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The number of described species of Brachycephalus has rapidly increased in the last decade (n = 22, which represents 56% of the total). Species of the genus Brachycephalus are mostly distributed in isolated mountaintops from Bahia (northeastern Brazil) to Santa Catarina states (southern Brazil), each one occupying only one or a few adjacent mountaintops. Herein, we described a new species of Brachycephalus of the B. pernix group, from Serra do Tabuleiro in Santa Catarina state, which also represents the southernmost known species. The new species can be distinguished from its congeners by a combination of characters, including the following: (1) "bufoniform" body; (2) small adult SVL: 9.57–11.10 mm for males and 10.88–12.70 mm for females; (3) head proportionally small (HL/SVL 19–28%) and eye proportionally large (ED/HL 36–56%); (4) dorsum texture rough; (5) snout shape rounded in dorsal and lateral views; (6) general dorsal body color olive green with head, arms and legs yellow-orangish scattered with olive green, and an orangish vertebral stripe spotted with white and brown colors; (7) skull and skeleton without hyperossification; (8) frontoparietal and sphenethmoid not fused; (9) advertisement with one or two high-frequency notes (6,115–6,562 Hz), and 2–4 pulses per note. The type locality is adjacent to Parque Estadual da Serra do Tabuleiro, a protected area, but we observed various agricultural activities in this locality, including the presence of exotic plants, which can change the amount and the quality of leaf litter, somehow compromising the population of the new species. Another aggravating factor is that the municipality of São Bonifácio has conflicts over land use with irregular occupation and unfinished expropriation processes in Parque Estadual da Serra do Tabuleiro. Considering that Brachycephalus sp. nov. is probably a mountaintop microendemic species, it is paramount that future studies quantifying the new species' full distribution and evaluating population trends to accurately assess its conservation status.

Seitdem im Jahr 1992 zur Prävention des plötzlichen Kindstodes die Empfehlung der Academy of Pediatrics veröffentlicht wurde, Säuglinge beim Schlafen auf dem Rücken zu lagern, ist weltweit eine deutliche Zunahme kindlicher lagebedingter Schädeldeformitäten zu verzeichnen. Eine bereits mehrfach untersuchte und erfolgreiche Therapieform ist die Korrektur der kindlichen Schädelform mittels individueller Kopforthese. Hierbei wird dem Kopfwachstum eine Wachstumsrichtung vorgegeben, um eine Schädelasymmetrie oder okzipitale zentrale Abflachung auszugleichen. Die Verfügbarkeit der Gießener Normperzentilen zur kindlichen Kopfform machte es nun erstmals möglich, Kinder mit abweichender Kopfform mit der „kraniofazialen Norm“ zu vergleichen und eine verlässliche Einschätzung asymmetrischer oder symmetrischer Schädeldeformitäten vor, während und nach einer Kopforthesentherapie vorzunehmen. Diese Studie vergleicht eine große Zahl von Kindern mit therapiebedürftigen, lagebedingten Schädeldeformitäten mit diesen Gießener Normperzentilen retrospektiv, da eine prospektive, randomisierte Studie an Säuglingen mit abnormalen Schädelformen nicht nur technisch schwierig, sondern insbesondere auch aus ethischer Sicht nicht durchführbar ist. In dieser Studie wurden deshalb über 1500 fortlaufend dokumentierte Fälle aus den Jahren 2009 bis 2014 hinzugezogen. Damit war die untersuchte Kohorte zu Beginn der Datensammlung die größte zu diesem Zeitpunkt untersuchte Gruppe und die Aussagekraft der erhobenen Daten erreicht ein hohes Maß an Validität. In der Gruppe der behandelten Kinder mit asymmetrischer Kopfdeformität (nicht synostotischer Plagiocephalie) zeigte sich, dass 93,4 % (CVAI) und 93,3 % (CVA) der Kinder vor Therapiebeginn oberhalb der 75. Normperzentile lagen und damit zumindest leicht von der „Norm“ abwichen. Bei Therapieabschluss hatten 67 % (CVAI) und 64,7 % (CVA) der mit Kopforthese therapierten Kinder den Sprung unter die 75. Perzentile geschafft und wiesen per definitionem normale anthropometrische Messparameter auf. Bei der Gruppe der mit einem nicht synostotischen „Kurzkopf“ (Brachycephalus) behandelten Kinder lagen prätherapeutisch 79,4 % der Kinder oberhalb der 75. Perzentile (CI), posttherapeutisch waren 31,0 % der behandelten Kinder unter die 75. Perzentile und damit auf das Niveau normaler Messwerte gefallen. Es war somit keine behandlungsbedürftige Deformität mehr nachweisbar. Unsere Daten zeigen, dass sich Indices, die asymmetrische Schädeldeformitäten bewerten sollen, auch durch das physiologische Kopfwachstum verbessern, während dies für absolute Differenzen (CVA) nicht der Fall ist. Folglich sollte bei der Festlegung des Schweregrades eines Plagiocephalus die Einschätzung eher anhand absoluter als anhand relativer Parameter, wie zum Beispiel des CVAI, erfolgen. Bei der Analyse der Nebenzielparameter zeigte sich, dass die Eltern der betroffenen Kinder intuitiv die Klassifikation nach dem äußeren Erscheinungsbild nutzen, also entsprechend dem Klassifikationsmodell von Argenta et al. (Argenta, David et al. 2004) zur „Laieneinschätzung“ einer lagebedingten Problematik der Kopfform. Die Frage nach vorausgegangenen, alternativen Behandlungsansätzen kann in diesem Studienmodell nur eingeschränkt beurteilt werden. Grund hierfür ist eine starke Selektion des behandelten Patientenkollektivs im Vorfeld der Erstvorstellung. Mehrlingsgeburten haben aufgrund des intrauterinen Platzmangels laut Studienlage ein erhöhtes Risiko zur Ausbildung eines „Liegeschädels“. Mit einem Anteil von 5,4 % an unserer Gesamtkohorte konnte dies im Rahmen unserer Studie nur bedingt bestätigt werden. Ursächlich hierfür ist am ehesten der oftmals frühere Beginn alternativer Therapiemaßnahmen. Basierend auf den in dieser Studie erhobenen Daten ergibt sich perspektivisch die Möglichkeit, kindliche lagebedingte Schädeldeformitäten anhand der Gießener Normperzentilen alters- und geschlechtsgebunden zu klassifizieren. Durch den Vergleich „krank“ zu „gesund“ bzw. „in der Norm“ zu „außerhalb der Norm“ konnte die klinische Reliabilität der Normperzentilen überprüft werden. Diese haben sich damit in der klinischen Anwendbarkeit bewährt.

