Search

The biodiversity collapse strongly affects the amphibian group and many factors have been pointed out as catalytic agents. It is estimated that several events in the amphibian population decline worldwide may have been caused by the interaction of multiple drivers. Thus, this study aimed to evaluate the stressful effects of the exposure to environmental doses of trichlorfon (TCF) pesticide (0.5 μg/L; and an additional 100-fold concentration of 50 µg/L) and ultraviolet radiation (UV) (184.0 kJ/m² of UVA and 3.4 kJ/m² of UVB, which correspond to 5% of the daily dose) in tadpoles of the Boana curupi species (Anura: Hylidae). The isolated and combined exposures to TCF happened within 24 h of acute treatments under laboratory-controlled conditions. In the combined treatments, we adopted three different moments (M) of tadpole irradiation from the beginning of the exposures to TCF (0 h - M1; 12 h - M2; and 24 h - M3). Then, we evaluated tadpole survival, change in morphological characters, induction of apoptotic cells, lipid peroxidation (LPO), protein carbonyl content (PCC), glutathione S-transferase (GST), non-protein thiols (NPSH), and acetylcholinesterase (AChE), as well as the induction of genomic DNA (gDNA) damage. UVB treatment alone resulted in high mortality, along with a high level of apoptosis induction. Both UVA, UVB, and TCF increased LPO, PC, and AChE, while decreased GST activity. Regarding co-exposures, the most striking effect was observed in the interaction between UVB and TCF, which surprisingly decreased UVB-induced tadpole mortality, apoptosis, and gDNA damage. These results reinforce the B. curupi sensitivity to solar UVB radiation and indicate a complex response in face of UVB interaction with TCF, which may be related to activation of DNA repair pathways and/or inhibition of apoptosis, decreasing UVB-induced tadpole mortality.

Leptodactylus kilombo sp. nov. (Figs. 2, 5d, 6f, 8) [http://zoobank.org/ urn:lsid:zoobank.org:act: E9291B2E-D63E-49AE-9CDD-122560774016] Leptodactylus sp. (aff. mystaceus): Silva et al. (2020) Holotype. CHUFPB 28204, an adult male from Bezerra stream (12.897899°S, 46.810419°W; 626 m above sea level [a.s.l.]), in Arraias, Tocantins State, northern Brazil, collected by L. A. da Silva on 7 November 2018. Paratypes. One adult male (CHUFPB 28205) and one adult female (CHUFPB 30409) collected with the holotype. Referred material. One genetic voucher (UFBA 7694) from Bom Jesus da Lapa, Bahia State (Fig. 1), originally assigned to L. mystacinus by Dória et al. (2018). Etymology. The Kimbundu word kilombo (quilombo in Portuguese), used as a noun in apposition, literally means war camp. Quilombos are hinterland settlements founded by people of African origin including the quilombolas (inhabitants of quilombos), maroons, and some other names and variations by which these communities recognize themselves. Most of the original inhabitants were runaway slaves. There are hundreds of recognized quilombos throughout Brazil and also several out of record by the federal government. Such settlements carry plenty of historical, cultural, and anthropological history back into the Brazilian Imperial Age (colonial Brazil), especially in the mid-17th century, related to the slavery in the country. The type locality of Leptodactylus kilombo is located nearby an ancient quilombola settlement named Chapada dos Negros in Arraias, in the south of Tocantins State, north central Brazil (Gualberto 2017). Diagnosis. Leptodactylus kilombo is characterized by the following combination of character states: (1) small to moderate size, male SVL = 37.6–41.3 mm, female SVL = 41.8 mm; (2) advertisement call composed of single, nonpulsed notes; (3) note duration varying from 30–56 ms; (4) note dominant frequency varying from 1098–1572 Hz; (5) frequency upsweep varying from 388–775 Hz. Comparisons with members of the L. mystaceus complex. Leptodactylus kilombo can be distinguished from other species by having the sole of the