Brachycephalus nanicus sp. nov.

https://zoobank.org/ B7A9AA10-0F6A-41FE-AFE3-AAF78FB87B4D

Figure 2

Chresonymy

Brachycephalus sp. Toledo et al. 2024

Holotype. Adult male (ZUEC-AMP 25581; field number LaHNAB 251) collected in Trilha do Ouro, Estação Ecológica de Bananal (22S 48’ 14”, 44W 22’ 17”, ~ 1.220m a.s.l.; WGS84 Datum; Figure 3), municipality of Bananal, state of São Paulo, Brazil by L.M. Botelho, L.A.O.S. Ferreira, E. Muscat, M. Moroti, T.M. Machado, and J.P. Bovolon on 28 March 2022.

Paratopotypes. ZUEC-AMP 25577–25579, 25610 and 25617 (Figure 4) adult males, and 25580 indetermined sex, all of them collected with the holotype by the same collectors; and ZUEC-AMP 25259, an adult female, also collected in the type-locality by J. Prado on 10 December 2021.

Etymology. The specific epithet nanicus an adjective in Latin (meaning those with reduced size) in allusion to the extremely small body size of the adult individuals of this Brachycephalus species (less than 1 cm).

Diagnosis. Brachycephalus nanicus can be assigned to the genus Brachycephalus by observed phalangeal reduction, and phylogenetic affinities. Member of the Brachycephalus puri species group (new group proposal), with the content of B. puri, B. nanicus and Brachycephalus sp. 8 (sensu Condez et al. 2020). The new species can be diagnosed from its congeners by the following combination of characters: leptodactyliform body; adult SVL 6.9–9.0 mm for males and 9.2–9.6 mm for females; smooth skin lacking hyperossification; snout rounded in dorsal view; finger II with pointed tips (arrow-shaped); toe I externally absent (lacking phalanges), and toes II, III, IV and V distinct; toe II with rounded tips; brown background with few golden spots scattered aleatory in the general coloration of dorsal surfaces and cloacal region; presence of a dark brown stripe along the flanks and the thigh; yellow blotches on the ventral surfaces; presence of dark brown strips on the legs. The new species is also genetic distinct.

Holotype description. Body slender and leptodactyliform (Figure 2); head as wide as long (HL/HW = 1.02%), narrower than the body; head length approximately 34% of SVL; snout shorter than the eye diameter, rounded in dorsal and lateral views; nostrils oval and protuberant, directed anterolaterally; canthus rostralis distinct; loreal region slightly concave; mouth nearly sigmoid; eye slightly protruding in dorsal and lateral views, 38.5% (ED/HW) and 37.5% (ED/HL); tympanum indistinct; vocal sac not externally expanded; vocal slights present; tongue longer than wide, posterior half not adherent to the floor of mouth; choanae relatively small and round; vomerine teeth absent. Arm and forearm slender; arm slightly shorter than forearm (UAL 15.4% and FAL 30.5% of SVL); hands shorter than the forearm and longer than the upper arm; externally, finger I and IV small, vestigial; fingers II and III robust and distinct; relative lengths of fingers I ≤ IV <II <III; inner and outer metacarpal tubercles absent; subarticular tubercles undefined in hand (Figure 2C). Leg relatively long, total leg length 81% of SVL; shank slightly shorter than thigh (SHL/SVL = 35% and THL/SVL = 45.8%); foot longer than tarsus and the shank; toe I externally not visible; toes II, III, IV and V distinct; relative toe length V <II <III <IV; metatarsal tubercles present, both oval, outer slightly larger than inner; subarticular tubercles undefined in foot (Figure 2D). Skin smooth without dermal ossifications. Measurements of the holotype (and the type series) are available in Table 1.