Made available in DSpace on 2021-06-25T12:22:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-19 CONICET ANPCyT Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Direct development implies transformations with respect to the anuran biphasic life cycle, including changes in embryonic anatomy. In the clade Brachycephaloidea, skeletal ontogeny is known inEleutherodactylus coquiof the basal family Eleutherodactylidae. In this work, we study it in four species representing the two other families in the group. We worked with developmental series ofOreobates barituensis,Haddadus binotatus,Ischnocnema henselii, andBrachycephalus ephippium. Specimens were prepared following protocols of clearing and staining and histology. In the cranium, results show an overall shared pattern that, as summarized forE. coqui, combines a partial recapitulation of aspects of the ancestral biphasic ontogeny with a profound remodeling that includes lost/novel structures and heterochronic shifts of developmental events. Among these transformations are the absence of suprarostrals and trabecular horns and the precocious ossification of jaw and suspensorium. In addition, each lineage shows particular features such that skull ontogeny varies interspecifically. In turn, the morphogenesis of the axial and appendicular skeleton is highly conserved, with main variations including the extent of ossification at hatching. Along with some external features such as the egg tooth and the enveloping tail with transversely arranged fins, an ossification sequence with extremely accelerated ossification of jaws and suspensorium could be distinctive of Brachycephaloidea. Fdn Miguel Lillo, Unidad Ejecutora Lillo, Consejo Nacl Invest Cient & Tecn, San Miguel De Tucuman, Tucuman, Argentina Consejo Nacl Invest Cient & Tecn, Inst Bio & Geociencias NOA, Rosario De Lerma, Salta, Argentina Univ Nacl Jujuy, Inst Ecorregiones Andinas, Consejo Nacl Invest Cient & Tecn, San Salvador De Jujuy, Jujuy, Argentina Univ Fed Sul & Sudeste Para, Museu Biodivers Tauari, Maraba, Para, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Biodivers, Campus Rio Claro, Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias, Ctr Aquicultura, Campus Rio Claro, Sao Paulo, Brazil Univ Fed Rio de Janeiro, Dept Vertebrados, Museu Nacl, Rio De Janeiro, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Biodivers, Campus Rio Claro, Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias, Ctr Aquicultura, Campus Rio Claro, Sao Paulo, Brazil CONICET: PIP 497 CONICET: PIP 747 CONICET: PIO 094 ANPCyT: PICT 2018-3349 FAPESP: 2019/04076-8 CAPES: 2014/05772-4

Brachycephalus bufonoides Miranda-Ribeiro, 1920 Brachycephalus ephippium var. bufonoides Miranda-Ribeiro, 1920. Brachycephalus ephippium: Cochran, 1955 (part). Brachycephalus bufonoides: Pombal, 2010. Lectotype. MZUSP 1459 (designated by Pombal 2010). Type locality: Serra de Maca��, Nova Friburgo, State of Rio de Janeiro, Brazil on September 1909 collected by Ernest Garbe. Paralectotype MZUSP 1458, collected with the lectotype. Referred specimens. The Serra Queimada trail (22��19���21������S, 42��16���19������W: datum WAG84; 1169 m above sea level), ��rea de Prote����o Ambiental (APA) de Maca�� de Cima, municipality of Nova Friburgo, state of Rio de Janeiro, souTHeasTern Brazil: MNRJ 91685 (male) anD ZUFRJ 15424���26 (males), 15428���30 (males) collecTeD on 3 OcTober 2015 by M. Folly anD L. C. Amaral; anD MNRJ 91686 (female), MNRJ 91687���88 (males), ZUFRJ 15525 (male), ZUFRJ 15527 (male), ZUFRJ 15528���29 (females), ZUFRJ 15530���31 (males), ZUFRJ 15534���35 (males), and ZUFRJ 15536 (female) collected on 20 November 2015 by Manuella Folly, Lucas C. Amaral, Sergio P. de Carvalho-e-Silva, and Jos�� P. Pombal Jr. Diagnosis. The specimens show absence of sternum; eight presacral vertebrae; palatal shelf of maxilla lacking pterygoid process; neopalatines and columella absents; arrow-shaped terminal phalanges; one phalange in Finger V, very short phalange in Toe I. These characters have been proposed as diagnostic of Brachycephalus (Hedges et al. 2008). Brachycephalus bufonoides belongs to this genus by the aforementioned characters and also by the small size (B. ephippium species group by: (1) bufoniform body shape; and (2) hyperossified skull and presence of hyperossified spinal processes of sacral and presacral vertebrae (intermediate condition sensu Clemente-Carvalho et al. 2012). Adults of B. bufonoides are distinguished from all of its congeners by the combination of the following characters: (1) skin on head and dorsum with dermal hyperossification; (2) skull with hyperossification of postorbital crests; (3) a pair of hyperossified bulges about equidistant between postorbital crests; (4) fourth presacral vertebra with transverse process hyperossified, ornamented and sacral diapophyses hyperossified, which can be seen externally (lineage of B. vertebralis sensu Condez et al. 2020); (5) presence of dermal ossification as separated bulges of each vertebrae; (6) general background color orange with different intensities of dark orange blotches on dorsum, including bordering of sacral region; (7) absence of osteoderms and presence of warts on the dorsolateral surface of body; (8) medium body size (SVL of adults: 12.0��� 14.5 mm for males and 14.7���16.3 mm for females; Table 1); (9) rough dorsum; (10) advertisement calls with 13 to 17 pulses; (11) presence of pulse period modulation; and (12) advertisement calls