foot covered with few, non-obvious tubercles (prominent tubercles in the other species; Heyer 1978, 1983; Heyer et al. 1996), except Leptodactylus notoaktites. The new species (male SVL = 37.6–41.3 mm, mean = 40.8; female SVL = 41.8 mm; Table 2) can be further distinguished from the other species of the L. mystaceus complex, except L. elenae and L. spixi, by a smaller size: L. didymus (male SVL = 45.9–52.2 mm, mean = 49.3; female SVL = 45.8–53.5 mm, mean = 49.8; Heyer et al. 1996), L. mystaceus (male SVL = 42.4–52.2 mm, mean = 47.4; female SVL = 44.8–56.1 mm, mean = 50.0; Heyer et al. 1996), L. notoaktites (male SVL = 42.5–54.2 mm, mean = 47.5; female SVL = 43.4–55.8 mm, mean = 48.0; de Sá et al. 2014). Members of the L. mystaceus complex are mainly distinguished from each other by their distinct advertisement calls (Fig. 6). Acoustic comparisons in the L. fuscus group. The advertisement call of Leptodactylus kilombo is composed of single notes (Fig. 6f), differing from the trill calls of L. cunicularius, L. cupreus, L. oreomantis, and L. plaumanni (Fig. 7; Carvalho et al. 2013). The new species is distinguished from species with pulsed calls (L. caatingae, L. fragilis, L. labrosus, L. mystaceus, and L. cf. mystaceus; Heyer 1978; Heyer & Juncá 2003; Heyer et al. 2006; Carvalho & Ron 2011) by having a nonpulsed call. From the remaining species of the L. fuscus group also having single-note, nonpulsed calls, L. kilombo is distinguished by a shorter note duration (30–56 ms) from L. bufonius, L. camaquara, L. didymus, L. elenae, L. laticeps, and L. spixi (> 100 ms; Table 3; Heyer & Scott 2006; Carvalho et al. 2013). The call of L. kilombo has a modest frequency upsweep (frequency modulation, FM = 388–775 Hz), whereas L. albilabris, L. fuscus, L. gracilis, L. jolyi, L. longirostris, L. notoaktites, L. sertanejo, and L. syphax have a pronounced frequency upsweep in their calls (FM> 1000 Hz; Heyer 1978; Giaretta & Costa 2007; Heyer et al. 2010), while that of L. poecilochilus has a negligible/slight frequency upsweep (FM Leptodactylus kilombo is distinguished by a lower dominant frequency (1098–1572 Hz) from L. furnarius, L. latinasus, L. marambaiae, L. mystacinus, L. tapiti, and L. troglodytes (> 2000 Hz) (Heyer 1978; Sazima & Bokermann 1978; Brandão et al. 2013). Description of holotype. Body robust. Snout sub-elliptical in dorsal and ventral views (Fig. 8 a–b), acuminate in lateral view (Fig. 8e). Canthus rostralis rounded; loreal region flat; tympanic annulus well-defined, circular (TD = 60.4% ED); tympanic membrane translucent (brownish); supratympanic fold from the posterior corner of the eye, passing over the dorsal edge of the tympanic annulus and ending at the base of the arm; vocal sac subgular; vocal slits present; vomerine teeth in two nearly straight rows medial and posterior to choanae and almost parallel to sagittal plane. Tongue rounded, free at its posterior third. Relative finger lengths IV Colors. In life, dorsal surfaces (body and limbs) reddish; snout whitish. Light brown blotches scattered on the dorsum (Fig. 5d). Upper portion of the loreal region dark brown with green specks, lower portion reddish on a cream-colored background, lips nonpigmented with light-colored spots and stains. Flank light gray; tubercles reddish and light cream. Throat white scattered with dark brown spots and dots. Chest and belly off-white; ventral surface of limbs cream-colored. Upper surface of thigh, tibia and tarsus brown with dark brown transversal bars. Posterior surface of thigh with brown spots and irregular longitudinal stripe light cream. In preservative, dorsal surfaces (body and limbs) brown with darker brown blotches (Fig. 8). The dorsal reddish coloration in life becomes brown or dark brown. Dark brown blotches and brown transversal bars on the upper surface of thigh and tibia are more conspicuous. Interorbital blotch poorly delimited posteriorly. Upper portion of the loreal region blackish brown, lower portion light brown, lips