Melanophryniscus setiba, Peloso & Faivovich & Grant & Gasparini & Haddad, 2012

Melanophryniscus setiba View in CoL , sp. nov.

Figures 2 – 12 View FIG View FIG View FIG View FIG View FIG View FIG View FIG View FIG View FIG View FIG View FIG , table 1

HOLOTYPE: CFBH 17036, male (figs. 3 – 4); from Parque Estadual Paulo César Vinha (20°36′25″S; 40°25′01″W, near sea level), approximately 1.5 km from the beach, restinga of Setiba, municipality of Guarapari, state of Espírito Santo, Brazil; collected on 10 December 2005 by P.L.V. Peloso, S.E. Pavan, N.B. Thomazini, and R. Montesinos. GoogleMaps

PARATOPOTYPES: CFBH 15727–33, three adult females, one adult male, one adult female, and two adult males, respectively, collected on 26 – 29 December 2005 by P.L.V. Peloso, R. Montesinos, and R.V. Peloso; CFBH 15734 – 41, one adult male, one adult female (15735, adult female cleared and double-stained), three adult males, one adult female, one adult male, and one subadult, respectively, collected on 08 – 24 January 2006 collected by P.L.V. Peloso, S.E. Pavan, R. Montesinos, B. Becacici, and J. Albino; CFBH 15742, MPEG 21940 – 41 three adult females, collected on 17–18 March 2006 by P.L.V. Peloso and R. Montesinos; MPEG 21944, adult male, collected in May 2006 by R. Kawada; CFBH 15743 – 45, three adult males (15745 cleared and double-stained), collected on 17 December 2006 by P.L.V. Peloso and S.E. Pavan; MPEG 21942 – 43, one adult male, and one adult female, respectively, collected by P.L.V. Peloso, D. Barbosa, P. Rubens, R. Kawada, and S.E. Pavan on 15 December 2007.

DIAGNOSIS: A small Melanophryniscus (SVL 13.8 – 16.1; table 1) with a moderately robust body that can be diagnosed by the combination of the following character states: (1) head wider than long; (2) snout round in dorsal view and slightly protruding in lateral view (profiles follow Heyer et al., 1990); (3) frontal macrogland (see Naya et al., 2004) absent; (4) fingers II, III, and V much reduced; (5) subarticular tubercles distinguishable on finger IV; (6) nuptial pad, with few enlarged, brown-colored keratinized spines at medial margin of finger II present in males; (7) finger and toe tips rounded, unexpanded; (8) toes I–IV connected by barely noticeable webbing; (9) frontoparietals heavily exostosed, fused, not diverging anteriorly; (10) pectoral girdle completely ossified, triangular; (11) sternum heavily mineralized and greatly reduced; (12) seven presacral vertebrae, the last fused with the sacrum; (13) ventral humeral crest prominent, forming a spinelike projection, more evident in males than females; (14) surfaces of head and dorsum slightly granular without keratinous spines; throat and chest smooth; and (15) color pattern: dorsum reddish brown to dark brown in life; two dorsal marks present on dor-sum—one in form of an “X” anteriorly, and one in form of a “Λ” posteriorly; venter light colored with variable amounts of brown markings and spots; a ventral dark brown stain usually present at midbody.

COMPARISONS WITH OTHER SPECIES: Melanophryniscus setiba differs from all remaining species of Melanophryniscus by the strong coossification of the dorsal skull elements. Males of the new species are readily distinguished from males of all remaining species of Melanophryniscus by the presence of a bifurcated humeral spine and by nuptial pads with a few enlarged, brown-colored spines on the base of finger II (taxonomic distribution of nuptial pads poorly known in Melanophryniscus , but, when present, composed of multiple minute, spines colored on its tips, on fingers II, III, and IV: see Discussion); presence of prominent ventral humeral crest, and reduced phalangeal formula of both hands and feet. The absence of a frontal macrogland readily separates the new species from all species in the M. tumifrons group (the macrogland is a putative synapomorphy of the M. tumifrons group; see Caramaschi and Cruz, 2002; Baldo and Basso, 2004; Naya et al., 2004). The new species further differs from all species in the M. moreirae and M. stelzneri groups by its almost smooth skin on dorsum and flanks (developed warts with an apical corneous spine in the M. moreirae group; and keratinized spines on skin in the M. stelzneri group). The diminutive size of M. setiba is rivaled only by M. vilavelhensis (12.8 – 17.2 mm; unassigned to any group; Steinbach-Padilha, 2008), with all other species> 20 mm (summary in Steinbach-Padilha, 2008: 104).