with notes longer than 0.2 s (0.22 to 0.31 s). Comparisons with the other species. Brachycephalus bufonoides (Fig. 2) has skin on the head and vertebrae with dermal ossification, and a hyperossified process of the fourth vertebra. These characteristics distinguish the species from B. pernix species group (B. actaeus, B. albolineatus, B. auroguttatus, B. boticario, B. brunneus, B. coloratus, B. curupira, B. ferruginus, B. fuscolineatus, B. izecksohni, B. leopardus, B. mariaeterezae, B. mirissimus, B. olivaceus, B. pernix, B. pombali, B. quiririensis, B. tridactylus, and B. verrucosus; Pombal et al. 1998; Ribeiro et al. 2005; Alves et al. 2006; Haddad et al. 2010; Garey et al. 2012; Pie and Ribeiro 2015; Bornschein et al. 2016; Monteiro et al. 2018a; Ribeiro et al. 2017; Pie et al. 2018) and from B. didactylus, B. hermogenesi, B. pulex, and B. sulfuratus that completely lack hyperossification (Izecksohn, 1971; Giaretta and Sawaya, 1998; Napoli et al. 2011; Clemente-Carvalho et al. 2012; Condez et al. 2016). By having a hyperossified dorsal shield or hyperossified spinal process of sacral and presacral vertebrae, Brachycephalus bufonoides belongs to B. ephippium species group. This species can be distinguished from B. darkside, B. ephippium, B. garbeanus, and B. margaritatus by its presence of dermal ossification on the vertebrae (bony shields on dorsum in these species: Pombal 2010; Pombal and Izecksohn 2011; Guimar��es et al. 2017). Brachycephalus bufonoides has a transverse process of the fourth presacral vertebra and sacral diapophyses hyperossified and ornamented, which is very distinct of the large bone plate exhibited by B. ephippium, B. darkside, B. garbeanus, and B. margaritatus; and also distinct from the comparatively less ornamented skeleton (intermediate condition sensu Clemente-Carvalho et al. 2012; B. vertebralis lineage sensu Condez et al. 2020); B. vertebralis lineage sensu Condez et al. 2020 of B. alipioi, B. crispus, B. guarani, B. nodoterga, B. pitanga, B. toby, and B. vertebralis (Pombal and Gasparini 2006; Haddad et al. 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014). Absence of osteoderms distinguishes B. bufonoides species from B. crispus, B. margaritatus, and B. nodoterga. Brachycephalus bufonoides has skin on its head with dermal ossification and the presence of dermal ossification on the vertebrae while B. atelopoide has no dorsal shields and cephalic ossification (Miranda-Ribeiro 1920; Pombal 2010).The orange coloration with different intensities of dark orange blotches on the dorsum of B. bufonoides differs from B. alipioi, B. pitanga, B. guarani, and B. toby (orange uniform in B. alipioi: Pombal and Gasparini 2006); orange with red irregular blotches in B. pitanga (Alves et al. 2009); orange with a dark brown vertebrate stripe in B. guarani (Clemente-Carvalho et al. 2012); orange with dorsal greenish in B. toby (Haddad et al. 2010). Further, B. bufonoides can be distinguished from B. crispus and B. nodoterga by its dorsum without dermal ossification like warts on the skin [with ossification is B. crispus and B. nodoterga (Condez et al. 2014)]. Redescription of the species. Measurements for 15 males and four females are given in Table 1. Vocal slits not observed in males; body robust and bufoniform (Figs. 1���3); head wider than long (HL/HW x = 0.51 in males and females); head length approximately 19% of SVL in males and 20% in females; snout short with length almost half of eye diameter (END/ED x = 0.56 in males and 0.57 in females), rounded in dorsal and lateral views; nostrils, directed anterolaterally, elliptical, not protuberant; canthus rostralis distinct, almost straight; loreal region vertical; eye not protruding dorsally, mean of eye diameter 57% of HL in males and 46% in females; tympanum not visible; supratympanic fold absent; a hyperossified postorbital crest; a pair of hyperossified bulges about equidistant between postorbital crest; tongue long and narrow, without indentation on its free posterior border; choanae elliptical, anterior to the eye; vomerine odontophores absent; premaxillary and maxillary teeth absent. Arm slightly shorter than forearm (AL/FAL x = 0.90 in males and 0.87 in females), slender; total arm length with mean of 42% SVL in males and 39% in females; hands with the same length in males (AL/HAL x = 1.0) and almost of the same length of upper arm in females (AL/HAL x = 0.95); Finger III and IV robust, distinct; IV longer than III; Finger II very short and Finger V vestigial; pointed tip of Finger III and IV; subarticular tubercles, inner, and outer metacarpal tubercles absent. Shank as long as thigh (SL/TL x = 0.96 in males and 0.95 in females); total leg length with mean of 84% of SVL in both males and females; thigh length larger than foot length (TL/FL x = 0.78 in males and 0.80 in females); Toe II, III, and IV distinct, robust; Toe I and V reduced; tip of Toe II rounded, tip of Toe III and IV pointed; relative lengTH of Toes I ��� V Coloration in life. General background coloration orange; dorsum with a dark orange blotch bordering the spine; protruding dorsal hyperossifications lighter orange than the dorsum color (Fig. 1); finger IV and toe V with black tips; a light-yellow line below the eye, eye black in color. Dorsum varies from orange to light orange blotches bordering to the spine varying in different intensities. Coloration in preservative and variation. Upper surfaces gray (Figs. 3 A���D); protruding dorsal