dark brown. Tympanic membrane light brown. Flank light gray. Throat white covered with dark brown spots and dots. Chest, belly, and ventral surface of limbs varying from cream-colored to off-white. Dark brown transversal bars on upper surface of thigh, tibia and tarsus. Posterior surface of thigh with scattered brown spots and irregular longitudinal stripe stripe light cream. Ventral surface of hand and foot brown interspersed with nonpigmented areas; Sole of foot with sparse white dots. Intraspecific variation. Lateral line of tubercles is complete in CHUFPB 28205. Posterior surface of tarsus can be smooth or covered with tiny tubercles (poorly visible in preservative). Tubercles on the sole of foot are more conspicuous in the examined female (CHUFPB 30409) than in male specimens. Advertisement call. Description based on 94 calls recorded from two males (Table 3). The call (Fig. 6f) consists of single, nonpulsed notes given at a rate of 109–122 per minute. Note duration varies from 30–56 ms. Rise time is 37–77% of note duration. The dominant frequency coincides with the fundamental harmonic at 1098–1572 Hz. Notes usually have a modest frequency upsweep throughout their duration, varying from 388–775 Hz. plex. Data are presented as mean SD (range). applicable to species with nonpulsed calls). Abbreviations: ND = note duration, RT = rise time, NR = note repetition rate, PP = pulses per note, PR = pulse repetition rate, DF = dominant frequency, and FM = frequency modulation. Habitat and natural history. At the type locality, we first found the species in breeding activity early in the rainy season (October 2018), when many calling males could be heard along a dried-up stream (Bezerra stream), which was humid in the sandy stream bed. On this occasion, most males were calling from underneath dense layer of leaf litter (see a calling male at: https://www.youtube.com/watch?v=SUyRQOiGReQ). During a second expedition to the type locality later in the rainy season (April 2019), we did not find or hear any specimen of the new species, suggesting that its breeding activity may be restricted to the early rainy season. Despite being located in a central region of the Cerrado domain, the type locality is more characterized by the presence of Seasonally Dry Tropical Forests (Werneck 2011) than typical savanna formations. Leptodactylus kilombo and L. cf. mystaceus are syntopic at the type locality (Fig. 1). Despite being quite similar to each other in overall morphology, these species can be easily distinguished by their advertisement calls (see “Comparisons with congeners” section). Besides L. cf. mystaceus, two other species of the L. fuscus group occur syntopically at the type locality (L. fuscus and L. troglodytes). All four species were observed calling simultaneously at the type locality. Distribution. Leptodactylus kilombo (Fig. 1) is known from two localities in the extreme southeast of Tocantins, the type locality (Arraias) and Conceição do Tocantins, from a single locality in northeastern Goiás (Flores de Goiás; see Appendix II) and a single locality in western Bahia (Bom Jesus da Lapa; see Appendix II). Remarks. Live specimens of Leptodactylus kilombo have a reddish-pink upper lip stripe. This color feature might be an additional diagnostic feature of the species in the L. mystaceus complex. Because of our small sample size of L. kilombo, further sampling is needed to confirm whether upper lip stripe coloration is an informative character in the diagnosis of this species. We examined three specimens collected from Conceição do Tocantins (field# RAB 1965–67), located approximately 85 km northwest of the type locality of L. kilombo. These specimens resemble L. kilombo in overall morphology and by having a reddish-pink upper lip stripe, thus assigned to L. kilombo based on morphological and color features from the preserved specimens and pictures in life, but acoustic and molecular data should also be obtained from this population.