DESCRIPTION OF THE HOLOTYPE (figs. 3–4): Body robust. Head large, wider than long; snout short, rounded in dorsal view and slightly protruding in lateral view (fig. 4A–B); nostrils small, not protuberant, directed anterolaterally, almost at tip of snout; frontal macrogland absent; canthus rostralis distinct, concave; loreal region nearly flat, vertical; eye diameter about half the interorbital distance, not protruding dorsally; postorbital crests absent; tympanum absent; supratympanic fold absent; vocal sac medial, not forming externally visible folds; vocal slits present, tongue long and narrow; premaxillary, maxillary, and vomerine teeth absent; choanae small, rounded. Strong coossification of the dorsal skull elements. Ventral humeral crest present, well developed, not projecting through the skin but externally visible as a protuberance. Arm and forearm hypertrophied (likely due to extensive development of musculature). Finger IV longest, followed by finger III; finger V reduced but distinguishable (the peculiar morphology of finger II and a much reduced finger V make judgments of relative finger lengths imprecise and unreliable); finger IV moderately robust; finger tips rounded; fingers connected by a thick web; nuptial pad with few enlarged, well-separated, brown-colored keratinized spines along the proximal medial margin of finger II (fig. 4C); subarticular tubercles present and conspicuous on fingers III and IV; inner thenar tubercle elliptical, larger than outer metacarpal tubercle; palmar tubercle present, large, rounded; supernumerary tubercles on palm of hand present but barely distinguishable (fig. 4C). Legs short, slender; toes robust; toes I, II, and V very reduced; toe IV the longest, followed by toe III (others too small to make a confident statement of relative length; fig. 4D); toe tips rounded; barely noticeable webbing connecting toes II–V; subarticular tubercles present, large; inner metatarsal tubercles large, elliptical, larger than inner metatarsal tubercle (fig. 4D). Skin on head, dorsum, dorsal surfaces of arms, and legs very lightly granular. Gular region, chest, and ventral surfaces of arms and legs smooth.

MEASUREMENTS OF HOLOTYPE: SVL 15.1, HL 3.9, HW 5.5, ED 1.5, IOD 3.2, END 1.1, THL 6.4, TBL 5.9, FL 4.0.

COLOR OF THE HOLOTYPE IN PRESERVATIVE: Dorsum brown; two dorsal marks present, one in the form of an “X” anteriorly and one in the form of a “Λ” posteriorly; marks on the dorsum dark brown; dorsal arms and legs grayish brown. Throat brown (usually more pigmented in males than females); chest dark brown; venter light brown with a large dark brown stain at midbody and many dark spots and minor stains all over the ventral surface; ventral arms and legs cream color; tips of fingers and toes beige.

COLOR IN LIFE (based on paratype CFBH 15475, male, fig. 2): Iris black; pupil ring golden. Dorsum reddish brown to dark brown; marks on the dorsum dark brown; a few white spots present dorsolaterally; arms and legs brown dorsally. Throat brown; chest dark brown; venter light orange with large dark brown blotch at midbody, ventral arms and legs light orange. Palm and sole brownish red; tips of fingers and toes reddish orange.

VARIATION AND SEXUAL DIMORPHISM: Measurements of the type series are given in table 1. Males have robust arms and forearms (likely due to muscular hypertrophy) while they are slender in females (fig. 5). The humeral processes and bifurcated spine are variably visible externally among male specimens (fig. 5B–D), never visible externally in females (fig. 5A). Females lack nuptial spines and have slender arms and forearms. One cleared and doublestained female (CFBH 15735) possesses a distinct humeral spine, although it is not bifurcated and is much less developed than that of the male specimen (CFBH 15745). No sexual dimorphism in SVL was detected among 14 males and 10 females (Student’s t- test; t = 0.139, df = 22, p = 0.891), although females have significantly shorter legs than males (t = -3.856, p <0.05 for THL; t = -2.796, p <0.05 for TBL; t = -2.021, p = 0.056 for FL). Bidder’s organ is apparently absent (not detected from direct observation under a dissecting microscope or from histological sections).