hyperossifications beige (Fig. 3E), flanks light brown; tips of Finger IV and toe V gray; a cream line below the eyes. Dorsal surfaces of body can be gray to brown, varying specially from below the head to the cloaca. In robust specimens, the ossified structures on the head, dorsum and diapophyses are less distinct (Fig. 3D) than thinner specimens that have such ossified structures much evident (Figs. 3A, C). The hyperossification in the spinal processes of presacral vertebrae is completely ossified forming an irregular (MNRJ 91688 and MNRJ 91687) or regular oval shape (ZU-FRJ 15428 and ZUFRJ 15529). Natural history. Specimens of Brachycephalus bufonoides were active during the day were found on forest floor or amidst leaf litter. Some individuals were found under fallen tree trunks where the moisture is higher. Specimens were observed walking slowly on the leaf-litter. Generally, males were observed and heard calling under the leaves of the leaf-litter or branches, but they can be found calling exposed on the surface of leaf-litter or timber (males observed on cloudy and rainy days). Individuals were seen calling from both above and under the leaf-litter, except for one individual, which was calling on a fallen tree trunk. Vocalizations. The individuals found calling in the field were recorded at air temperature of 20.8 oC, and relative humidity of the air of 78%. Calls were emitted as a regular series of low-intensity buzzes (around 120 dB at about 1-meter recording distance). The advertisement call duration is of up to 3 minutes (specimens usually stopped calling when disturbed), and consists of a series of regularly emitted notes, with constant general amplitude (Fig. 4). Notes consisted of a number of pulses, ranging from 13 to 17 pulses/note (x = 15.05 �� 0.88; = 15; Mo = 16; n = 352), with most of them consisting of 14 to 16 pulses (96.7%). Amplitude modulation increased until the second quarter, with a mild descending modulation from end of second quarter until the end of the note. Note duration ranges from 222.09 to 308.53 ms (x = 271.33 �� 18.70; = 276.00; Mo = 290.07; n = 352). Inter-note interval duration ranges from 147.58 to 265.00 ms (x = 209.05 �� 24.68; = 219.00; Mo = 232.00; n = 298). Note period ranges from 401.82 to 545.00 ms (x = 482.10 �� 35.61; = 495.00; Mo = 503.00; n = 298). Note repetition rate ranges from 1.98 to 2.43 note/s (x = 2.15 �� 0.20; = 2.04; n = 3). Pulse period ranges from 9.89 to 25.13 ms (x = 18.18 �� 3.39; n = 729). There is a pulse period modulation to shorter duration of the periods starting through the second third of the note, and continuing modulation to longer duration of the periods through the final third of the note. Pulse repetition rate ranges from 47.55 to 71.28 pulses/s (x = 56.20 �� 4.81; = 55.01; Mo = 51.27; n = 352). Pulse envelope with decay steeper than attack. Dominant frequency of the call ranges from 4.13 to 4.88 kHz (x = 4.55 �� 0.14; = 4.5; Mo = 4.5; n = 344); minimum frequency ranges from 2.49 to 4.24 kHz (x = 3.38 �� 0.37; = 3.43; Mo = 3.37; n = 98), and maximum frequency ranges from 5.58 to 7.05 kHz (x = 6.268 �� 0.37; = 6.25; Mo = 5.94; n = 98). Comparison with advertisement calls of species of Brachycephalus. Brachycephalus bufonoides differs from the other species of Brachycephalus by a number of distinct advertisement call parameters. Brachycephalus bufonoides has greater number of pulses (range = 13���17) than the species belonging to the B. pernix group: B. actaeus 2���3 pulses (Monteiro et al. 2018a); B. albolineatus 1���3 pulses (Bornschein et al. 2017); B. mirissimus 1���3 pulses (Pie et al. 2018); B. olivaceus 2���3 (Monteiro et al. 2018b); B. pernix: 3 pulses (Wistuba 1998); B. quiririensis 3���4 pulses (Monteiro et al. 2018b); B. tridactylus 1 pulse (Garey et al. 2012), and also presents longer note duration in B. bufonoides: x = 271.33 ms, while in B. actaeus x = 4 ms (Monteiro et al. 2018a); B. albolineatus x = 20 ms (Bornschein et al. 2017); B. olivaceus x = 38 ms (Monteiro et al. 2018b); B. pernix x = 60 ms (Wistuba 1998); B. quiririensis x = 50 ms (Monteiro et al. 2018b); B. tridactylus x = 110 ms (Garey et al. 2012). It also differs acoustically from most of them by presenting shorter inter-note interval in B. bufonoides x = 0.27 s, while in B. actaeus x = 5.42 s (Monteiro et al. 2018a); B. albolineatus x = 6.66 s (Bornschein et al. 2018); B. mirissimus x = 5.83 s (Pie et al. 2018); B. olivaceus: x = 10.69 s (Monteiro et al. 2018b); B. quiririensis: x = 12.58 s (Monteiro et al. 2018b), and lower dominant frequency in B. bufonoides x = 4.6 kHz, while in B. actaeus x = 6.9 kHz (Monteiro et al. 2018a); B. albolineatus x = 6.4 kHz (Bornschein et al. 2018); B. mirissimus x = 6.7 kHz (Pie et al. 2018); B. olivaceus: x = 6.8 kHz (Monteiro et al. 2018b); B. quiririensis x = 6.3 kHz (Monteiro et al. 2018b). The dominant frequency of B. tridactylus x = 4.8 kHz (Garey et al. 2012) overlaps with the range of B. bufonoides (4.1���4.9 kHz), and so does B. pernix 4.5���6.7, x = 5.6 kHz (Wistuba 1998). Values for pulse repetition rate and note repetition rate also vary greatly. The species from Brachycephalus ephippium group have an overall similar structure of the call, consisting in constant repetition of pulsed notes. Brachycephalus bufonoides can be distinguished from the other species of the B. ephippium group by a combination of different call structures. If differs most from B. darkside for presenting greater