Leptodactylus watu sp. nov. (Figs. 2, 4a, 4c, 5h, 6h, 10) [http://zoobank.org/ urn:lsid:zoobank.org:act: 5D79A081-C50C-463A-8101-04351E87AD23] Leptodactylus aff. spixi: Guimar��es et al. (2019) Holotype. UFMG 21332, an adult male from Parque Estadual do Rio Doce (19.753922��S, 42.633047��W, approximately 280 m a.s.l.), in Marli��ria, Minas Gerais State, southeastern Brazil, collected by H. Thomassen on 21 November 2017. Paratypes. Seven adult males (UFMG 21327���21331 and 21337���21338) and five adult females (UFMG 21333��� 21336 and 21339) collected from the type locality by H. Thomassen from November 2017 to February 2018. Additional material. One genetic voucher (UFMG 16995) from Santa B��rbara do Leste, Minas Gerais State (Fig. 1). Etymology. The word watu, a noun in apposition, is derived from the dialect of the Bor��n indigenous people and is a reference to the Doce River (Costa-Reis & Genovez 2013). In 2015, the Doce River experienced the worst environmental disaster that ever took place in Brazil, the collapse of a mining tailings dam owned by Samarco (and co-owned by the Brazilian Vale and Australian BHP Billiton). The catastrophic dam failure released around 60 million cubic meters of iron ore tailings (toxic slurry) directly into the Doce River watershed, killing 20 people and affecting biodiversity across hundreds of kilometers around the river drainage, riparian lands, and the Atlantic coast, in addition to a severe contamination of the soil and water table within the affected region. The epithet is a tribute to the resistance of the Bor��n people and to the Watu (Doce River) in southeastern Brazil. Diagnosis. Leptodactylus watu is characterized by the following combination of character states: (1) small to moderate size, male SVL = 38.5���42.2 mm, female SVL = 40.5���47.9 mm; (2) sole of foot with few, non-obvious tubercles; (3) advertisement call composed of single, pulsed notes; (4) note duration varying from 27���70 ms; (5) note dominant frequency varying from 495���1098 Hz; (6) frequency upsweep varying from 0���560 Hz. Comparisons with members of the L. mystaceus complex. Leptodactylus watu is distinguished from other species belonging to the L. mystaceus complex, except L. barrioi, L. kilombo and L. notoaktites, by having the sole of the foot covered with few, non-obvious tubercles, whereas L. didymus, L. elenae, L. mystaceus, and L. spixi have prominent tubercles (Heyer 1978, 1983; Heyer et al. 1996). The new species can be further distinguished from some species by a smaller size (Table 2): L. didymus (male SVL = 45.9���52.2 mm, mean = 49.3; female SVL = 45.8���53.5 mm, mean = 49.8; Heyer et al. 1996), L. mystaceus (male SVL = 42.4���52.2 mm, mean = 47.4; female SVL = 44.8��� 56.1 mm, mean = 50.0; Heyer et al. 1996), and L. notoaktites (male SVL = 42.5���54.2 mm, mean = 47.5; female SVL = 43.4���55.8 mm, mean = 48.0; de S�� et al. 2014). Members of the L. mystaceus complex are mainly distinguished from each other by their distinct advertisement calls (Fig. 6). Acoustic comparisons in the L. fuscus group. The advertisement call of Leptodactylus watu is composed of single, pulsed notes (Table 3; Fig. 6h), differing from species with trill calls (L. cunicularius, L. cupreus, L. oreomantis, and L. plaumanni; Fig. 7; Carvalho et al. 2013) and species with nonpulsed calls (L. albilabris, L. bufonius, L. camaquara, L. didymus, L. elenae, L. furnarius, L. fuscus, L. gracilis, L. jolyi, L. kilombo sp. nov., L. laticeps, L. latinasus, L. longirostris, L. marambaiae, L. mystacinus, L. notoaktites, L. poecilochilus, L. sertanejo, L. spixi, L. syphax, L. tapiti, and L. troglodytes). From other species with pulsed calls, L. watu is distinguished by having its call formed by partly fused pulses, whereas the calls of L. barrioi, L. mystaceus and L. cf. mystaceus are formed by complete pulses (Fig. 6). From species also having calls with partly fused pulses (L. caatingae, L. fragilis, and L. labrosus), L. watu is distinguished by a shorter note (27���70 ms) from L. fragilis (> 100 ms; Heyer et al. 2006). The calls of L. watu, L. caatingae, and L. labrosus are the most similar to each other in the L. fuscus group, but these species can be