The X- and Λ- shaped marks on the dorsum vary in intensity, but this variation does not seem to be ontogenetic or sexually dimorphic; some specimens have a light-brown dorsum while in others it is dark brown (colors in preservative, but such variations do occur in live specimens; reddish brown/dark brown). A few individuals have white spots on the dorsolateral surface near the thigh in life (fig. 2); in preservative these white spots tend to disappear. Ventral pattern of brown markings variable among individuals (fig. 5); throat uniformly dark colored (brownish) or light (creamish) with brown spots and blotches; ventral midbody usually with a brown blotch, highly variable in shape and size, light or dark brown.

OSTEOLOGY: Description based on male CFBH 15745 and female CFBH 15735. Skull slightly longer than wide in dorsal view. Frontoparietals, prootics, exooccipitals, sphenethmoid complex, nasals, palatines, and, if actually present, vomers, forming a continuous, single, fused structure with unclear limits between individual endochondral bones (fig. 6A–D); dorsal surface of nasals and frontoparietals heavily exostosed with irregularly shaped outgrowths, allowing limits to be inferred (fig. 6B). Frontoparietals fused to each other and nasals, sphenethmoid, prootic, and ossified tectum synoticum. Frontoparietals with nearly triangular posterolateral processes covering posterior corner of orbit and anteromedial portion of otic capsule, extending laterally to anterolateral tip of otic capsule. Occipital groove covered along most of its length, exposed in some areas. Nasals fused medially, longer than wide, reaching anteriorly the tip of the snout; maxillary process short, not reaching pars facialis of the maxilla laterally. Premaxillae edentulous and in close contact with each other and with the maxillae (fig. 6C–D). Alary process of premaxilla laminar, expanded distally; directed slightly anteriorly in profile. Pars palatina well developed; pars dentalis nearly laminar. Palatine process pointed in ventral view, conspicuously thicker than pars palatina; maximum length about twice the width of the pars palatina. Maxilla edentulous; pars facialis of the maxilla with irregular free margin. Quadratojugal small and restricted to anteroventral part of quadrate (fig. 6E), contiguous with distal portion of ventral ramus of the squamosal; anterior process short and widely separated from the maxilla; quadrate unossified. Parasphenoid indistinguishably fused with sphenethmoid and prootic (fig. 6C). Vomers absent as discrete elements (i.e., either fused to underlying sphenethmoid complex or absent); dentigerous process absent; prechoanal process well developed; postchoanal process reduced. Palatines absent as discrete elements, likely fused to the sphenethmoid complex, as suggested by a distinct thickening of the bone in the corresponding area. Pterygoid triradiate; anterior ramus in contact with maxilla, reaching base of antorbital process, almost touching lateral tip of sphenethmoid complex; medial ramus in bony contact with otic capsule. Squamosal lacking zygomatic ramus; otic ramus with broad otic plate abutting crista parotica; ventral arm laterally flattened in its anterior section, resulting in bony sheath with pointed ventral process. Sphenethmoid extensively ossified, forming sphenethmoid complex due to fusion with several ossified nasal structures, including complete septum nasi. Orbitosphenoid cartilage completely ossified. Prootic fused with parasphenoid, exooccipital, and with the sphenethmoid complex anteriorly. Epiotic eminences low. Crista parotica broad, completely ossified. Occipital condyles broadly separated. Columellae and tympanic ring absent. Operculum cartilaginous, about two thirds of its volume ossified, with prominent point of attachment for the m. opercularis (fig. 6F). Fenestra ovalis with bony margins, except for the lateral wall in the male, which is cartilaginous.