number of pulses (B. bufonoides 13���17 pulses, x = 15) in B. darkside 5���8 pulses, x = 6 (Guimar��es et al. 2017), while is similar or overlaps parts of the range with the other species: B. crispus 7���12 pulses, x = 10 (Condez et al. 2014); B. ephippium 5���15 pulses, x = 12 (Pombal et al. 1994); B. pitanga: 7���14 pulses, x = 11 (Ara��jo et al. 2012; Tandel et al. 2014). The note duration also is close to or overlaps some of its range with two of the species (B. bufonoides: 221���309, x = 271 ms): B. crispus x = 280 ms (Condez et al. 2014); B. pitanga: 150���250, x = 190 ms (Tandel et al. 2014), x = 170 ms (Ara��jo et al. 2012), but the value is lower in B. darkside x = 111 ms: Guimar��es et al. 2017) and B. ephippium (x = 112 ms: Pombal et al. 1994). The interval between notes of B. bufonoides (148���265, x = 209 ms) differs mostly from that of B. crispus (x = 350 ms: Condez et al. 2014), but overlaps slightly with the other species (B. darkside 122���215, x = 159.5 ms: Guimar��es et al. 2017); B. ephippium (123���149, x = 134 ms: Pombal et al. 1994); B. pitanga (200���430, x = 280 ms: Tandel et al. 2014). The pulse repetition rate (x = 56.20 pulses/s in B. bufonoides) is distinct from that of B. crispus x = 17.4 pulses/s: Condez et al. 2014) but is similar to the rate of B. darkside x = 56.9 pulses/s: Guimar��es et al. 2017) and B. pitanga x = 62.4 pulses/s: Ara��jo et al. 2012). Furthermore, note repetition rate of B. bufonoides (x = 2.15 notes/s, or 129 notes/min) is similar to the rate of B. crispus x = 1.67 notes/s: Condez et al. 2014) and B. pitanga x = 159 notes/min: Ara��jo et al. 2012) but differs from that of B. darkside x = 211 notes/min: Guimar��es et al. 2017). Brachycephalus bufonoides can be further distinguished from B. darkside for its higher dominant frequency (B. bufonoides: 4.1���4.9 kHz, x = 4.6 kHz) while in B. darkside: 2.8���3.8 kHz (Guimar��es et al. 2017), but differ less from the other species (B. ephippium: x = 3.9 kHz (Goutte et al. 2017), lowest frequency = 3.4 kHz, highest frequency = 5.3 kHz (Pombal et al. 1994), and overlaps with the range of B. crispus: x = 4.6 kHz: Condez et al. 2014 and B. pitanga: x = 4.9 kHz (Ara��jo et al. 2012) and x = 4.8 kHz (Tandel et al. 2014). The advertisement call of B. hermogenesi (x = 6.8 kHz: Verdade et al. 2008) and B. sulfuratus (x = 6.7 kHz: Condez et al. 2016) differ from B. bufonoides by presenting higher dominant frequency. Some overlap between the note duration of B. sulfuratus (131���233, x = 195 ms: Condez et al. 2016) and B. bufonoides (221���309 ms) was noticed, however a greater number of pulses per note in B. bufonoides (x = 15 pulses) than in B. sulfuratus 7���11 pulses, x = 9 (Condez et al. 2016) was observed, which results in difference in the pulse rate between B. bufonoides (x = 56.2 pulses/s) and B. sulfuratus (6.1���12.3, x = 9.3 pulses/s: Condez et al. 2016). The note repetition rate of B. bufonoides (x = 2.15 notes/s) is greater than B. hermogenesi (, Published as part of Folly, Manuella, Amaral, Lucas Coutinho, Carvalho-E-Silva, Sergio Potsch De & Pombal Jr, Jos�� P., 2020, Rediscovery of the toadlet Brachycephalus bufonoides Miranda-Ribeiro, 1920 (Anura: Brachycephalidae) with osteological and acoustic descriptions, pp. 265-294 in Zootaxa 4819 (2) on pages 269-283, DOI: 10.11646/zootaxa.4819.2.3, http://zenodo.org/record/4396900, {"references":["Miranda-Ribeiro, A. (1920) Os Brachycephalideos do Museu Paulista (com tres especes novas). Revista do Museu Paulista, 12, 307 - 316. https: // doi. org / 10.5962 / bhl. part. 22255","Cochran, D. M. (1955) Frogs of southeastern Brazil. Bulletin of the United States National Museum, 206, 1 - 423. https: // doi. org / 10.5479 / si. 03629236.206.1","Pombal, J. P. Jr. (2010) A posicao taxonomica das variedades de Brachycephalus ephippium (Spix, 1824) descritas por Miranda- Ribeiro, 1920 (Amphibia, Anura, Brachycephalidae). Boletim do Museu Nacional (N. S.), Zoologia, 526, 1 - 12.","Hedges, S. B., Duellman, W. E. & Heinicke, M. P. (2008) New World direct-development frogs (Anura: Terrarana): Molecular phylogeny, classification, biogeography, and conservation. Zootaxa, 1737 (1), 1 - 182. https: // doi. org / 10.11646 / zootaxa. 1737.1.1","Clemente-Carvalho, R. B. G., Giaretta, A. A., Condez, T. H., Haddad, C. F. B. & dos Reis, S. F. (2012) A new species of miniaturized toadlet, genus Brachycephalus (Anura: Brachycephalidae), from the Atlantic forest of Southeastern Brazil. Herpetologica, 68 (3), 365 - 374. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00085.1","Condez, T. H., Haddad, C. F. B. & Zamudio, K. R. (2020) Historical biogeography and multi-trait evolution in miniature toadlets of the genus Brachycephalus (Anura: Brachycephalidae), Biological Journal of the Linnean Society, 129 (3), 664 - 686. https: // doi. org / 10.1093 / biolinnean / blz 200","Pombal, J. P. Jr., Wistuba, E. M. & Bornschein, M. R. (1998) A new species of brachycephalid (Anura) from the Atlantic Rain Forest of Brazil. Journal of Herpetology, 32 (1), 70 - 74. https: // doi. org / 10.2307 / 1565481","Ribeiro, L. F., Alves, A. C. R., Haddad, C. F. B. & Reis, S. F. dos (2005) Two new species of Brachycephalus Gunther, 1858 from the State of Parana, southern Brazil (Amphibia, Anura, Brachyephalidae). Boletim do Museu Nacional, New Series, Zoologia, 519, 1 - 18.","Alves, A. C. R., Ribeiro, L. F., Haddad, C. F. B. & Reis, S. F. (2006) Two new species of Brachycephalus (Anura: Brachycephalidae) from the Atlantic forest in Parana State, southern Brazil. Herpetologica, 62 (2), 221 - 233. https: // doi. org / 10.1655 / 05 - 41.1","Haddad, C. F. B., Alves, A. C. R., Clemente-Carvalho, R. B. G. & dos Reis, S. F. (2010) A new species of Brachycephalus from the Atlantic Rain Forest in Sao Paulo State, southeastern Brazil (Amphibia: Anura: Brachycephalidae). Copeia, 2010 (3), 410 - 420. https: // doi. org / 10.1643 / CH- 09 - 102","Garey, M. V., Lima, A. M. X., Hartmann, M. T. & Haddad, C. F. B. (2012) A new species of miniaturized toadlet, genus Brachy- cephalus (Anura: Brachycephalidae), from southern Brazil. Herpetologica, 68 (2), 266 - 271. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00074.1","Ribeiro, L. F., Bornschein, M. R., Belmonte-Lopes, R., Firkowski, C. R., Morato, S. A. A. & Pie, M. R. (2015) Seven new microendemic species of Brachycephalus (Anura: Brachycephalidae) from southern Brazil. PeerJ, 3, e 1011. https: // doi. org / 10.7717 / peerj. 1011","Bornschein, M. R., Ribeiro, L. F., Blackburn, D. C., Stanley, E. L. & Pie, M. R. (2016) A new species of Brachycephalus (Anura: Brachycephalidae) from Santa Catarina, southern Brazil. PeerJ, 4 (e 2629), 1 - 19. https: // doi. org / 10.7717 / peerj. 2629","Monteiro, J. P. C., Condez, T. H., Garcia, P. C. A., Comitti, E. J., Amaral, I. B. & Haddad, C. F. B. (2018 a) A new species of Brachycephalus (Anura, Brachycephalidae) from the coast of Santa Catarina State, southern Atlantic Forest, Brazil. Zootaxa, 4407 (4), 483 - 505. https: // doi. org / 10.11646 / zootaxa. 4407.4.2","Ribeiro, L. F., Blackburn, D. C., Stanley, E. L., Pie, M. R. & Bornschein, M. R. (2017) Two new species of the Brachycephalus pernix group (Anura: Brachycephalidae) from the state of Parana, southern Brazil. PeerJ, 5, e 3603. https: // doi. org / 10.7717 / peerj. 3603","Pie, M. R., Ribeiro, L. F., Confetti, A. E., Nadaline, M. J. & Bornschein, M. R. (2018) A new species of Brachycephalus (Anura: Brachycephalidae) from southern Brazil. PeerJ, 6 (e 5683), 1 - 27. https: // doi. org / 10.7717 / peerj. 5683","Izecksohn, E. (1971) Novo genero e nova especie de Brachycephalidae do Estado do Rio de Janeiro, Brasil (Amphibia, Anura). Boletim do Museu Nacional, Boletim do Museu Nacional (N. S.), Zoologia, New Series, Zoologia, 280, 1 - 12.","Giaretta, A. A. & Sawaya, R. J. (1998) Second species of Psyllophryne (Anura: Brachycephalidae). Copeia, 1998 (4), 985 - 987. https: // doi. org / 10.2307 / 1447345","Napoli, M. F., Caramaschi, U., Cruz, C. A. G. & Dias, I. R. (2011) A new species of flea-toad, genus Brachycephalus Fitzinger (Amphibia: Anura: Brachycephalidae), from the Atlantic rainforest of southern Bahia, Brazil. Zootaxa, 2739 (1), 33 - 40. https: // doi. org / 10.11646 / zootaxa. 2739.1.3","Condez, T. H., Monteiro, J. P. C., Comitti, E. J., Garcia, P. C. A., Amaral, I. B. & Haddad, C. F. B. (2016) A new species of flea-toad (Anura: Brachycephalidae) from southern Atlantic Forest, Brazil. Zootaxa, 4083 (1), 40 - 56. https: // doi. org / 10.11646 / zootaxa. 4083.1.2","Pombal, J. P. Jr. & Izecksohn, E. (2011) Uma nova especie de Brachycephalus (Anura: Brachycephalidae) do estado do Rio de Janeiro. Papeis Avulsos de Zoologia, 51 (28), 443 - 451. https: // doi. org / 10.1590 / S 0031 - 10492011002800001","Guimaraes, C. da S., Luz, S., Rocha, P. C. & Feio, R. N. (2017) The dark side of pumpkin toadlet: a new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Brigadeiro, southeastern Brazil. Zootaxa, 4258 (4), 327 - 344. https: // doi. org / 10.11646 / zootaxa. 4258.4.2","Pombal, J. P. Jr. & Gasparini, J. L. (2006) A new Brachycephalus (Anura: Brachycephalidae) from the Atlantic Rainforest of Espirito Santo, southeastern Brazil. South American Journal of Herpetology, 1 (2), 87 - 93. https: // doi. org / 10.2994 / 1808 - 9798 (2006) 1 [87: ANBABF] 2.0. CO; 2","Condez, T. H., Clemente-Carvalho, R. B. G., Haddad, C. F. B. & dos Reis, S. F. (2014) A new species of Brachycephalus (Anura: Brachycephalidae) from the highlands of the Atlantic Forest, Southeastern Brazil. Herpetologica, 70 (1), 89 - 99. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 13 - 00044","Alves, A. C. R., Sawaya, R., Reis, S. F. & Haddad, C. B. F. (2009) A new species of Brachycephalus (Anura: Brachycephalidae) from the Atlantic Rain Forest in Sao Paulo State, Southeastern Brazil. Journal of Herpetology, 43 (2), 212 - 219. https: // doi. org / 10.1670 / 0022 - 1511 - 43.2.212","Bornschein, M. R., Ribeiro, L. F., Rollo Jr., M. M., Confetti, A. E. & Pie, M. R. (2017) Advertisement call of Brachycephalus albolineatus (Anura: Brachycephalidae). PeerJ, 6 (e 5273), 1 - 18. https: // doi. org / 10.7287 / peerj. preprints. 3461 v 1","Monteiro, J. P. C., Condez, T. H., Garcia, P. C. A. & Haddad, C. F. B. (2018 b) The advertisement calls of two species of Brachycephalus (Anura: Brachycephalidae) from southern forest Brazil. Zootaxa, 4415 (1), 183 - 188. https: // doi. org / 10.11646 / zootaxa. 4415.1.10","Wistuba, E. M. (1998) Historia natural de Brachycephalus pernix Pombal, Wistuba and Bornschein, 1998 (Anura) no Morro Anhangava, Municipio de Quatro Barras, Estado do Parana. M. Sc. dissertation, Universidade Federal do Parana, Curitiba, 89 pp.","Pombal, J. P. Jr., Sazima, I. & Haddad, C. F. B. (1994) Breeding behavior of the pumpkin toadlet, Brachycephalus ephippium (Brachycephalidae). Journal of Herpetology, 28 (4), 516 - 519. https: // doi. org / 10.2307 / 1564972","Araujo, C. B., Guerra, T. J., Amatuzzi, M. C. O. & Campos, L. A. (2012) Advertisement and territorial calls of Brachycephalus pitanga (Anura: Brachycephalidae). Zootaxa, 3302 (1), 66 - 67. https: // doi. org / 10.11646 / zootaxa. 