distinguished, although value ranges partially overlap, by differences in the dominant frequency (L. watu: 495���1098 Hz, mean = 763; L. caatingae: 1148���1430 Hz, mean = 1315, in our sample; 943���1620 Hz, mean = 1423 Hz, in the original description; Heyer & Junc�� 2003) or duration (L. watu: 27���70 ms, mean = 40; L. labrosus: 64���133 ms, mean = 100; Carvalho & Ron 2011) of their calls. Still, these species differ markedly in morphology and size (see previous paragraph; Heyer 1978; Heyer & Junc�� 2003). Description of holotype. Body robust. Snout sub-elliptical in dorsal and ventral views (Fig. 10 a���b), acuminate in lateral view (Fig. 10e). Canthus rostralis rounded; loreal region flat; tympanic annulus well-defined, circular (TD = 62.4% ED); supratympanic fold from the posterior corner of the eye, passing over the dorsal edge of the tympanic annulus, and ending at the base of the arm; vocal sac subgular; vocal slits present; vomerine teeth in two nearly straight rows, almost connected, medial and posterior to choanae and almost parallel to sagittal plane. Tongue rounded, free at its posterior third. Relative finger lengths II = IV Colors. In life, dorsal surfaces (body and limbs) light brown with dark gray blotches poorly delimited (Figs. 4a, c, 5h). Upper region of the loreal region black, lower region light cream, lips dark brown. Black stripe on the supratympanic fold, delimited dorsally by a white line. Light cream stripe on the dorsolateral fold, more conspicuous posteriorly. Flank light gray. Tympanic membrane dark brown on the center and upper rim, light brown laterally and on ventral rim. Throat immaculate white with nonpigmented portions near the jaw. Ventral surface of arms, legs (except tarsus), and belly nonpigmented; tarsus with scattered dark brown stains. Weakly defined dark brown transversal bars on the upper surface of hindlimbs and forearm. Posterior surface of thigh with a yellow longitudinal stripe bordered by two black stains; upper portion of with black spots and stains. Ventral surface of hand, foot, and tarsus dark brown interspersed with nonpigmented areas. In preservative, dorsal surfaces (body and hindlimbs) light gray with dark gray blotches poorly delimited; forearms light cream with scattered dark gray dots and spots (Fig. 10). Upper portion of the loreal region black, lower half cream-colored, lips blackish gray. Black stripe on the supratympanic fold. White stripe on the dorsolateral fold, more conspicuous posteriorly. Flank light gray. Tympanic membrane light brown with the center and upper edge dark gray. Ventral surfaces cream-colored. Throat with few gray dots and stains near the jaw laterally. Ventral surface of hindlimbs with few dark gray spots. Dark brown transversal bars on the dorsal surface of hindlimbs and forearm. Posterior surface of thigh with a white longitudinal stripe bordered by two black stains. Ventral surface of hand, foot, and tarsus dark brown interspersed with nonpigmented areas. Intraspecific variation. In life, dorsal coloration varies from brown, sometimes conspicuous reddish shades, or dark gray. The dorsal light brown coloration in life becomes light gray in preservative. Dark brown blotches on the dorsum can be present, more conspicuous in preserved specimens. Dark brown transversal bars on the upper surface of thigh and tibia are more evident in preserved species. Lateral line of tubercles is poorly evident in most specimens. Inner metacarpal tubercle is rounded in UFMG 21329. Advertisement call. Description based on 129 calls recorded from two males (Table 3). The call (Fig. 6h) consists of single, pulsed notes given at a rate of 124���275 per minute. Note duration varies from 27���70 ms. Rise time varies from 37���80% of note duration. Notes are formed by 2���7 partly fused pulses emitted at a rate of 57���153 per second. The dominant frequency coincides with the fundamental harmonic at 495���1098 Hz. Notes have no frequency modulation or a modest frequency upsweep throughout their duration, varying from 0���560 Hz. Habitat and natural history. At the type locality, Leptodactylus watu was found calling during the rainy season in the region from late October to late February. Calling