Angulosplenial extended from the jaw articulation almost to mentomekelian bones. Dentary widest at anterior termination of angulosplenial, overlapping mentomeckelian. Mentomeckelian elements small and syndesmotically united. Distal portion of each half of the jaw, including mentomekelian, and distal portions of dentary and angulosplenial, curved ventrally, forming a wide V-shape in the area around the symphysis in frontal view.

Hyoid plate ( fig. 7 View FIG ) about five times longer than wide (measured at narrowest point); not mineralized in female, slightly mineralized in male; hyalia moderately long, anterior processes oriented medially; hyalia inserted at limit between prootic and basal process. Anterolateral process present and broadly expanded, about 55% as long as hyoid plate; posterolateral processes absent. Posteromedial processes long, well ossified, noticeably curved dorsally. Larynx of male and female similar sized; female with complete cricoid; esophageal process small, triangular; bronchial processes long and slender (cricoid and bronchial processes of male damaged during preparation).

Seven procoelous presacral vertebrae ( fig. 8 View FIG ); first six vertebrae free, seventh fused with sacral vertebra, as evidenced by the intervertebral foramina between fused centra. Vertebrae II–VI with imbricated neural arches. Cotylar facets of atlas broadly separated. Transverse processes of presacral II directed anteriorly; transverse process of presacral III broader than those of presacrals II, IV–VI; processes with irregular flanges along its free margins. Neural arches of presacrals II–VI with irregular posterior margins; neural arches of presacrals III–V ornamented with elevated, anteriorly directed triangular shape with irregular base, those of presacrals VI and VII + sacrum ornamented with an irregular sagittal flange. Sacral diapophyses flattened, broadly expanded; irregular flanges along anterior and posterior margins. Iliosacral articulation with elongate sesamoid. Sacro-urostilar articulation bicondilar. Anterior half of urostyle bearing low, posteriorly tapered dorsal crest ( fig. 8A View FIG ); anterior portion of the crest with two parasagittal flanges; middle portion of urostyle with two lateral flanges, tapering and disappearing by the distal fifth of the bone.

Pectoral girdle ( fig. 9 View FIG ) lacking omosternum. Clavicles straight and robust, fused to scapulae and coracoid. Partes acromialis larger than partes glenoidiales. Coracoids broadly dilated proximally. Epicoracoid cartilages heavily mineralized and reduced to a single, thin sliver of cartilage joining clavicles and coracoids. In the female the median section of the medial margin of the

coracoids is concave, and the epicoracoid discontinuous, establishing a fenestra between both coracoids. Sternum cartilaginous and heavily mineralized, round, greatly reduced. Cleithrum and ossified suprascapula indistinguishably fused.

Pelvic girdle ( fig. 10 View FIG ) V-shaped. Ischia, pubis, and ilia fused. Ilial shaft cylindrical, elongate; dorsal crest developed along proximal 60% of ilium; dorsal prominence directed dorsolaterally. Preacetabular angle obtuse.

Humerus slender. In the male, it bears prominent ventral, medial, and lateral crests; ventral crest nearly 40% as long as humerus, bearing distinct, bicapitate, spinelike process near distal end of free margin (fig. 11); lateral and medial crests about 50% as long as humerus; medial crest higher than lateral crest. In the female, medial and lateral crests inconspicuous, forming low ridge; ventral crest present but less developed than in the male, lacking distinct spinelike process.

Carpus of right hand of female composed of radiale, ulnare, distal carpal 3 – 4 – 5, distal carpal 2, element Y, proximal element of prepollex, one sesamoid on the dorsal surface between radioulna and radiale, and one sesamoid on the ventral surface of distal carpal 3 – 4 – 5. Carpus of left hand differing in that proximal element of prepollex is absent as a discrete element, with equivalent space occupied by element Y; we consider it likely that in this hand the prepollex is fused with element Y. Terminal phalanges knoblike; reduced in digits II, III, V; elongate and slightly expanded in digit IV; penultimate phalanx of digit V reduced. Metacarpal II enlarged, bearing a peculiar, longitudinal bony outgrowth on medial margin, not ossified distally. Carpus of male ( fig. 12A View FIG ) with same morphology as female, with the addition that metacarpal II is even more enlarged medially due to hypertrophy of the bony outgrowth, which is fully ossified and has a distal process that covers the mediodistal section of metacarpal II. The relatively depressed longitudinal space delimited by this process and the element Y–prepollex coincident with space occupied by nuptial spines. Phalangeal formula of hand 1 – 2 – 3 – 2.

