A new species of Neblinaphryne (Anura: Brachycephaloidea: Neblinaphrynidae) from Serra do Imeri, Amazonas state, Brazil Author Fouquet, Antoine Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT & 118 route de Narbonne, 31062 Toulouse cedex Author Moraes, Leandro J. C. L. 0000-0001-6704-898X Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT Author Grant, Taran 0000-0001-6704-898X Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT Author Recoder, Renato 0000-0001-6704-898X Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT Author Camacho, Agustín 0000-0003-2978-792X Université Toulouse III - Paul Sabatier, Bât. Author Ghellere, José Mário 0000-0001-6704-898X Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT Author Barutel, Alexandre Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT & 118 route de Narbonne, 31062 Toulouse cedex Author Rodrigues, Miguel Trefaut 0000-0001-6704-898X Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT & Centre de Recherche sur la Biodiversité et l’Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT & 118 route de Narbonne, 31062 Toulouse cedex text Zootaxa 2024 2024-09-25 5514 1 73 90 http://dx.doi.org/10.11646/zootaxa.5514.1.5 journal article 303916 10.11646/zootaxa.5514.1.5 7e2f7382-29d2-42bf-91a9-d0e6e893ebd7 1175-5326 13849425 2DE0DD0B-7C78-4557-BF0C-0261DE60827D Neblinaphryne imeri sp. nov. Holotype ( Fig. 4 ). MZUSP 160479 ( MTR 30991 ), an adult male from Serra do Imeri , Parque Nacional Pico da Neblina , Santa Isabel do Rio Negro , Amazonas state, Brazil ( 0.470833 N , 65.33889 W , ca. 1800 m elevation), collected 16 November 2022 by M . T . Rodrigues; A . Camacho; A . Fouquet; J . M . B . Ghellere; T . Grant; L . Moraes; R . Recoder. Paratopotypes ( n = 9) ( Fig. 5 ). MZUSP 160471 ( MTR 30853 ) , MZUSP 160474–160475 , ( MTR 30895 , MTR 30928 ) , MZUSP 160477–160478 ( MTR 30988 , MTR 30990 ) , and MZUSP 160480 ( MTR 31003 ) , six adult males, and MZUSP 160476 ( MTR 30964 ) , MZUSP 160472–160473 ( MTR 30864 , MTR 30892 ) , three adult females, all collected between 8 and 16 November 2022 by the same collectors. Etymology. The specific epithet “ imeri ” is a noun in apposition and refers to the Imeri mountain massif where the species occurs. Generic placement. The new species is assigned to the genus Neblinaphryne based on its phylogenetic relationship as assessed by molecular data ( Fig. 3 ) and its pointed (not T–shaped) terminal phalanges and minuscule septomaxillae, two osteological characters that were identified as diagnostic of the genus by Fouquet et al. (2024) . Definition. A minute brachycephaloid frog characterized by: (1) SVL 15.9 ± 1.1 mm in males, 19.8 ± 0.5 mm in females ( Table 1 ); (2) head wider than long, narrower than body; (3) tympanum indistinct, covered by skin and dorsally by a supratympanic fold, columella present ( Fig. 6 ); (4) one prominent tubercle on each eyelid (creamcolored in life) and in the humeral region (white-tipped, Fig. 4 ); (5) frontoparietal smooth, cranial crests absent; (6) dentigerous process of vomer absent, vomers free of contact with any other bone; (7) subgular vocal sac, vocal slits present in males; (8) fingers fully developed without subarticular tubercles, finger tips not expanded, rounded on Finger I , pointed or mucronate on II and IV, mucronate on Finger III, without discs and circumferential grooves, terminal phalanges pointed; (9) outer metacarpal tubercle flat, drop-shaped, other tubercles on hands and fingers absent; (10) Finger I slightly shorter than Finger II; relative lengths of fingers: I <II <IV <III; nuptial pads absent; (11) toes fully developed, toe tips not expanded, pointed or rounded on Toes I and V , mucronate on Toes II –IV, terminal phalanges pointed on Toes I and V and slightly knobbed on Toes II–IV; (12) relative lengths of toes: I <II <III< V <IV, Toe V only slightly longer than Toe III; (13) lateral fringes and webbing absent on fingers and toes; (14) toe subarticular tubercles absent; supernumerary tubercles absent; (15) outer metatarsal tubercle round, large, protruding, inner metatarsal