New species of flea-toad, genus Brachycephalus (Anura: Brachycephalidae) from the Atlantic Forest of Espírito Santo, Brazil Author Almeida-Silva, Diego Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Av. dos Estados, 5001, Santo André, 09210 - 580, São Paulo, Brazil. Author Silva-Soares, Thiago Universidade Federal do Espírito Santo, Museu de História Natural do Sul do Estado do Espírito Santo, Jerônimo Monteiro, 29550 - 000, Espírito Santo, Brazil. & Herpeto Capixaba, Enseada Azul, Guarapari, 29206 - 090, Espírito Santo, Brazil. Author Rodrigues, Miguel Trefaut Universidade de São Paulo, Instituto de Biociências, Caixa Postal 11461, CEP 05422 - 970, São Paulo, Brazil. Author Verdade, Vanessa Kruth 0000-0001-8990-0571 Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Av. dos Estados, 5001, Santo André, 09210 - 580, São Paulo, Brazil. & vanessa. verdade @ ufabc. edu. br; https: // orcid. org / 0000 - 0001 - 8990 - 0571 vanessa.verdade@ufabc.edu.br text Zootaxa 2021 2021-11-18 5068 4 517 532 journal article 3397 10.11646/zootaxa.5068.4.3 582fe5b8-d9a7-4b8c-b100-44f4b0d96dde 1175-5326 5709572 2CCF622B-005C-48DF-A1A4-41F81BD66177 Brachycephalus puri sp. nov. ( Figs. 3–4 ) Brachycephalus sp. : Zornosa-Torres et al . 2020 Holotype . MZUSP 135709 , adult male ( Figs. 3–4 ), collected on 7 November 2005 by MT Rodrigues , D Pavan , FF Curcio , and J Cassimiro at Córrego do Calçado ( 20°28’00” S , 41°44’03” W , Datum WGS 84; 1140 m elevation; field number MTR 11564), Parque Nacional do Caparaó , Patrimônio Santa Marta , municipality of Ibitirama , state of Espírito Santo , southeastern Brazil . TABLE 1. Percentage (%) of uncorrected pairwise distances ( p-distances ) for 16S mtDNA gene between 18 Brachycephalus species used in bayesian inference (BI). We ran this analysis on MEGA X, considering 342 sites after complete deletion of gaps and missing data. Minimum and maximum p-distances are reported for each pair of species. We give the number of individuals for each species in parentheses, as well as the intraspecific distances.

1	2	3	4	5	6	7	8	9
1	B. alipioi	0 (3)
2	B. bufonoides	1	NA (1)
3	B. crispus	1.2	1.8	0 (2)
4	B. didactylus	5.3–5.8	5.6–6	6.2–6,7	0–0.4 (3)
5	B. ferruginus	12.8	13.3	13.9	12.5	0 (2)
6	B. garbeanus	1.8	2	3	5.4–5.8	14.2	0 (2)
7	B. guarani	0.8	1.4	0.4	5.8–6.2	13.3	2.6	0 (4)
8	B. hermogenesi	11.4	11.3	12.4	11.8	8.9	12.2	11.8	NA (1)
9	B. izecksohni	11.1	11.6	12.7	10.7	6	11.9	12.1	10.1	NA (1)
10	B. margaritatus	2.4	2.2	3.6	5.8–6.2	13.6	1.8	3.2	10.9	11.6
11	B. nodoterga	0.4	1.4	1.2	5.3–5.8	13.3	2	0,8	11,9	11,6
12	B. pitanga	0.8	1.4	0.4	5.8–6.2	13.3	2.6	0	11.8	12.1
13	B. pulex	21.2	21.9	23.3	20.2–20.5	16.7	21.8	22.5	14	18.9
14	B. puri	10.5–11.3	10.7–11.5	11–11.8	10.3–11.6	14.7–16.2	11.5–12.4	10.4–11.3	14–15	14.5–16.1
15	B. rotenbergae	2.4	2.2	3.6	5.8–6.2	13.6	1.8	3.2	10.9	11.6
16	B. sulfuratus	11.2	11.4	11.9	10.6–10.7	7.2	12	11.4	10.1	7.4
17	B. toby	0.4	1.4	1.2	5.3–5.8	13.3	2	0.8	11.9	11.6
18	B. vertebralis	0.8	1.4	0.8	5.8–6.2	13.3	2.6	0.4	11.3	12.1

......continued on the next page TABLE 1. (Continued)

