Brachycephalus dacnis Toledo, Botelho, Carrasco-Medina, Gray, Ernetti, Gama, Lyra, Blackburn, Nunes & Muscat, 2024, sp. nov.

Brachycephalusdacnis sp. nov. (Fig. 1). Brachycephalus sp. (Bornschein et al., 2021). LSID: urn:lsid:zoobank.org:act: 18F93070-2CAE-4817-8CCD-48C0C5DA5210. Etymology –The specific epithet name ‘ dacnis’ honors the Projeto Dacnis private reserve and NGO that has supported biodiversity research since 2010 in the municipalities of São José dos Campos, Miracatu and Ubatuba (where the new species was discovered), state of São Paulo, Brazil. The name is used as an invariable noun in apposition to the generic name. Holotype – Adult male (ZUEC-AMP 24982) collected and recorded (FNJV 51341) at Projeto Dacnis reserve, municipality of Ubatuba, state of São Paulo, Brazil, on 18 May 2021 by A. Mariano (Fig. 1; Table 1). We also sequenced the DNA of this individual. Paratypes – Eleven adults, only considered those confirmed by DNA and/or call traits: 03–09 August 2019 by R. Mitsuo and R.C. Menegucci (HCLP-A 267–68; heard, but not recorded); 10 March 2021 by E. Muscat and L.M. Botelho (ZUEC-AMP 24978; sequenced); 05 May 2021 by L.M. Botelho (ZUEC-AMP 24984; sequenced); 18 May 2021 by A. Mariano (ZUEC-AMP 24979; recorded); 24 May 2021 by E. Muscat and L.M. Botelho (ZUEC-AMP 24981; recorded and sequenced); 21, 22 February 2022 by L.M. Botelho (ZUEC-AMP 25270; 25272; 25274–75; sequenced); 14 April 2022 by E. Muscat, L. M. Botelho, L.A.O.S Ferreira (adult male; ZUEC-AMP 25612; Fig. 2; recorded). All paratypes were collected at the same locality as the holotype. Sex determination was not possible in most cases, except for the individual that was dissected (a female; see below) and the calling males. Diagnosis –The new species is assigned to the genus Brachycephalus because of its miniature body size, fewer phalanges and toes than a typical frog, fingers and toe tips not expanded but apically pointed, and toes lacking circumferential grooves. The new species can be diagnosed from its congeners by the following combination of characters: (1) “leptodactyliform” body shape; (2) adult body length (SVL) smaller than 1 cm; (3) distinct and functional toes II and V; (4) presence of vestigial fingers Iand IV; (5) distinct iris; (6) absence of dark markings on the skin over the pectoral region; (7) dark black or pale brown marbled venter with small white blotches in preserved specimens; (8) and advertisement call composed of one or two multi-pulsed (3–7 pulses) note with dominant frequency between 8.01 and 8.44 kHz, note duration between 0.03– 0.08 s (when isolated), up to 0.41 s (when in pairs), and absence of attenuated notes. Holotype description –Body slender and leptodactyliform (Fig. 1; SVL = 7.55 mm); head as wide as long (1.78 mm; Table 1), and one-fourth of total body length (HW/SVL = HL/ SVL = 23.6%); snout short and slightly mucronate in dorsal view and between rounded and vertical in lateral view; nostrils oval, not protuberant and directed anterolaterally; canthus rostralis distinct; loreal region slightly concave; lips nearly sigmoid; eyes slightly protruding in dorsal and lateral view, and directed anterolaterally, 27.5% ED/HW and ED/HL (as HW = HL); tympanum indistinct; vocal sac not externally expanded; vocal slits present; tongue longer than wide, with posterior half free; choanae small and rounded; vomerine teeth absent. Forearm more than twice (2.3 times) length of upper arm length; hand length shorter than forearm or upper arm lengths; hands with all fingers distinct; fingers Iand IV externally vestigial; fingers II and III robust; tips of fingers Iand IV (when visible) rounded, fingers II and III pointed; relative finger length I ≤ IV <II <III; subarticular tubercles absent; inner and outer metacarpal tubercles absent. Leg relatively long, total leg length (TBL + THL + FL = 12.1 mm) 160% of SVL (7.6 mm); shank slightly shorter than thigh (SHL/SVL = 40.6% and THL/SVL = 47.5%); foot longer than tarsus and the shank; toe I externally absent; toes II, III, IV and V externally distinct and functional. Skin smooth without dermal ossifications. Measurements of holotype presented in Table 1. Color in life of the holotype –The general coloration of the dorsal body surface is yellowish-brown. Adark brown stripe extends laterally from the tip of the snout to the flanks. Dorsal surfaces of arms and legs interrupted by dark brown stripes. An x-shaped or dorsal mark is present. The lateral view of the body is dark brown; the region overlying the upper jaw is dark brown with distinct white spots. The ventral body surface is brown with small white blotches. The ventral surface of the hands and feet are dark brown with white blotches over each phalanx of the fingers and toes. The pupil is black, and the iris is bronze. Color variation –Color and ornamentation patterns in this species vary considerably among individuals. Some frogs have a leaf-like dorsal coloration, while others have a rock-like dorsal coloration (Fig. 3). We did not observe significant variation in the