is strongly associated with rainy days or few days after heavy pours. Males were found calling around flooded areas or close to moist ground in both open areas (transitional marshlands between the forest and lakes) and inside the forest. All calling males were hidden either under dense vegetation, ground leaf litter or inside underground chambers. They were never found calling in pastures. The congeneric species found calling in syntopy with L. watu were L. fuscus, L. latrans, and L. labyrinthicus. Distribution. Leptodactylus watu is only known from two localities in a region characterized by lowland interior Atlantic forests associated with the upper-middle Doce River drainage, in the Brazilian state of Minas Gerais (Fig. 1): (1) the type locality, at Parque Estadual do Rio Doce, and (2) Santa B��rbara do Leste, approximately 70 km southeast from the type locality. Remarks. The identity of populations assigned to Leptodactylus aff. mystaceus (Santana et al. 2010), L. aff. spixi (Ferreira et al. 2019), and L. spixi (R��dder et al. 2007; Silva-Soares & Scherrer 2013; Pereira-Ribeiro et al. 2019) from different localities in southeastern and northeastern Brazil should be addressed using calls and/or DNA sequences, as these records could correspond to L. barrioi or L. watu, or additional candidate new species., Published as part of Da Silva, Leandro A., Magalh��es, Felipe M., Thomassen, Hans, Leite, Felipe S. F., Garda, Adrian A., Brand��o, Reuber A., Haddad, C��lio F. B., Giaretta, Ariovaldo A. & De Carvalho, Thiago R., 2020, Unraveling the species diversity and relationships in the Leptodactylus mystaceus complex (Anura: Leptodactylidae), with the description of three new Brazilian species, pp. 151-189 in Zootaxa 4779 (2) on pages 167-170, DOI: 10.11646/zootaxa.4779.2.1, http://zenodo.org/record/3833431, {"references":["Guimaraes, C. S., Assis, C. L., Thomassen, H., Leite, F. S. F. & Feio, R. N. (2019) Anfibios do Parque Estadual do Rio Doce, Minas Gerais. Universidade Federal de Vicosa, Departamento de Biologia Animal, Museu de Zoologia Joao Moojen, Vicosa, Minas Gerais, 57 pp.","Costa-Reis, R. & Genovez, P. F. (2013) Territorio sagrado: exilio, diaspora e reconquista Krenak no vale do Rio Doce, Resplendor, MG. Boletim Goiano de Geografia, 33, 1 - 15. https: // doi. org / 10.5216 / bgg. v 33 i 1.23628","Heyer, W. R. (1978) Systematics of the fuscus group of the frog genus Leptodactylus (Amphibia, Leptodactylidae). Science Bulletin, Natural History Museum of Los Angeles County, 29, 1 - 85.","Heyer, W. R. (1983) Clarification of the names Rana mystacea Spix, 1824, Leptodactylus amazonicus Heyer, 1978, and a description of a new species, Leptodactylus spixi (Amphibia: Leptodactylidae). Proceedings of the Biological Society of Washington, 96, 270 - 272.","Heyer, W. R., Garcia-Lopez, J. M. & Cardoso, A. J. (1996) Advertisement call variation in the Leptodactylus mystaceus species complex (Amphibia: Leptodactylidae) with a description of a new sibling species. Amphibia-Reptilia, 17, 7 - 31. https: // doi. org / 10.1163 / 156853896 X 00252","de Sa, R. O., Grant, T., Camargo, A., Heyer, W. R., Ponssa, M. L. & Stanley, E. (2014) Systematics of the Neotropical genus Leptodactylus Fitzinger, 1826 (Anura: Leptodactylidae): Phylogeny, the relevance of non-molecular evidence, and species accounts. South American Journal of Herpetology, 9, S 1 - S 128. https: // doi. org / 10.2994 / SAJH-D- 13 - 00022.1","Carvalho, T. R., Leite, F. S. F. & Pezzuti, T. L. (2013) A new species of Leptodactylus Fitzinger (Anura, Leptodactylidae, Leptodactylinae) from montane rock fields of the Chapada Diamantina, northeastern Brazil. Zootaxa, 3701 (3), 349 - 364. https: // doi. org / 10.11646 / zootaxa. 