tubercle ovoid, flat; (16) dorsolateral folds absent; (17) discoidal fold absent; (18) septomaxillae minuscule; (19) iris copper with dark reticulations in life, pupillary ring thin and often discontinuous, horizontal large and diffuse dark band and vertical thin, and sharp dark band reaching the pupil only on the lower portion; (20) m. iliacus externus originating via a single head anteriorly and extending in parallel alongside the entire length of the iliac shaft; (21) m. tensor fasciae latae originating anteriorly on the iliac shaft, immediately posterior to the sacral diapophyses ( Fig. 7 ); (20) Testis immaculate white in males and oviduct expanded, convoluted and large white oocytes in females; (21) call consisting of long (414–592 ms) Type 1 notes formed by series of 31–70 pulses emitted with dominant frequency 3,010 –3,957 Hz generally followed by shorter (76–180 ms) Type 2 notes that have otherwise the same basic structure ( Fig. 8 ). FIGURE 4. Holotype of Neblinaphryne imeri sp. nov. MZUSP 160479 (MTR 30991, adult male). (A) Dorsal and (B) ventral views in preservative; (C) palmar view of the left hand and (D) plantar view of the left foot; (E) dorsolateral and (F) ventral views in life. The humeral tubercle is indicated by white arrows on (A) and (E). FIGURE 5. Dorsolateral and ventral views of paratypes of Neblinaphryne imeri sp. nov. in life. (A) MZUSP 160471 (MTR 30853); (B) MZUSP 160474 (MTR 30895); (C) MZUSP 160477 (MTR 30988); (D) MZUSP 160478 (MTR 30990); (E) MZUSP 160480 (MTR 31003); (F) MZUSP 160472 (MTR 30864); (G) MZUSP 160473 (MTR 30892); (H) MZUSP 160476 (MTR 30964). Description of the holotype ( Fig. 4 ). Adult male with a ventral incision on the right side below the chest, SVL 16.3 mm , head slightly wider than long, head length 25% SVL; head width 38% SVL; snout short, rounded in dorsal and lateral views; upper eyelid width 62% of interorbital distance; eye to nostril distance 60% of eye diameter; nostrils ovoid. Pupil horizontal ( Cervino et al. 2021 ). Tympanum indistinct, its dorsal portion concealed by a supratympanic fold. Head without enlarged tubercles or folds except the two enlarged tubercles on each eyelid. Cranial crests and dentigerous processes of vomers absent. Vocal slits present, one on each side, from the jaw angle to the lateral base of the tongue; vocal sac subgular; tongue ovoid, free posteriorly for nearly half its length; choanae small, round, located laterally and anteriorly. Skin of dorsum, throat, and upper and lower limbs smooth with few small, scattered tubercles, notably on eyelids, dorsum, and legs; skin of belly finely granular. Prominent white-tipped tubercle present on the humeral region. Axillary membrane absent. Fingers without discs and circumferential grooves absent, tips unexpanded, rounded on Finger I, pointed on II and IV, mucronate on III; fingers lacking fringes and webbing. Finger I slightly shorter than Finger II; relative lengths of fingers I <II <IV <III. Outer metacarpal tubercle flat, drop-shaped; other tubercles on hand and fingers (e.g., subarticular, supernumerary) absent. Nuptial pads absent. Toes without discs and circumferential grooves, toe tips unexpanded, pointed on Toe I, mucronate on Toes II–IV, rounded on Toe V; toes lacking lateral fringes and webbing. Toe V slightly longer than Toe III; relative lengths of toes I <II <III <V <IV. Inner metatarsal tubercle ovoid, flat. Outer metatarsal tubercle rounded, prominent, slightly larger than inner one. Subarticular and supernumerary tubercles absent. FIGURE 6. Volume renderings of a male paratype (MZUSP 160474 / MTR 30895) in dorsal, ventral, lateral, and frontal views. Minuscule septomaxillae are indicated by red circles in the frontal view. Coloration of holotype in life. Dorsum reddish brown with irregular dark brown blotches and small white dots. Interorbital region with dark brown bar delimited anteriorly by cream line. Canthus rostralis, tip of snout, lips, and supratympanic folds with dark brown blotches and a few white dots. Coloration of flanks forming series of oblique stripes delimited by