Tarsus ( fig. 12B View FIG ) composed of tibiale, fibulare, and three individual elements, including distal tarsal 2 – 3, an element that we interpret as distal tarsal 1 + element Y, and prehallux. Terminal phalanges knoblike, reduced on digits I–III and V, elongate on digit IV; penultimate phalanx on digit III reduced in the female. One sesamoid occurring in tarso-metatarsal joint at level of metatarsal IV, other between the proximal heads of tibiale and fibulare. Phalangeal formula of foot 1 – 2 – 2 – 4 – 2 (male), 1 – 2 – 3 – 4 – 2 (female).

DISTRIBUTION AND NATURAL HISTORY: Melanophryniscus setiba is known only from the type locality, where it was found in leaf litter of forested areas. Specimens were usually found in mata seca (“dry forest”). Several individuals were observed at the forest edge, less than 1 m away from an adjacent formation known as “open Clusia formation.” The open Clusia formation occurs in sandy soil and is characterized by discontinuities in the vegetation with small and large shrubs forming a mosaic structure ( fig. 13 View FIG ).

Melanophryniscus setiba , like most other Melanophryniscus (see Santos and Grant, 2011), is apparently a diurnal species with a peak of activity recorded in late afternoon. One specimen was collected at night while walking on the leaf litter, while 10 others were collected when daylight was available (all others collected in pitfall traps). Additional specimens were seen, but not collected, during the day (P.L.V.P., personal obs.). The species is a walking toad, with locomotion similar to that previously observed in other Melanophryniscus spp. and similar bufonids ( McDiarmid, 1971; Baldo and Basso, 2004).

Despite a major, concentrated effort to obtain data on the reproductive biology of this species (see Material and Methods), no reproductive activity (e.g., calling males or amplectant pairs) or clues that could point to breeding site (e.g., eggs, tadpoles) were observed in the field. A dissected female (CFBH 15735) contained eight ova (ca. 2.0 mm each) with a pigmented animal pole and several other small immature eggs. The small ovarian complement and large size of ovarian eggs are suggestive of increased incubation time, and/or hatching in advanced developmental stages ( Bradford, 1990; Summers et al., 2006, 2007). The type locality has a large quantity of bromeliad plants that could serve as a potential breeding site for M. setiba - phytotelm breeding is present at least in two Melanophryniscus species ( M. alipioi Langone et al., 2008 ,

and M vilavelhensis Steinbach-Padilha, 2008 ). Nevertheless, we note that the diameter of oviposited ova in water- breeding Melanophryniscus are 1.7–2.0 mm in M. krauczuki ( Baldo and Basso, 2004 ), 2.0 in M. montevidensis ( Langone et al., 2008), 2.0– 2.5 in M. moreirae ( Bokermann, 1967; Starret, 1967), 1.4– 2.0 in M. stelzneri ( Echeverria, 1998; Bustos Singer and Gutiérrez, 1997), and egg diameter in the phytotelm-breeding M. alipioi is 2.5–3.0 mm ( Langone et al., 2008). The presence of vocal slits and a vocal sac in males suggests that this spe- cies vocalizes. However, no calling males were detected, despite several targeted searches at the type locality by the senior author.

Stomachs of two specimens (CFBH 15735, female, and CFBH 15745, male) contained several ants and one mite. The diets of previously studied species

of Melanophryniscus consist predominantly of ants and mites as well (reviewed by Daly et al., 2008; see also Bonansea and Vaira, 2007; Quiroga et al., 2011). Mites and ants are probably the dietary source of lipophilic alkaloids found in defensive skin secretions of Melanophryniscus ( Saporito et al., 2011) , which suggests that M. setiba might also secrete these alkaloids.