3302.1.5","Tandel, M. C. F. F., Loibel, S., Oliveira, E. G. & Haddad, C. F. B. (2014) Diferenciacao de 3 tipos de vocalizacoes (cantos) na especie Brachycephalus pitanga. Revista da Estatistica da Universidade Federal de Ouro Preto, 3, 374 - 386.","Goutte, S., Mason, M. J., Christensen-Dalsgaard, J., Montealegre-Z, F., Chivers, B. D., Sarria-S, F. A., Antoniazzi, M. M., Jared, C., Sato, L. A. & Toledo, L. F. (2017) Evidence of auditory insensitivity to vocalization frequencies in two frogs. Scientific Reports, 7 (12121), 1 - 9. https: // doi. org / 10.1038 / s 41598 - 017 - 12145 - 5","Verdade, V. K., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. C. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta and Sawaya, 1998) (Anura: Brachycephalidae). Journal of Herpetology, 42 (3), 542 - 549. https: // doi. org / 10.1670 / 07 - 287.1","Bokermann, W. C. A. (1966) Lista anotada das localidades tipo de anfibios brasileiros. Servico de Documentacao, Sao Paulo, 183 pp.","Ihering, H. V. & Ihering, R. V. (1911) O Museu Paulista nos anos de 1906 a 1909. Revista do Museu Paulista, 8, 1 - 22. https: // doi. org / 10.5962 / bhl. part. 20998","Campos, L. A., Silva, H. R. & Sebben, A. (2010) Morphology and development of additional bony elements in the genus Brachycephalus (Anura: Brachycephalidae). The Biological Journal of the Linnean Society, 99 (4), 752 - 767. https: // doi. org / 10.1111 / j. 1095 - 8312.2010.01375. x","Alberch, P. & Gale, E. A. (1985) A developmental analysis of an evolutionary trend: digital reduction in amphibians. Evolution, 39, 8 - 23. https: // doi. org / 10.1111 / j. 1558 - 5646.1985. tb 04076. x","Fabrezi, M. (2001) A survey of prepollex and prehallux variation in anuran limbs. Zoological Journal of the Linnean Society, 131 (2), 227 - 248. https: // doi. org / 10.1111 / j. 1096 - 3642.2001. tb 01316. x","Andersen, M. L. (1978) The comparative myology and osteology of the carpus and tarsus of selected anurans. Ph. D. dissertation, University of Kansas, Lawrence, Kansas, 295 pp."]}

Submitted by Moreno Barros (moreno@ct.ufrj.br) on 2020-10-23T02:06:06Z No. of bitstreams: 1 CaioMarcioSorrentinoDeFreitasFariasDosSantos.pdf: 4740649 bytes, checksum: 1d32e98e21d25dc914793c3742d2ebde (MD5) Made available in DSpace on 2020-10-23T02:06:06Z (GMT). No. of bitstreams: 1 CaioMarcioSorrentinoDeFreitasFariasDosSantos.pdf: 4740649 bytes, checksum: 1d32e98e21d25dc914793c3742d2ebde (MD5) Previous issue date: 2019-03 A Floresta Atlântica brasileira abriga muitas espécies do gênero Brachycephalus, e, dentre elas, a espécie Brachycephalus ephippium. A microtomografia computadorizada por raios X é uma técnica não destrutiva que permite a visualização e análise de microestruturas internas de amostras diversas. Esta técnica já foi e continua sendo aplicada nas áreas biológicas para o estudo destes animais, permitindo uma minuciosa descrição e caracterização de novas espécies. A espécie usada nesse estudo é particularmente especial, uma vez que foi a primeira do gênero a ser descoberta, tornando-a de certa forma a base comparativa entre espécies. O crânio é de fundamental interesse, sendo muito usado nos trabalhos de aplicação de microCT. Dentre as estruturas de interesse no crânio, algumas são da ordem do milímetro, podendo chegar a menos de meio milímetro. Os efeitos do tamanho de voxel de imagem então podem tornar-se relevantes, e foi isso que foi investigado nesse trabalho, tanto visualmente quanto quantitativamente. Foram usados dois métodos de segmentação de imagens bem conhecidos, o Global (manual) e o de Otsu (automático), e oito parâmetros morfométricos foram analisados. Verificou-se que a variação do tamanho de voxel implicou em perda substancial de informações nas imagens para a maioria dos parâmetros estudados, enquanto a variação quanto ao método de segmentação foi verificada somente para os parâmetros Tb.Pf e Obj.N. The Brazilian Rainforest inhabits many species of the Brachycephalus genre and, among them, the Brachycephalus ephippium species. X-ray computed tomography is a nondestructive imaging technique which allows for the visualization and analysis of internal microestructures of various samples. This technique was and still is applied on biological research for the study of these animals, enabling thorough description and characterization of new species. The species used in this study is of particular interest, since it was the first of this genre to be discovered, which makes it in a way the interspecies comparative basis. The cranium is of fundamental interest and it’s very used in microCT application works. Amid structures of interest in the cranium, some are of the order of the milimeter, sometimes less than half a milimeter. The effects of the voxel size of microCT images can therefore be relevant, and this is what was investigated in this work, both visually and quantitatively. Two well-known segmentation methods were used: Global (manual) and Otsu (automatic), and eight morphometric parameters were analyzed. There was significant variation due to voxel size alone, which led to information loss on the images for most evaluated parameters, while segmentation did not represent significant variation for most parameters, only for Tb.Pf. and Obj.N.