grayish lines, anteriorly dark brown, gray in middle, and posteriorly with oblique dark brown lateral stripe. Axillary and inguinal regions bright orange. Throat, belly and ventral surface of legs dark gray with many small white spots, with diffuse bright orange blotches posteriorly. Dorsal surface of arms and legs same as dorsum but with dark brown blotches forming transverse bands. Inner surface of shank and feet bright orange. Iris copper with dark brown vertical bar on its upper and lower portions, only reaching the pupil on the lower part. Variation. Morphometric variation is summarized in Table 1 . Females are larger than males ( SVL 19.2–20.1 mm in females and 14.2–17.2 mm in males). The coloration is highly variable, especially the distribution of dark brown blotches and the extent of orange blotches. Only the dark brown supratympanic blotch and the interorbital bar are found in all the specimens. Females have fewer dark brown blotches, but the variation of bright orange marks does not seem to be related to sex. The orange marks vary from tiny blotches in the inguinal regions and blotches on shanks ( MZUSP 160480 / MTR 31003 ) to being extensively distributed on the throat and belly and the entirety of the ventral sides of the arms and thighs, axillae, inguinal regions, and shanks ( MZUSP 160472 / MTR 30864 ). One female ( MZUSP 160473 / MTR 30892 ) has wide, well-defined, light brown dorsolateral lines. Two males ( MZUSP 160477 / MTR 30988 , MZUSP 160480 / MTR 31003 ) have less conspicuous dorsolateral lines. A single male ( MZUSP 160478 / MTR 30990 ) has many dark brown blotches on the belly . FIGURE 7. Photograph of the posterior side of the thigh of Neblinaphryne imeri sp. nov. (paratype male, MZUSP 160477 / MTR 30988) showing the m. iliacus externus (ile) and the m. tensor fasciae latae (tfl). FIGURE 8. Spectrogram and oscillogram of the advertisement call of Neblinaphryne imeri sp. nov. (holotype MZUSP 160479/ MTR 30991) consisting of one Type 1 note and two Type 2 notes. Osteology. The following description is based on the skeleton examined by volume rendering of MZUSP 160474 (male paratype , field number MTR 30895; Fig. 6 ). Two additional specimens were examined and agree with this description. The osteology is very similar to N. mayeri that has been extensively described by Fouquet et al. (2024) . Therefore, we only mention the observed differences below: Cranium larger than long; palatine slightly curved and expanding towards sphenethmoid; alary processes of premaxilla with omega-shaped ridge posterodorsally; pterygoid long, reaching neopalatine anteriorly; quadratojugal very short; angulosplenial only slightly sigmoid; neural arches of presacrals well developed, bearing large, projecting neural crests on presacrals III–IV only; calcanea and astragali partly fused with only a narrow gap medially; absence of fabella sesamoid ( Abdala et al. 2019 ). Osteological variation (Appendix 3). The osteological features of two additional specimens have been examined (MZUSP 160473/ MTR 30892; MZUSP 160478 / MTR 30990). We did not observe any noteworthy deviation from MZUSP 160474 (MTR 30895). Advertisement call. The call consists of two types of notes ( Fig. 8 ; Table 2 ). Type 1 notes are long (414–592 ms) series of pulses (31–70 pulses) emitted at increasing rate (silence between pulses 15–34 at the beginning of the call vs 9–17 ms at the end of the call) within the note and downward frequency modulation (starting dominant frequency 3,349 –3,957 Hz; ending dominant frequency 3,010 –3,882 Hz). A harmonic structure is visible as high as ca. 13,000 Hz (not shown) with the most intense harmonics being ca. 7,000 –7,500 and 9,500 –10,000 Hz (not shown). Type 1 notes are often emitted alone, whereas series of 2– 4 Type 2 notes are generally emitted after Type 1 notes. Type 2 notes have the same basic structure and dominant frequency ( 3,356 –4,023 Hz) as Type 1 notes but are shorter (76–180 ms) and have fewer pulses (7–20). TABLE 2. Acoustic variables across four recorded males (one measurement of each variable per recorded male).