10	11	12	13	14	15	16	17	18
1	B. alipioi
2	B. bufonoides
3	B. crispus
4	B. didactylus
5	B. ferruginus
6	B. garbeanus
7	B. guarani
8	B. hermogenesi
9	B. izecksohni
10	B. margaritatus	0 (4)
11	B. nodoterga	2,8	0 (2)
12	B. pitanga	3.2	0.8	NA (1)
13	B. pulex	20.2	21.8	22.5	0 (2)
14	B. puri	11.5–12.4	10.9–11.8	10.4–11.3	23.6–24.9	0.2–1.8 (3)
15	B. rotenbergae	0	2.8	3.2	20.2	11.5–12.4	0 (3)
16	B. sulfuratus	12.2	11.7	11.4	18.5	14.6–15.6	12.2	0 (2)
17	B. toby	2.8	0	0.8	21.8	10.9–11.8	2.8	11.7	NA (1)
18	B. vertebralis	3.2	0.8	0.4	22.2	10.4–11.3	3.2	11.4	0.8	0 (3)

FIGURE 2. Bayesian Inference phylogenetic tree including species from Brachycephalus didactylus and B. ephippium groups occurring northward the state of São Paulo, based on 12S + 16S (1,152 bp, 42 terminals) genes, under GTR+G model of nucleotide substitution. We highlight the three phenotypic groups. Tip labels of Brachycephalus puri sp. nov. are in bold. Numbers close to nodes are Bayesian posterior probabilities (BPP). Values below 0.7 not shown. Paratypes . (N=4): MZUSP 135710 , adult female, collected on 8 November 2005 by MT Rodrigues and J. Cassimiro , field number MTR11579 . MZUSP 139152 , adult female, collected on 31 October 2006 by MT Rodrigues , J Cassimiro , LD Mattos , and VK Verdade , field number MTR12612 . MZUSP 139153-54 , adult males, collected on 1 November 2006 by MT Rodrigues , J Cassimiro , LD Mattos , and VK Verdade , field numbers MTR12615-16. All collected at the type locality. Diagnosis. (1) A small-sized species of Brachycephalus (adult male SVL 8.9–10.0 mm, adult female SVL 10.2 mm ), (2) body shape “leptodactyliform”, (3) skin smooth, without hyperossification or dorsal shield, (4) linea masculinea absent, (5) inverted V-shape dorsal dark mark present, (6) Fingers I and IV vestigial, (7) Toe I externally absent, Toe II reduced but functional, Toe V vestigial; (8) Toes III and IV with pointed tips; (9) venter uniformly brown. Description of the holotype . Diminutive “leptodactyliform” body, head length 33.7 % of SVL, as long as wide, and slightly narrower than body; snout length smaller than the diameter of eye; snout rounded in dorsal and lateral views; nostrils ellipsoid, directed anterolaterally, slightly protuberant, long axis shorter than END; canthus rostralis indistinct in frontal view; loreal region straight, vertical; eye medium sized (eye diameter 35% of head length), slightly protruding dorsolaterally; tympanum not visible externally, supratympanic fold absent, temporal region protruded; tongue longer than wide; dentigerous processes of vomers, pre-maxillary and maxillary teeth absent; curved grooves under the eye and nostrils. FIGURE 3. Dorsal (A) and ventral (B) views of the Brachycephalus puri sp. nov. , holotype (adult male, MZUSP 135709; SVL 9.2 mm). Scale = 1 mm. Vertebral column slightly protruding. Skin on top of the head, throat and chest smooth; dorsum of body, flanks, and dorsum of limbs slightly wrinkled and granular. Skin on the venter and ventral surfaces of legs smooth; skin granular on ventrolateral surfaces of the body and in the area around cloacal opening. Hyperossification and dorsal dermal shield absent. Arms slender. Upper arm is approximately the same length as the hand; both are slightly shorter than the forearm. Second and third fingers robust, pointed on their tips; first and fourth fingers vestigial but clearly visible; third finger twice as long as the second; relative lengths of fingers IV ≈ I <II <III; subarticular tubercles absent; inner and outer metacarpal tubercles inconspicuous. Legs short [(TL + THL)/SVL = 0.88], moderately robust; toes robust; first toe externally absent, second toe reduced, fifth vestigial; relative lengths of toes V <II <III <IV; third and fourth toe tips pointed, second slightly rounded; subarticular tubercles absent; inner and outer metatarsal tubercles inconspicuous. Measurements of the holotype (in mm) . SVL = 9.2, HW = 3.4, HL = 3.1, IOD = 1.6, ED = 1.1, IND = 1.3, END = 0.5, SL = 1.0, ND = 0.3, TL = 3.8, THL = 4.3, FOL = 3.3, HAL = 1.5, FLL = 1.6, UAL = 1.4. Coloration in preservative. The general coloration of dorsal surfaces is brown, darkened by black or brown dots; inverted V-shaped dorsal dark mark present in mid-dorsum; no linea masculinea . Arms and legs with dark brown stripes. Ventral surface uniformly pale brown, as well as most of the lateral surfaces, except by some darker spots located in the dorsolateral area. Presence of an M-shaped dark mark dorsally to the cloacal aperture. FIGURE 4. Brachycephalus puri sp. nov. , holotype (adult male, MZUSP 135709). (A) Lateral view of head, ventral right views of (B) hand and (C) foot, and (D) cloacal adornment (M-shaped black mark). Scale = 0.5 mm. Variation. No sexual dimorphism detected in body measurements ( Table 2 ) or color pattern, though females seem more robust than males. The dorsal background color of some specimens may vary from brownish to pale brown, with narrow stripes that may form a complete black X from the inverted V-shaped dark mark of mid-dorsum. In these specimens, anterior portion of the X-shaped mark is discontinuous, consisting of two independent weak stripes. TABLE 2. Measurements taken from specimens of the Brachycephalus puri sp. nov. type series. Values are in millimeters. Measurements and abbreviations follow Watters et al . (2016) ; forearm length (FLL) corresponds to the FAL in literature for the genus (e.g. Condez et al . 2016 , 2021; Nunes et al . 2021 ). See Material and Methods for details. Holotype in bold. Some measurements were not taken in specimens that were poorly preserved.