3701.3.5","Heyer, W. R., Heyer, M. M. & de Sa, R. O. (2006) Leptodactylus fragilis. Catalogue of American Amphibians and Reptiles, 830, 1 - 26.","Heyer, W. R. & Junca, F. A. (2003) Leptodactylus caatingae, a new species of frog from eastern Brazil (Amphibia: Anura: Leptodactylidae). Proceedings of the Biological Society of Washington, 116, 317 - 329.","Carvalho, T. R. & Ron, S. R. (2011) Advertisement call of Leptodactylus labrosus Jimenez de la Espada, 1875 (Anura, Leptodactylidae): an unusual advertisement call within the L. fuscus group. Herpetology Notes, 4, 325 - 326.","Santana, D. J., Sao Pedro, V. A., Hote, P. S., Roberti, H. M., Sant'Anna, A. C., Figueiredo-de-Andrade, C. A. & Feio, R. N. (2010) Anurans in the region of the High Muriae River, state of Minas Gerais, Brazil. Herpetology Notes, 3, 1 - 10.","Ferreira, R. B., Monico, A. T., Silva, E. T., Lirio, F. C. F., Zocca, C., Mageski, M. M., Tonini, J. F. R., Beard, K. H., Duca, C. & Silva- Soares, T. (2019) Amphibians of Santa Teresa, Brazil: the hotspot further evaluated. ZooKeys, 857, 139 - 162. https: // doi. org / 10.3897 / zookeys. 857.30302","Rodder, D., Teixeira, R. L., Ferreira, R. B., Dantas, R. B., Pertel, W. & Guarniere, G. J. (2007) Anuran hotspots: the municipality of Santa Teresa, Espirito Santo, southeastern Brazil. Salamandra, 43, 91 - 110.","Silva-Soares, T. & Scherrer, P. V. (2013) Amphibians of Parque Estadual do Forno Grande, State of Espirito Santo, Southeastern Brazil: Species composition and conservation. North-Western Journal of Zoology, 9, 113 - 120.","Pereira-Ribeiro, J., Ferreguetti, A. C., Linause, T. M., Cozer, J. S., Bergallo, H. G. & Rocha, C. F. D. (2019) Diversity and distribution of anurans from Mata das Flores State Park, Espirito Santo, southeastern Brazil. Oecologia Australis, 23, 292 - 300. https: // doi. org / 10.4257 / oeco. 2019.2302.08"]}

Leptodactylus barrioi sp. nov. (Figs. 2, 4e, 5a, 6g, 9) [http://zoobank.org/ urn:lsid:zoobank.org:act: 4948F138-66DC-41DD-8426-0DE12B0FED67] Holotype. AAG-UFU 502, an adult male from Duas Barras (22.062862°S, 2.520780°W, approximately 625 m a.s.l.), Rio de Janeiro State, southeastern Brazil, collected by T. R. de Carvalho and L. B. Martins on 10 September 2011. Paratypes. Three adult males (AAG-UFU 503, 506, and 694) and two adult females (AAG-UFU 504–505) collected at the type locality from 10 September to 7 December 2011. One adult male (AAG-UFU 1102) from Chiador (21.921698°S, 42.926676°W, nearly 495 m a.s.l.), Minas Gerais State, southeastern Brazil, collected by T. R. de Carvalho on 8 March 2012. One adult male (AAG-UFU 6183) from the District of Povoação (19.551215°S, 39.846788°W, nearly 20 m a.s.l.), in Linhares, Espírito Santo State, southeastern Brazil, collected by T. R. de Carvalho, B. F. V. Teixeira, and L. B. Martins on 6 December 2017. Additional material. Genetic vouchers from Ibitirama, Espírito Santo State (UFMG 17268), and Cataguases, Minas Gerais State (AAGARDA 3300–3301); see Fig. 1. Etymology. The epithet is a homage to Avelino Barrio, who was a pioneer in characterizing calls of Leptodactylus and Physalaemus species in two comparative studies (Barrio 1965a, b). This researcher was very attentive to the relevance of acoustic data for species discrimination in both leptodactylid genera. Diagnosis. Leptodactylus barrioi is characterized by the following combination of character states: (1) sole of foot with few, non-obvious tubercles; (2) advertisement call composed of single, pulsed notes; (3) note duration varying from 62–126 ms; (4) note dominant frequency varying from 586–1383 Hz; (5) frequency upsweep varying from 141–750 Hz. Comparisons with members of the L. mystaceus complex. Leptodactylus barrioi can be distinguished by having the sole of the foot covered with few, non-obvious tubercles, whereas the other species have prominent tubercles (Heyer 1978, 1983; Heyer et al. 1996), except L. kilombo and L. notoaktites. The new species cannot be distinguished from other members of the L. mystaceus complex by morphometric traits (Table 2), but differs from the other species in acoustic traits (Fig. 6). Acoustic comparisons in the L. fuscus group. The advertisement call of Leptodactylus barrioi is composed of single, pulsed notes (Table 3; Fig. 6g), differing from the trill calls of L. cunicularius, L. cupreus, L. oreomantis, and L. plaumanni (Fig. 7; Carvalho et al. 2013), and from the nonpulsed calls of L. albilabris, L. bufonius, L. camaquara, L. didymus, L. elenae, L. furnarius, L. fuscus, L. gracilis, L. jolyi, L. kilombo, L. laticeps, L. latinasus, L. longirostris, L. marambaiae, L. mystacinus, L. notoaktites, L. poecilochilus, L. sertanejo, L. spixi, L. syphax, L. tapiti, and