Fig 1.
Holotype of Brachycephalus lulai sp. nov. (MHNCI 11592), male.
(A) Dorsal view of the body. (B) Lateral view of the head. (C) Ventral view of right hand. (D) Ventral view of right foot. The specimen’s image was projected using a stereomicroscope with a camera lucida, and the illustration was rendered in black ink using the pointillism technique Drawing by Verônica R. Apolônio.
Fig 2.
Holotype of Brachycephalus lulai sp. nov. (MHNCI 11592), male, in life.
(A) Anterolateral view. (B) Ventral view. In B, white arrow indicates the presence of the linea masculinea. Photographs by Luiz F. Ribeiro.
Fig 3.
Holotype of Brachycephalus lulai sp. nov.
(MHNCI 11592), male, in preservative. (A) Dorsal view. (B) Ventral view.
Fig 4.
Variation in coloration of paratypes of Brachycephalus lulai sp. nov. Column one and column three show specimens in dorsal view in life and in preservative, respectively.
Column two and column four show specimens in ventral view in life and in preservative, respectively. A1–A4 = MHNCI 11612. B1–B4 = MHNCI 11598. C1–C4 = MHNCI 11596. D1–D4 = MHNCI 11599. E1–E4 = MHNCI 11600. F1–F4 = MHNCI 11594. Scale bars equal 5 mm. Photographs by Luiz F. Ribeiro.
Table 1.
Measurements of the 15 variables (in mm) of the type series of Brachycephalus lulai sp. nov.
Table 2.
Comparison of the diagnostic features of Brachycephalus lulai sp. nov. in relation to remaining species of B. pernix group. In bold we highlight the variables that are diagnostic in relation to B. lulai sp. nov. Sample size refers to the number of individuals in which the authors observed each variable or number of individuals from which one or more calls where recorded and analyzed by the authors.
Fig 5.
Divergent color variation of Brachycephalus species.
A1–A3 = B. actaeus MHNCI 11625 (Forte Marechal Luz, Ilha de São Francisco, municipality of São Francisco do Sul, Santa Catarina). B1–B3 = B. auroguttatus MHNCI 11768 (trail to Pedra da Tartaruga, municipality of Garuva, Santa Catarina). C1–C3 = B. fuscolineatus MHNCI 11599 (Morro do Baú, municipality of Ilhota, Santa Catarina [type locality]). D1–D3 and E1–E3 = B. tridactylus MHNCI 10852 and MHNCI 11767, respectively (Torre Embratel, Parque Estadual do Rio Turvo, municipality of Cajati, São Paulo). Abbreviation: MHNCI = Museu de História Natural Capão da Imbuia, Curitiba, Paraná. Scale bars equal 5 mm. Photographs: A1–D3 = Luiz F. Ribeiro; E1–E3 = Marcos R. Bornschein.
Fig 6.
High-resolution computed tomography (CT) scans of a paratype of Brachycephalus lulai sp. nov. (MHNCI 11601) showing key osteological features.
(A) Dorsal view of the skeleton; (B) dorsal, (C) lateral (without the lower jaw), and (D) ventral views of the skull; (E) pectoral girdle in ventral view; (F) ilium in lateral view; (G) left hand in palmar view; (H) left foot in plantar view. While the vomer is fused to surrounding elements, we have highlighted its approximate boundaries. Abbreviations: qj = quadratojugal; sq = squamosal; v = vomer. Scale bars equal 2 mm.
Table 3.
Measurement (µm) of the dermis thickness and mineralized dermal layer (MDL) of the skin of the medial and caudal regions of the dorsal body of Brachycephalus lulai sp. nov.
Fig 7.
Photomicrography of the dorsal skin of Brachycephalus lulai sp. nov.
(A and B) Specimens from Pico Garuva. (C and D) Specimens from Monte Crista. The red arrows indicate the mineralized dermal layer in the dermis. The black line indicates the extent of the dermis. In A and C, Hematoxylin and Eosin staining; in B and D, von Kossa staining.
Table 4.
Structure of the advertisements calls (AC) of Brachycephalus lulai sp. nov. and other members of the B. pernix species group.
Table 5.
Call parameters described in the advertisement call of Brachycephalus lulai sp. nov.
Fig 8.
Example of advertisement call parameters of Brachycephalus lulai sp. nov.
(A) Entire advertisement call (MHNCI 226). (B) Isolated note with one pulse (MHNCI 225). (C) Isolated note with three pulses (MHNCI 224). (D) Note group with three and two pulses (MHNCI 232). (E) Attenuated note (black circle) preceding an isolated note with three pulses (MHNCI 226). Spectrograms are produced with a FFT size of 8192 points, Hann window, and overlap of 90% in A and FFT 512 points, Hann window, and overlap of 90% in B–E.
Fig 9.
Example of an attenuated note (smaller, on the left) of Brachycephalus lulai sp. nov. followed by an isolated note with three pulses (larger, on the right).
Sound taken from MHNCI 227 and generated with 90 overlap and 256 FFT size by R package Soundshape.
Table 6.
Pairwise genetic distance (%) between the studied Brachycephalus species for all loci (upper diagonal) and 16S alone (lower diagonal).
Fig 10.
Relationships between Brachycephalus lulai sp. nov. and some species of the B. pernix group based on three loci.
Phylogenetic analysis was carried out using Bayesian inference and values above branches correspond to node posterior probabilities. Nodes with posterior probabilities lower than 50% were collapsed.
Fig 11.
Habitat at the type locality of Brachycephalus lulai sp. nov., Pico Garuva, municipality of Garuva, Santa Catarina, southern Brazil.
(A) Middle and low strata of the forest (Floresta Ombrófila Densa Montana) at 750 m above sea level. (B) East slope of Pico Garuva, 1,260 m above sea level. Photographs by Luiz F. Ribeiro.
Fig 12.
Location of specimens examined of species from Brachycephalus pernix group, in southeastern and southern Brazil.
(A) All specimens. (B) Detail of the distribution of B. lulai sp. nov. Abbreviations: SP = São Paulo; PR = Paraná; SC = Santa Catarina. Urban areas are displayed in gray, open vegetation areas in light green, and dense vegetation in dark green. Basemaps: OpenStreetMap, under Open Database License (ODbL), available at https://www.openstreetmap.org/.
Fig 13.
Brachycephalus lulai sp. nov. (MHNCI 11593) parasitized by tapeworms (Ophiotaenia sp).
(A) The arrows point to the locations of the parasites at the gular region and the left thigh. (B) The arrow points to the location of the parasite on the left thigh. Edema regions can also be seen in these images, such as the left leg and right thigh. (C) Ophiotaenia sp. soon after removal from the gular region of the collected frog. (D) Ophiotaenia sp. isolated from the B. lulai sp. nov. Photographs by Luiz F. Ribeiro.
Table 7.
Conservation status of species of the Brachycephalus pernix group, southeastern and southern Brazil.
Table 8.
Green Status of species of the Brachycephalus pernix group, southeastern and southern Brazil.
Fig 14.
Limits of the Refúgio de Vida Silvestre Serra do Quiriri, integral protection conservation unit proposed for Brachycephalus lulai sp. nov., B. auroguttatus, and B. quiririensis.
Abbreviations: PR = Paraná; SC = Santa Catarina; RVS = Refúgio de Vida Silvestre. Basemaps: Natural Earth, under Public Domain, available at https://www.naturalearthdata.com, and OpenStreetMap, under Open Database License (ODbL), available at https://www.openstreetmap.org/.