Comparison with other species. The leptodactyliform body of Brachycephalus nanicus distinguishes it from B. clarissae and the species belonging to the groups of B. ephippium, B. pernix, and B. vertebralis which have bufoniform phenotype (Almeida-Silva et al. 2021; Condez et al. 2021; Folly et al. 2021, 2024; Lyra et al. 2021; Nunes et al. 2021; Mângia et al. 2023). Brachycephalus nanicus adult female size (SVL 9.55 mm) is smaller than that of B. clarissae (female SVL 10.1–10.9 mm; Folly et al. 2022), B. didactylus (female SVL 10.2 mm; Izecksohn 1971), B. tabuleiro (female SVL 10.9–12.7 mm; Mângia et al. 2023), and B. puri (female SVL 10.2 mm; Almeida-Silva et al. 2021). Brachycephalus nanicus can also be distinguished from B. clarissae by the smooth dorsal skin [dorsum with a median row of small light-colored (yellow in life, cream in preservative) tubercles; Folly et al. 2022], HL/SVL = 34%, SHL/SVL = 35%, and abdominal muscles transparent also can distinguish Brachycephalus nanicus from B. dacnis (HL/SVL = 23.6%, SHL/SVL = 40.6%, and muscles with a slight dark-brown coloration – melanocytes; Toledo et al. 2024). Brachycephalus nanicus has toe I externally not visible, and toes II, III, IV and V distinct. This condition distinguishes the new species from B. didactylus (toe I not visible, toes II and III distinct and functional, and toe IV and V vestigial; Izecksohn, 1971; Napoli et al. 2011), B. hermogenesi (toes IV [externally not visible] and V reduced; Napoli et al. 2011), B. pulex (toes I, II, and V not visible, and toes III and IV distinct and functional; Napoli et al. 2011), and B. puri (toe I not visible, II, III, and IV functional, and V vestigial; Almeida-Silva et al. 2021). The new species can be diagnosed from B. dacnis, B. didactylus, B. hermogenesi, B. pulex, B. puri, and B. sulfuratus by having brown background with few golden spots scattered randomly in the general coloration of dorsal surfaces and cloacal region [dark ornamentations (some with x-shaped mark) in these species; Izecksohn 1971; Giaretta & Sawaya 1998; Napoli et al. 2011; Condez et al. 2016; Almeida-Silva et al. 2021; Toledo et al. 2024)]. Brachycephalus nanicus can also be distinguished from B. didactylus, B. hermogenesi, B. puri, and B. pulex by having yellow blotches on the ventral surfaces (absent in these species; Izecksohn 1971; Giaretta & Sawaya 1998; Almeida-Silva et al. 2021). The new species can also be diagnosed from B. puri by the rounded snout in dorsal view, finger II with pointed tips (arrow-shaped), and the presence of a dark brown stripe along the flanks and of the thigh (mucronate snout, finger II with rounded tips, and dark brown stripe absent in B. puri; Pie & Ribeiro 2015; Almeida-Silva et al. 2021; Folly 2022). The new species can also be diagnosed from B. didactylus by toe II with pointed tips (toe II with rounded tips in B. didactylus; Izecksohn 1971). The new species can also be diagnosed from B. pulex by the dark brown strips on the legs (reddish brown stripes in B. pulex; Napoli et al. 2011).

The smooth skin lacking discernible osteoderms can diagnose Brachycephalus nanicus from B. alipioi, B. bufonoides, B. crispus, B. ephippium, B. garbeanus, B. guarani, B. herculeus, B. margaritatus, B. nodoterga, B. pitanga, B. toby, and B. vertebralis (which present hyperossification: Clemente-Carvalho et al. 2009; Haddad et al. 2010; Pombal 2010; Clemente-Carvalho et al. 2012; Condez et al. 2010, 2014; Folly et al. 2021, 2024). Though many flea-toads are often externally similar looking, there is substantial variation in the extent of reductions and patterns of co-ossification in their skeletons. In contrast to the extensively synostosed cranium of B. nanicus, the cranial bones are not coossified in B. dacnis or B. sulfuratus, but is similarly synostosed in B. clarissae, B. didactylus, and B. hermogenesi, (for comparisons, see Figure 7 of Toledo et al., 2024); in B. hermogenesi, the nasals are not synostosed to the composite cranium. Like B. nanicus, B. clarissae and B. didactylus are edentulous, whereas B. dacnis and B. sulfuratus have a handful of small teeth or odontoids on the maxillae and premaxillae, and teeth are well developed and more numerous in B. hermogenesi. The squamosal of B. nanicus has an anterior flange that incorporates a small zygomatic ramus and is like that of B. dacnis, B. didactylus, B. hermogenesi, and B. sulfuratus, whereas B. clarissae has only a small anterior flange along the body of the squamosal. The quadratojugal of B. nanicus is like that of B. didactylus in being small, restricted to the distal end of the squamosal, and lacking an anterior process, whereas there is a thin, rodlike anterior process in B. clarissae, B. dacnis, B. hermogenesi, and B. sulfuratus. The pterygoid of B. nanicus is less robust than in B. dacnis, and B. hermogenesi, but more robust than the thin rod-like pterygoid of B. clarissae and B. didactylus. In contrast to the vertebral fusions observed in a skeletally mature B. nanicus (ZUEC-AMP 25610; none observed in the smaller ZUEC-AMP 25580), there are no vertebral fusions in B. clarissae, B. dacnis, B. hermogenesi, or B. sulfuratus; in B. didactylus, presacral vertebrae VI, VII, and VIII are co-ossified. Among the flea-toads examined, the urostyle of B. didactylus is unique in having laterally expanded flanges extending along its length. In B. nanicus, the single phalanx of fingers I and IV are tiny elements (similar to B. clarissae), whereas these are slightly larger in B. dacnis and B. sulfuratus, even more robust in B. hermogenesi, and lacking completely in B. didactylus.

