Unraveling the species diversity and relationships in the Leptodactylus mystaceus complex (Anura: Leptodactylidae), with the description of three new Brazilian species Da Silva, Leandro A. Magalhães, Felipe M. Thomassen, Hans Leite, Felipe S. F. Garda, Adrian A. Brandão, Reuber A. Haddad, Célio F. B. Giaretta, Ariovaldo A. De Carvalho, Thiago R. Zootaxa 2020 2020-05-19 4779 2 151 189 Da Silva & Magalhães & Thomassen & Leite & Garda & Brandão & Haddad & Giaretta & De Carvalho, 2020 Da Silva & Magalhães & Thomassen & Leite & Garda & Brandão & Haddad & Giaretta & De Carvalho 2020 [151,409,441,468] Amphibia Leptodactylidae Leptodactylus Animalia Anura 14 165 Chordata species 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 ma.s.l.), Rio de Janeiro State, southeastern Brazil, collected by T. R. de Carvalhoand L. B. Martinson  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 ma.s.l.), Minas Gerais State, southeastern Brazil, collected by T. R. de Carvalhoon  8 March 2012. One adult male ( AAG-UFU 6183) from the District of Povoação( 19.551215°S, 39.846788°W, nearly  20 ma.s.l.), in Linhares, Espírito Santo State, southeastern Brazil, collected by T. R. de Carvalho, B. F. V. Teixeira, and L. B. Martinson  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  Leptodactylusand  Physalaemusspecies 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 barrioiis 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. mystaceuscomplex.  Leptodactylus barrioican 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. kilomboand  L. notoaktites. The new species cannot be distinguished from other members of the  L. mystaceuscomplex by morphometric traits ( Table 2), but differs from the other species in acoustic traits ( Fig. 6).  Acoustic comparisons in the  L. fuscusgroup.The advertisement call of  Leptodactylus barrioiis 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. barrioiis 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. labrosusare formed by partly fused pulses ( Heyer 1978; Heyer & Juncá 2003; Carvalho & Ron 2011). The only other congeners of the  L. fuscusgroup with the call formed by complete pulses are  L. mystaceusand  L.cf. mystaceus, from which  L. barrioiis 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. 9a–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 <II <I <III; fingers without lateral fringing or webbing; finger tips rounded, un- expanded ( Fig. 9c). Inner metacarpal tubercle rounded, single; outer metacarpal tubercle cordiform; outer tubercle twice the maximum width of the inner. Subarticular tubercles rounded; supernumerary tubercles absent. Dorsal surfaces of body and limbs smooth, shagreened on flank. Dorsolateral fold from the posterior corner of the eye, extending posteriorly to the groin. Lateral line of tubercles restricted to posterior flank. Dorsal surface of hindlimbs and posterior surface of tarsus with tiny tubercles, poorly visible in preservative. Ventral surface of body and limbs smooth, areolate underside of thigh. Relative toe lengths I <II <V <III <IV; toes without lateral fringing or webbing; toe tips unexpanded; inner metatarsal tubercle rounded and at least twice the maximum length of the nearly rounded outer metatarsal tubercle ( Fig. 9d). Tarsal fold extending 2/3 of tarsus length, from the inner metatarsal tubercle towards the heel; subarticular tubercles rounded; inconspicuous supernumerary tubercles.  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.   FIGURE 9.Holotype of  Leptodactylus barrioi(AAG-UFU 502; SVL = 43.5 mm). (a) Dorsal and (b) ventral body; (c) palm of hand; (d) sole of foot; (e) head in profile. Scale bars = 5 mm.  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 typelocality, 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 barrioiis 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). 2620582305 2011-09-10 T, R de Carvalho & L. B. Martins Brazil 625 -22.062862 Duas Barras 1 -2.52078 14 165 AAG-UFU 502 1 Rio de Janeiro holotype 2620582306 2011-09-10 2011-12-07 2011-09-10 AAG-UFU 14 165 AAG-UFU 503, 506, AAG-UFU 504-505 1 paratype 2620582302 2012-03-08 T, R de Carvalho Brazil 495 -21.921698 Chiador 1 -42.926678 14 165 AAG-UFU 1102 1 Minas Gerais paratype 2620582301 2017-12-06 T, R, V de Carvalho, B. F. & Teixeira & L. B. Martins Brazil 20 -19.551214 District of Povoacao 1 -39.846786 Linhares 14 165 AAG-UFU 6183 1 Espirito Santo paratype