Melanoprhyniscus setiba seems to be locally abundant, although it is difficult to observe due to its small size and cryptic coloration. Most activity was recorded from December through March, which coincides with the rainy season. Other leaf-litter frogs captured in the same forest patch with the new species were Chiasmocleis carvalhoi , Leptodactylus natalensis , and Physalaemus cf. crombiei . Of these, C. carvalhoi and P. cf. crombiei were captured simultaneously with the new species in pitfall traps. Some hylids were also detected in the area where we found the new species, especially associated with bromeliads: Aparasphenodon brunoi , Phyllodytes luteolus , Scinax agilis , S. alter and S. argyreornatus . Among these, S. argyreornatus was the only one ever found in the leaf litter.

ETYMOLOGY: The specific name, setiba , is derived from the Tupi Guarani language and means “seashells in abundance.” Setiba is the popular name of the region where the type locality is located, likely due to the high number of mollusk shells found in the beaches of the region.

SUGGESTED COMMON NAMES: Restinga toadlet (English); sapinho-da-restinga (Portuguese).

DISCUSSION

Consistent with previous findings (e.g., Graybeal, 1997; Darst and Cannatella, 2004; Frost et al., 2006; Pramuk, 2006; Van Bocxlaer et al., 2010; Pyron and Wiens, 2011), our phylogenetic analysis recovered Melanophryniscus as the sister taxon of all other bufonids. Melanophryniscus setiba, in turn, was placed as the sister taxon of the remaining exemplars of Melanophryniscus. Although our taxon sampling does provide a rigorous test of the placement of M. setiba among bufonids and other anurans, 7 it leaves the question of its placement among the species of Melanophryniscus largely unanswered. Of the 26 currently recognized species, only three were included in the present analysis. Following Cruz and Caramaschi (2003), Melanophryniscus klappenbachi and M. fulvoguttatus are exemplars of the M. stelzneri group, and M. devincenzii is an exemplar of the M. tumifrons groups. Unfortunately, we lack exemplars of the M. moreirae group and were also unable to include the recently named phytotelm breeders M. alipioi (Langone et al., 2008) and the diminutive M. vilavelhensis (unassigned to any group, Steinbach-Padilha, 2008). Importantly, we also failed to include Truebella (presumably related to Melanophryniscus; Pramuk, 2006). Sampling within Melanophryniscus must be greatly increased to test the placement of M. setiba relative to other species of this clade 7 The number of recognized bufonid genera is a matter of ongoing discussion (see Frost et al., 2006, 2008, 2009; Pauly et al., 2009; Pyron and Wiens, 2011). We follow Frost (2011), who lists 48 genera, with the addition of Amazophrynella (Fouquet et al., 2012a, 2012b). We included 30 out of the 49 genera in our analysis. Most of the exclusions are unlikely to influence the position of the new taxa (e.g., the African genera Altiphrynoides , Churamiti ), but some exclusions are, nonetheless, regrettable (see text). Our analysis was performed with the sole purpose of positioning the new species and we reserve ourselves to not comment any further on outgroup relationships and on the relationships within Bufonidae .

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McDiarmid (1971) proposed several diagnostic features of Melanophryniscus , and Graybeal and Cannatella (1995) further listed as putative synapomorphies: the “absence of the zygomatic ramus of the squamosal, exostosed frontoparietals that diverge anteriorly, ossified orbitosphenoid cartilage, frontoparietals always fused posteriorly, and parasphenoid fused to the underlying chondrocranium….” Most of those synapomorphies were found in the specimens examined by us, including M. setiba . The only inconsistency we observed regards the “frontoparietals that diverge anteriorly.” McDiarmid (1971) had already shown that the frontoparietals may not diverge anteriorly in some Melanophryniscus and this was confirmed by us when analyzing M. moreirae . The frontoparietals also do not diverge anteriorly in M. setiba . The apparent absence of a Bidder’s organ in M. setiba is consistent with Echeverria’s (1998) observations of M. stelzneri . The food items found on stomachs of two specimens of M. setiba (ants and mites) are the same food-item classes found on other studied species of Melanophryniscus ( Filipello and Crespo, 1994; Daly et al., 2008, Quiroga et al., 2011).