L. troglodytes. From other species with pulsed calls, L. barrioi is distinguished by having its call formed by complete pulses (i.e., with silent gaps in between pulses), whereas those of L. caatingae, L. fragilis, and L. labrosus are formed by partly fused pulses (Heyer 1978; Heyer & Juncá 2003; Carvalho & Ron 2011). The only other congeners of the L. fuscus group with the call formed by complete pulses are L. mystaceus and L. cf. mystaceus, from which L. barrioi is distinguished by a shorter note duration (L. barrioi: 62–126 ms; L. mystaceus: 182–218 ms; L. cf. mystaceus: 136–260 ms; Table 3). Description of holotype. Body robust. Snout sub-elliptical in dorsal and ventral views (Fig. 9 a–b), acuminate in lateral view (Fig. 9e). Canthus rostralis rounded; loreal region flat; tympanic annulus well-defined, circular (TD = 60.8% ED); supratympanic fold from the posterior corner of the eye, passing over the dorsal edge of the tympanic annulus, and ending at the base of the arm; vocal sac subgular; vocal slits present; vomerine teeth in two nearly convex rows medial and posterior to choanae and almost parallel to sagittal plane. Tongue ovoid, free at its posterior third. Relative finger lengths IV Colors. In preservative (Fig. 9), dorsal surfaces of body light grayish brown with light brown blotches and black spots. Upper portion of the loreal region black, lower portion cream-colored, lips blackish brown. Black stripe on the supratympanic fold delimited dorsally by a white line. Flank light gray. Tympanic membrane dark brown in the middle and upper rim, light brown laterally and lower rim. Throat, ventral surface of forelimbs, hindlimbs, and belly cream-colored. Brown transversal bars on the upper surfaces of forearms and hindlimbs. Posterior surface of thigh with a white longitudinal stripe bordered by black stains; upper portion of the posterior surface of thigh with gray spots and irregular stripe. Ventral surface of hand, foot, and tarsus dark gray interspersed with nonpigmented areas. Intraspecific variation. In life, dorsal coloration varies from light gray to reddish, dark brown. Reddish brown shades become light gray or light brown in preservative. Lateral line of tubercles most times restricted to posterior flanks (AAG-UFU 503) or poorly evident (AAG-UFU 505). Females (AAG-UFU 504–505) and most males (AAG- UFU 503, 506, 694, and 1102) have tiny white tubercles on the sole of foot, whereas the male AAG-UFU 6183 has the sole of foot nearly smooth. In most specimens, throat predominantly light-colored without dark gray spots and dots concentrated near the jaw (dark gray spots and dots present in AAG-UFU 6183). Advertisement call. Description based on 522 calls recorded from seven males (Table 3). The call consists of single, pulsed notes given at a rate of 145–196 per minute (Fig. 6g). Note duration varies from 62–126 ms. Rise time varies from 26–94% of note duration. Notes are formed by 6–12 complete pulses emitted at a rate of 75–125 per second. The dominant frequency coincides with the fundamental harmonic at 586–1383 Hz. Notes usually have a modest frequency upsweep throughout their duration, varying from 141–750 Hz. Habitat and natural history. At the type locality, individuals were found calling from inside underground chambers in open areas always adjacent to the forest edge, where water was accumulated as small puddles. The surroundings can be described as grass tufts in poorly drained marshy soil. Although all three study sites are characterized as moderately to highly modified by anthropogenic activities, small fragments of Atlantic forest were present in these areas. In the District of Povoação (Linhares, Espírito Santo), calling males were heard from underneath dense leaf litter in cacao plantations. Distribution. Leptodactylus barrioi is known to occur within the interior Atlantic forests of southeastern Minas Gerais, west of the Serra do Mar mountain range in Rio de Janeiro State, in the northern sector of the Mantiqueira mountain range in extreme south of Espírito Santo, and also in the coastal Atlantic forests alongside the lower Doce River in the same Brazilian state (all regions within southeastern Brazil; Fig. 1).