas Gerais. Only three specimens (in 1987, 1992, and 2016; J Pombal Jr., personal communication; CFB Haddad, personal observation) were collected at the type locality after the earlier 1970´s [5]. The scarce biological information and the apparent small population size lead researchers to consider the conservation status of S. pinima as "Data Deficient" in IUCN Red List [183]. However, we identified several populations of S. pinima farther south, previously reported as S. uruguayus (or Hyla uruguaya) from the Brazilian states of Paraná, Santa Catarina, and Rio Grande do Sul (e.g., [11,17,18]; see also Fig 10). Therefore, based on our new observations about geographic distribution, conspicuousness, and habitats of occurrence, we suggest including S. fontanarrosai sp. n. and S. pinima in the "Least Concern" category of the IUCN Red List. Despite this, it is necessary to study the status of the northernmost population of S. pinima to better understand the dynamics of its fluctuation, and if it entails any risk of local extinction. In regard to S. uruguayus, although considered rare by some authors [78,84] it is a fairly abundant species ( [6]; this work) and we recommend inclusion in the "Least Concern" category of the IUCN Red List.