Osteology. Based on ZUEC-AMP 25610 (sex unknown). The skeleton of B. nanicus is 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) seems to be osteologically mature with ossified mesopodials and complete, ossified distal long bones.

The skull is compact, slightly longer than wide, and lacking ornamented dermal roofing bones.The frontoparietals, sphenethmoids, prootics, exoccipitals, parasphenoid, nasals, and ossified nasal cartilages are completely synostosed. There are well-defined and bony margins to the optic fenestrae and prootic foramina as well as within the sphenethmoid and nasal cartilages, including for the olfactory nerve. The premaxillae are broad, clearly separated from one another, and lack teeth; each has a robust and deep pars dentalis, and a robust club-like alary process that is taller than wide and widely separated from the nasal. In ventral view, the maxillae are nearly straight and lack teeth and odontoids. The quadratojugals are compact, restricted to the ventral end of the squamosal, and lack a rostrally projecting process; there is a small medially projecting process articulating with the squamosal. The pterygoids are slender, with a flattened subtriangular posterior ramus adjacent to but not articulating with the ventral ramus of the squamosal, and a short medial ramus that abuts the prootic each; in this specimen, the right anterior ramus is a short process whereas the right ramus is straight and long, approaching but not articulating with the adjacent maxilla. Distinct neopalatines are not observable and, if present, these might be synostosed to the sphenethmoid; in the other CT-scanned specimen (ZUEC-AMP 25580), which is skeletally immature and lacks cranial synostosis, there is no distinct neopalatine. If present, the vomers are synostosed to the ossified nasal cartilages and sphenethmoid. The septomaxillae appear to be synostosed to the anterior margin of the ossified n nasal cartilages. The parasphenoid is broad with a long, rectangular anterior ramus that is synostosed to the sphenethmoid and prootics. The squamosals are robust, and each has a prominent sheet-like flange along its anterior margin and a hook-like slender posterior ramus that is adjacent but not fused to the prootic. The prootic is well ossified and has a well-defined fenestra ovalis, but neither an operculum nor a columella is present. The posteromedial processes of the hyoid are ossified and slender. The arytenoid cartilages are not obviously mineralized.

There are eight procoelous, imbricating presacral vertebrae with well-developed and intercalating neural spines. The atlas lacks transverse processes and cotyles that are widely separated, a distance more than twice the posterior diameter of the atlas centrum. Transverse processes of presacral II are distinctly shorter than those of presacral III– IV, which are synostosed. The broad, fused transverse processes of III–IV have a large ventral foramen, presumably for the passage of spinal nerves. Presacral VIII is weakly synostosed to the sacrum; in dorsal view, these vertebrae appear continuous but in ventral view there are gaps between them. The sacrum has stout transverse processes that are approximately as long as those of III–IV. There is a prominent sesamoid at the distal transverse process of the sacrum at its articulation with the ilium. The urostyle is shorter than the presacral vertebral column and bears a dorsal ridge that decreases in height posteriorly along the anterior two-thirds of the urostyle.

The pectoral girdle is well ossified. The coracoids are expanded medially and appear to have met at the midline; they appear to be out of articulation from preservation and dissection. The coracoids are broad and articulated at the midline. The scapula is short with a well-developed anterior process and a prominent supraglenoid foramen. The epicoracoid and procoracoid cartilages are heavily mineralized and form solid elements that are synostosed to the coracoid and clavicle creating a larger composite pectoral girdle.

The pelvic girdle is a robust element composed of synostosed ilium, pubis, and ischium; the pubis and ischium are completely ossified. The circular acetabulum is well ossified with well-defined margins. The shaft of the ilium is stout and mostly straight in both lateral and dorsal views and has both a well-developed dorsal protuberance that is continuous with a prominent dorsal crest. There is a broad, plate-like ventral acetabular expansion comprising the ilium and pubis.

The forearm is somewhat shorter than the humerus. The distal carpals (Element Y and II–V) are fused. The radiale and ulnare are large and subequal in size. The phalangeal formula for the manus is 1–2–3–1, with the phalanges of I and IV being small and barely discernable. There appears to be a single, minute ossified prepollex. There is one palmar sesamoid, which is larger than the phalanges of I and IV. The tips of the terminal manual phalanges are arrow-shaped in digits II and III but small triangular blobs in I and IV. The tibiofibula is slightly shorter than the femur. There are two large distal tarsals that are shorter than the tibiofibular. There is no ossified fabellar or tarsal sesamoids. The phalangeal formula for the pes is 0–2–3–4–1 and there is a single small ossified prehallux. On the plantar surface, there are no ossified medial sesamoids, but laterally there are two large sesamoids. The tips of the terminal pedal phalanges are expanded and blunt (rounded) in in digits III–IV and rounded in digit II; the phalanx of digit V is small round ossification.