ventral pattern of the individuals. Color in preservative of the holotype (less than 1 year in preservative)–The general coloration of the dorsal surface is reddish-brown (Fig. 1). The white spots overlying the upper jaw and ventral region can become pale cream; no linea masculina is visible. The stripes on the arms and legs become more evident. The extremities of the fingers and toes become pale cream. Internal anatomy: organs (ZUEC-AMP 25273; female)–After cutting the skin, the transparent abdominal muscles were observed to have a slight dark-brown coloration (melanocytes). Beneath the musculature, the liver and small intestine were also observed with similar dark-brown pigmentation. However, it was more prominent in the intestine than in the ventral musculature and liver (Fig. 4A). After removing the abdominal muscles and opening the pericardium, we observed the heart to have melanocytes scattered throughout the atria, ventricle, conus arteriosus, and truncus arteriosus. After cutting the pectoral girdle in the medial region of the weakly mineralized procoracoid cartilages, we observed that the dark pigmentation extends from the cranial region of the heart to the carotid and systemic arteries. We did not see this pigmentation in the pulmocutaneous artery (Figs. 4B, 4C). Aslight extension of this dark pigmentation occurs in the musculature lateral to the systemic and carotid arteries (Figs. 4B, 4C). Near the bifurcation of the internal and external jugular arteries and between the systemic and carotid arteries, there were two black, ovoid parathyroid glands on each side (Fig. 4C). Less black pigmentation was observed in the thoracic region; however, a few small spots were observed close to the white eggs and in the oviduct. The oviduct was transparent and between the left and right oviduct two mature eggs were observed, and the oviducts joined near the pubis (Figs. 4D, 4E). Internal anatomy: skeleton (ZUEC-AMP 24978; undetermined sex). The skeleton of B. dacnis is broadly similar to that of other small species in the genus, including by lacking osteoderms and having unornamented dermal skull bones that are not co-ossified to the skin. This individual (Fig. 5) is osteologically mature with ossified mesopodials and complete, ossified distal long bones; the specimen is missing the right leg, which was removed for DNA analysis. The skull is compact, slightly longer than wide, and lacking ornamented dermal roofing bones. The frontoparietals, sphenethmoids, prootics, exoccipitals, and parasphenoid are incompletely synostosed with each other and often have clear separations between the bones. There are well-defined and bony margins to both the optic fenestrae and prootic foramina. The premaxillae are broad, clearly separated from one another, and have teeth; each has a robust pars dentalis, and a robust alary process that is approximately as tall as wide and widely separated from the nasal. In ventral view, the maxillae are nearly straight and bear odontoids on the anterior third. The quadratojugals are thin and angled dorsally, and do not articulate with the maxillae. The pterygoids are slender, each with a long, straight, and forked anterior ramus that approaches but does not articulate with the adjacent maxilla, a short, subtriangular posterior ramus adjacent to but not articulating with the ventral ramus of the squamosal, and a short, broad medial ramus that is well separated from the prootic. Distinct neopalatines are not observable and likely not synostosed to the sphenethmoid. Distinct, small c-shaped vomers are present bordering the anterior margins of the choanae. Tall, thin, and curved septomaxillae are present at the anterior margin of the nasal capsule. The parasphenoid is triradiate with a long, rectangular anterior ramus. The squamosals are robust, and each has a prominent zygomatic ramus that is expanded ventrally as a sheet-like flange along its anterior margin and a long, slender posterior ramus that is adjacent but not fused to the prootic. The portions of the prootic are not completely synostosed and the broad fenestra ovalis contains a small, irregularly shaped operculum; a stapes (or columella) is not present. The posteromedial processes of the hyoid are ossified and slender. The arytenoid cartilages are not obviously mineralized. There are eight distinct, procoelous, non-imbricating presacral vertebrae that arenot fused to one another. The atlas lacks transverse processes and cotyles that are widely separated, a distance greater than the posterior diameter of the atlas centrum. Transverse processes of presacral II are distinctly shorter than those of III–VIII. The sacrum is procoelous with stout transverse processes. Asesamoid at the distal transverse process of the sacrum is present at its articulation with the ilium. The urostyle bears a tall dorsal ridge that decreases in height posteriorly along the anterior two-thirds of the urostyle. The pectoral girdle is not heavily ossified. The coracoids are expanded medially but do not meet at the midline.