MZUSP 135709 (male)	MZUSP 135710 (female)	MZUSP 139152 (female)	MZUSP 139153 (male)	MZUSP 139154 (male)
SVL	9.2	10.2	9.2	10.0	8.9
HW	3.4	3.5	3.5	3.6	3.3
HL	3.1	3.5	2.9	3.1	3.1
IOD	1.6	1.7	2.1	1.4	1.5
ED	1.1	1.4	1.5	1.2	1.2
IND	1.3	1.3	–	1.2	1.3
END	0.5	0.5	0.4	0.4	0.4
SL	1.0	1.1	–	0.9	1.0
ND	0.3	0.3	–	0.3	0.2
UAL	1.4	1.8	–	1.4	1.6
FLL	1.6	2.0	–	1.9	1.8
HAL	1.5	1.8	1.7	1.4	1.6
THL	4.3	4.4	4.1	4.0	4.5
TL	3.8	4.1	3.7	4.1	3.8

Comparison to other species. Brachycephalus puri is promptly diagnosed from members of the B. ephippium and B. pernix species groups by the combination of “leptodactyliform” body shape, dull-coloration, absence of hyperossification or dorsal bony shield, and absence of linea masculinea . Except for B. brunneus and B. curupira , all other species in the B. ephippium and B. pernix species groups are brightly colored varying from uniform bright yellow to reddish-orange, sometimes including dark blotches or stripes in life. Additionally, all species in the B. ephippium species group present hyperossification and, in some species, a dorsal shield (absent in B. puri ). All species in B. pernix group present linea masculinea (absent in B. puri ). Brachycephalus brunneus and B. curupira are dull colored members of B. pernix group. However, additionally to the presence of linea masculinea , the toes of both species are more severely reduced, Toe I and II are absent, and Toe V is vestigial, barely seen (Toe I absent, but Toe II present and functional, and Toe V vestigial in B. puri ). Brachycephalus didactylus , B. hermogenesi , B. pulex and B. sulfuratus are dull-colored members of the genus that do not present hyperossification, dorsal shield or linea masculinea . Brachycephalus pulex promptly differ from other members of the group by presenting fingers and toes more severely reduced: Fingers I and IV are externally absent (vestigial in B. puri ); Toes I and II are absent, and Toe V is vestigial, barely seen (Toe I absent, but Toe II present and functional, and Toe V vestigial in B. puri ). Brachycephalus didactylus has a large trapezoidal black spot on the venter that represents an aggregation of skin glands (see Izecksohn 1971 ; Giaretta & Sawaya 1998 ) (absent in B. puri ), and the cloacal region is usually not ornamented nor presents a rounded black mark (M-shaped black mark in B. puri ). Brachycephalus hermogenesi and B. sulfuratus have a distinct and functional Toe V (vestigial in B. puri ). Additionally, live individuals of B. sulfuratus often present yellow spots on the ventral region (venter uniformly brown in B. puri ). Also, Brachycephalus didactylus , B. hermogenesi , and B. pulex often show an X-shaped dorsal mark (usually inverted V-shaped in B. puri ). A comparative summary between B. puri and the other representatives of B. didactylus species (including B. pulex ) group is shown in Table 3 . Etymology. The specific name honors the indigenous people from the Puri tribe, who lived from the 16th to 19th centuries in the territory encompassing the river basin of Paraíba do Sul and the boundaries between the Rio Grande and Rio Doce river basins ( IPHAN 2017 ). The name proposed represents our respect and sorrow for the indigenous lives lost throughout Brazil’s history, including the recent ones caused by the Covid-19 pandemic. Common name. Due to presenting the flea-toad phenotype, we designate to Brachycephalus puri the common English name “Caparaó Flea-toad” and, in Portuguese, “sapo-pulga-do-Caparaó”. Distribution and natural history. Brachycephalus puri seems to be endemic to the oriental portion of the Parque Nacional do Caparaó, being currently known only for the type locality, the surroundings of Córrego do Calçado, municipality of Ibitirama, in the western part of State of Espírito Santo ( Fig. 1 ). All specimens were captured in 500 ml pitfall traps installed in dense and preserved rainforest around 1,200 m of elevation.