Variable	Mean±s.d. (Min–Max)
Call length (ms)	1809 ± 128.8 (1689–2001)
Note length 1 (ms)	592 ± 81.9 (414–592)
Note length 2 (ms)	142.5 ± 44.6 (76–180)
Silence between Type 1 and 1st Type 2 notes (ms)	475 ± 56.9 (431–558)
Silence between 1st and 2nd Type 2 notes (ms)	455 ± 70.9 (400–570)
Number of pulses/Type 1 notes	70 ± 17.5 (31–70)
Number of pulses/Type 2 notes	11 ± 5.8 (7–20)
Number of interpulse silence start Type 1 notes (ms)	18 ± 8.5 (15–34)
Number of interpulse silence end Type 1 notes (ms)	9 ± 3.9 (9–17)
Fundamental start Type 1 (Hz)	2843 ± 250 (2564–3150)
Dominant start Type 1 (Hz)	3957 ± 265 (3349–3957)
Fundamental end Type 1 (Hz)	2371 ± 409 (2371–3144)
Dominant end Type 1 (Hz)	3324 ± 393 (3010–3882)
Delta Dominant frequency Type 1 (Hz) Dominant frequency Type 2 (Hz)	633 ± 358 (-72–723) 3799 + 286 (3356–4023)

Comparison with other species. Neblinaphryne imeri sp. nov. is distinguished from N. mayeri (in parentheses) by the following traits: (1) coloration characterized by dark brown blotches scattered on dorsal surface of the body, whitish dots and orange marks on the inguinal region, inner surface of arms, legs and venter (almost uniformly brown on dorsum, without white dots and orange marks); (2) one cream colored (white in life) tubercle on each eyelid (absent); (3) prominent white-tipped, humeral tubercle present (absent); (4) palatine slightly curved, expanding towards sphenethmoid (straight, tapering towards sphenethmoid); (5) pterygoid long, reaching palatine anteriorly (not reaching); (6) alary processes of premaxilla with omega-shaped ridge posterodorsally (without ridge); (7) presacral neural arches bearing large, projecting neural crests on presacrals III–IV (present on presacrals II–IV); (8) calcanea and astragali partly fused with only a narrow gap medially (large gap); (9) fabella sesamoid absent (fabella sesamoid present, large). Neblinaphryne imeri sp. nov. can be distinguished from all the brachycephaloids examined by Fouquet et al. (2024 ; in parentheses) except N. mayeri by its pointed terminal phalanges (T–shaped) and minuscule septomaxillae (large). It can be further distinguished from the four other brachycephaloid lineages occurring in Pantepui— Adelophryne ( Eleutherodactylidae , Phyzelaphryninae ), Pristimantis ( Strabomantidae , Pristimantinae), Ceuthomantis ( Ceuthomantidae ), and Dischidodactylus (incertae sedis)—as follows (character states of other taxa in parentheses): from Adelophryne by its fully developed fingers and toes (reduced Finger IV), with mucronate tips on Fingers II–IV and Toes II–IV only (most digits with mucronate tips), and without a lateral groove (with groove); lack of vomerine teeth (present), and from Ceuthomantis , Pristimantis , and Dischidodactylus by its mucronate tips on Fingers II–IV and Toes II–IV (rounded) and m. tensor fasciae latae originating anteriorly on the iliac shaft (posterior or middle). It can be further distinguished from Ceuthomantis and Dischidodactylus by its m. iliacus externus originating anteriorly on the iliac shaft (vs. originating on the posterior half or three-fourths of the iliac shaft) and from Dischidodactylus by the lack of vomerine teeth (present). Distribution ( Fig. 1 ). Neblinaphryne imeri sp. nov. is known only from the type locality, in the Serra do Imeri, Parque Nacional do Pico da Neblina, Santa Isabel do Rio Negro, Amazonas state, Brazil . Based on the spatial distribution of calling males, the species seems quite abundant in both the open vegetation covering the top of the massif, at elevations between 1,800 and 1,940 m , and in the adjacent mountain forests. Below 1,800 m the species becomes rarer, and no calling males were heard at 1,700 m . Natural history. In both open and forested habitats Neblinaphryne imeri is very difficult to locate and capture. Females were opportunistically found when searching for calling males (e.g. MZUSP 160472/MTR 30864), suggesting that they were reproductively active, which is corroborated by large oocytes (oviduct expanded and oocytes white, two oocytes measured 1.5–1.7 mm diameter in MZUSP 160472, other smaller oocytes not counted to avoid damaging the specimen), possibly attracted by the calling male. In open habitat, the species calls beneath herbaceous vegetation or in dry leaves and stems of rosette-forming plants, such as giant bromeliads. In forested habitat, individuals call from deep inside the layers of moss. The holotype MZUSP 160479 (MTR 30991) was calling from inside a theraphosid (tarantula) burrow. Based on the calling activity, Neblinaphryne imeri sp. nov. is diurnal, vocalizing throughout the day but with peak activity during the first hours of light in the morning and the end of day until the first hour of darkness.