Color in life. The general coloration of dorsal surfaces is brown with a few golden spots scattered aleatory in the dorsum surface and in the cloacal region (Figure 4). A golden stripe extends laterally from the tip of the snout to the flanks. The dorsal surfaces of arms and legs have dark brown stripes. The lateral view of the body is dark brown; the maxilla is dark brown with distinct white spots. The ventral surface of the body is brown with small white blotches concentrated in the cloacal region. The ventral surface of the hand and feet are light brown with dark brown blotches in the fingers and toes. The pupil is black, and the iris is golden.

Color in preservative. The general coloration of the dorsal surfaces is brown, darkened in the nostril and eyes region; the golden spots became pale cream. In recently preserved individuals, the stripes in the arms and legs became less distinct and difficult to discern, becoming more visible in individuals that have been preserved for a long time. The extremities of fingers and toes became pale cream. The ventral background coloration is cream and tinged light brown; the linea masculine absent. There are white spots in the maxillae, ventral, and cloacal regions. Eyes are black.

Internal anatomy. An adult female (ZUEC-AMP 25259) and an adult male (ZUEC-AMP 25578) of B. nanicus were dissected. In the female, after removing the skin, we observed that the abdominal muscles were transparent, without pigmentation, and we could see through them the ventral abdominal vein, pelvic veins, liver, gallbladder, and heart (Figure 6A and B). After cutting the abdominal muscle, cutting, and separating the procoracoid bone, we observed the internal organs (Figure 6B). The liver was yellowish white (Figure 6A and B). The heart presented slight pigmentation on the left side of the ventricle in ventral view. The species presented pigmentation near the carotid artery. Similarly, dark pigmentation, probably melanocytes, was observed at the ventral abdominal vein, near the point of origin of the right and left branches of the abdominal vein (Figure 6C). Such dark pigmentation can also be observed in the external jugular vein. Behind the liver, we observed two large eggs in the oviduct, which (the oviduct) was darkly pigmented by melanocytes (Figure 6D). The white abdominal pigmentation was observed in the male of B. nanicus, the dorsal white musculature was pigmented in B. nanicus male. The parathyroid glands were ovoid, transparent, presented a minor blood supply, and were single on each side of the body, near the bifurcation of the external and internal carotid arteries (Figure 6C).

The male’s abdominal musculature was triangular shaped, white pigmented, mainly covering the rectus abdominalis muscle.The pars abdominalis of m. pectoralis did not present such white coloration and was transparent (Figure 6E). In the female all the abdominal musculature was transparent. The liver was brown colored (Figure 6F), different from the female coloration. The left lobe of the liver was bilobed in males and females, but in males, the liver was relatively smaller than in females. In females, the left lobe covered the stomach almost completely (Figure 6B), whereas in males it slightly exceeded half the stomach size (Figure 6F). The right lobe was also smaller in the male than in the female. The dorsal musculature presented a slight dark pigmentation in the male (Figure 6G), not observed in the female. The heart and parathyroid glands were similar in male and female (Figure 6C and H).

Variation. The specimens are alike. We only have noticed some color variation among individuals with some frogs having a darker dorsal coloration.

Natural history. During the study we found six individuals of B. nanicus inhabiting a forested area with a deep leaf litter cover. Although the air humidity was over 98% in all samples, the individuals were always hidden under the leaf litter and were found among it or using roots and rotten trunks as shelter. The specimens displayed different defensive behaviors upon our approach: motionless, crouching down posture, or fleeing by jumping away. We also observed a congeneric syntopic species, B. margaritatus Pombal & Izecksohn, (2011) (ZUEC-AMP 25239), in the same area as B. nanicus. However, the B. margaritatus individuals were using the steepest areas of the ravines, whereas B. nanicus was all over the area.

We identified a total of 13 consumed prey items in the stomachs of one individual of B. nanicus (ZUEC-AMP 25610), belonging to five taxa divided between mites (Arachnida) and hexapods (Hexapoda) (Table 2; Figure 7). Mites were the most frequent (n = 12) prey item in the diet of B. nanicus, relative to the other prey categories found. Specifically, the order Mesostigmata was the most frequent item (n = 7), followed by the order Sarcoptiformes (n = 4), and Trombidiformes (n = 1). The Hexapoda prey species were from the order Psocoptera (n = 1; Table 2).

Geographic distribution. The new species is only known for its type-locality: Estação Ecológica de Bananal (22S 48’ 14”, 44W 22’ 17”, ~ 1.220 m a.s.l.; WGS84 Datum), municipality of Bananal, state of São Paulo, Brazil.