In addition to putative synapomorphies M. setiba shares with other species of Melanophryniscus , it has a number of unusual character states:

(1) Nuptial pad structure. In addition to our report of nuptial pads in M. setiba , the taxonomic distribution of nuptial pads in Melanophryniscus is poorly known. Several papers dealing with the taxonomy of the genus have omitted any reference to nuptial pads (e.g., Klappenbach, 1968; Caramaschi and Cruz, 2002; Kwet et al., 2005; Di-Bernardo et al., 2006; Steinbach-Padilla, 2008), with the notable exceptions of Cei (1980), Baldo and Basso (2004), Langone et al. (2008), and Baldo et al. (2012), who mention its presence but do not discuss taxonomic distribution. Nuptial pads are known to be present in M. alipioi ( Langone et al., 2008) , M. cambaraensis ( Santos et al., 2010) , M. krauczuki ( Baldo and Basso, 2004) , M. pachyrhynus ( Baldo et al., 2012) , M. rubriventris ( Vaira, 2005) , and M. stelzneri ( Cei, 1980) , and we have observed them in adult males of M. devincenzii , M. moreirae , M. simplex , and M. tumifrons . In all these species, the pad occurs on finger II and most frequently on III or even IV (the case of M. rubriventris ). Our observations indicate that the pad is composed of many minute, keratinized spines. In contrast, the nuptial pad of M. setiba has only a few, enlarged keratinized spines at medial margin of finger II (fig. 4C). The enlarged spines present in the pad of M. setiba are extremely infrequent in bufonids and are most reminiscent to those described and illustrated by Duellman and Ochoa (1991) in Nannophryne corynetes (as Bufo corynetes ).

(2) Presence of anterior process of hyoid. The anterior process is absent in M. moreirae , M. rubriventris , M. krauczuki , and M. pachyrhynus ( McDiarmid, 1971, 1972; Baldo and Basso, 2004; Baldo et al., 2012) but present and well developed in M. setiba . An anterior process on the hyale is also present in some species of Atelopus, ( McDiarmid, 1971) and Frostius ( Cannatella, 1986) .

(3) Small ovarian complement. McDiarmid (1971) mentioned clutches with 78– 237 eggs with black animal pole and light vegetal pole for Melanophryniscus ; Baldo and Basso (2004) reported more than 109 eggs per clutch and up to 401 eggs per breeding event in M. krauczuki . We observed an ovarian complement of eight ova in Melanophryniscus setiba , the same number of eggs reported for a clutch in M. vilavelhensis ( Steinbach-Padilha, 2008) .

(4) Low phalangeal formula. Previously studied species of Melanophryniscus present phalangeal formulae of 2-2-3-3 and 2-2-3-4-3 for hands and feet, respectively ( McDiarmid, 1971; Baldo and Basso, 2004). In contrast, the phalangeal formulae are 1-2-3-2 for hands and 1-2-2- 4-2 or 1-2-3-4- 2 in feet for M. setiba . Phalangeal loss, involving fingers II and V, and toes I, III, and V follows the general pattern described by Alberch and Gale (1985) in anurans (see also Yeh, 2002). Phalangeal losses occur also occur in other bufonids, such as some species of Atelopus , Dendrophryniscus , Didynamipus , Incilius , Mertensophryne , and Osornophryne ( Savage and Kluge, 1961; McDiarmid, 1971; Ruiz-Carranza and Hernández-Camacho, 1976; Grandison, 1981).

(5) Spinelike humeral process. The highly developed ventral crest of the humerus is apparently a novelty among bufonids. While a notable ventral crest was reported by Perret (1972) and Boistel and Amiet (2001) for Wolterstorffina mirei and W. chirioi respectively, it is not elaborated into a spine. Similar processes, in shapes that vary from an elevated ridge to a sharply pointed, curved spine, are present at least in Centrolenidae ( Cisneros-Heredia and McDiarmid, 2007) , Hylidae ( Bokermann, 1965) , Microhylidae ( Matsui, 2009) , Telmatobiidae ( Lavilla and Sandoval, 1991) , and Rhacophoridae ( Kuramoto and Joshy, 2003) .