Made available in DSpace on 2018-11-26T16:04:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-01 Fundacao Grupo Boticario de Protecao a Natureza Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Despite encompassing a relatively small geographical area, montane regions harbour disproportionately high levels of species diversity and endemism. Nonetheless, relatively little is known about the evolutionary mechanisms that ultimately lead to montane diversity. In this study, we used target capture of ultraconserved elements to investigate the phylogenetic relationships and diversification patterns of Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae), two frog genera that occur in sky islands of the southern Atlantic Forest of Brazil. Specifically, we tested whether diversification of montane species in these genera could be explained by a single climatic shift leading to isolation in sky islands, followed by climatic stability that maintained populations in allopatry. In both genera, the topologies inferred using concatenation and coalescent-based methods were concordant and had strong nodal support, except for a few recent splits, which nevertheless tended to be supported by more informative loci. Estimation of divergence time of a combined dataset using both genera is consistent with a concordant timing of their diversification. These results support the scenario of diversification by isolation in sky islands and suggest that allopatry attributable to climatic gradients in montane regions is an important mechanism for generating species diversity and endemism in these regions. Univ Fed Parana, Dept Zool, Curitiba, Parana, Brazil Mater Nat Inst Estudos Ambientais, Curitiba, Parana, Brazil Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA Louisiana State Univ, Museum Nat Sci, Baton Rouge, LA 70803 USA Pontificia Univ Catolica Parana, Escola Ciencias Vida, Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Sao Paulo, Brazil Occidental Coll, Moore Lab Zool, Los Angeles, CA 90041 USA Univ Estadual Paulista, Inst Biociencias, Sao Paulo, Brazil Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014 CNPq: 301636/2016-8

Submitted by Maurício Humberto Vancine (mauricio.vancine@gmail.com) on 2018-08-29T16:03:04Z No. of bitstreams: 1 dissertacao_vancine_2018_08_d29.pdf: 4299716 bytes, checksum: 0da83f2f1fb91c3955a011cc7705a7d1 (MD5) Approved for entry into archive by Ana Paula Santulo Custódio de Medeiros null (asantulo@rc.unesp.br) on 2018-08-30T11:50:16Z (GMT) No. of bitstreams: 1 vancine_mh_me_rcla_par.pdf: 684124 bytes, checksum: 1d009d13de69a423df4ab316d03141e6 (MD5) Made available in DSpace on 2018-08-30T11:50:17Z (GMT). No. of bitstreams: 1 vancine_mh_me_rcla_par.pdf: 684124 bytes, checksum: 1d009d13de69a423df4ab316d03141e6 (MD5) Previous issue date: 2018-06-29 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) As incertezas acerca da diversidade e distribuição de espécies é um dos principais desafios para a biologia da conservação. Dentre os hotspots mundiais de biodiversidade, a Mata Atlântica é considerada um dos biomas mais ameaçados devido, principalmente, à perda e fragmentação de habitat, mudanças climáticas e espécies invasoras. Entre essas ameaças, as projeções de cenários de mudanças climáticas têm se mostrado o principal agente de redução da biodiversidade, particularmente entre os anfíbios. Esse grupo é considerado um dos táxons mais sensíveis a essas alterações, devido às suas características particulares de morfologia, fisiologia e ecologia. Dessa forma, buscamos atingir dois objetivos nesse trabalho: 1) apresentar uma ampla compilação e avaliação da composição e distribuição das comunidades de anfíbios já inventariadas para o Bioma da Mata Atlântica, e 2) utilizando parte dos dados das comunidades, inferir como as mudanças climáticas poderão afetar a distribuição de anuros do gênero Brachycephalus, grupo considerado altamente sensível às alterações climáticas. Realizamos análises descritivas e espaciais das amostragens das comunidades de anfíbios para a Mata Atlântica e fizemos uso de Modelos de Nicho Ecológico para inferir a distribuição do gênero frente aos impactos das mudanças climáticas. No primeiro objetivo, não tivemos uma hipótese pré-definida, já que se tratou de uma compilação de dados. No segundo objetivo, nossa hipótese foi de que no futuro, haverá um aumento dos valores de adequabilidade aos Brachycephalus em regiões de maiores altitudes, onde provavelmente haverá pouca floresta disponível para a manutenção das populações das espécies desse gênero. Os resultados da compilação das amostragens de anfíbios gerou um panorama geral da diversidade, composição, métodos e esforço amostral das comunidades desse táxon para a Mata Atlântica. Em relação aos efeitos das mudanças climáticas, nossos resultados indicaram drásticas mudanças de adequabilidade do gênero Brachycephalus em distintas áreas do bioma. Quando relacionamos os valores de adequabilidade (atual e futura) com os valores de altitude, observamos que as regiões de altitude mais elevadas terão um aumento dos valores de adequabilidade, quando comparadas com as áreas atuais. No entanto, essas regiões mais elevadas (geralmente acima de 1500 m) não possuirão habitat florestal, colocando diversas espécies em sério risco de redução do tamanho populacional e/ou até mesmo extinções locais, principalmente as espécies com distribuição restritas. Uncertainties about species diversity and distribution are one of the major challenges for conservation biology. Among the world's biodiversity hotspots, the Atlantic Forest is considered one of the most threatened biomes, mainly due to the loss and fragmentation of habitats, climate changes and invasive species. Among these threats, projections of climate change scenarios have proven to be the main agent of biodiversity reduction, particularly among amphibians. This group is considered one of the most sensitive taxa to these alterations, due to their particular characteristics of morphology, physiology and ecology. Thus, we aim to achieve two objectives in this work: 1) to present a comprehensive compilation and evaluation of the composition and distribution of the amphibian communities already inventoried to the Atlantic Forest Biome, and 2) to use part of the data from the communities, to infer how climate change may affect the distribution of anurans of the genus Brachycephalus, a group considered highly sensitive to climate change. We performed descriptive and spatial analyzes of amphibian community samples for the Atlantic Forest and used Ecological Niche Models to infer the distribution of the genus in face of the impacts of climate change. In the first objective, we did not have a pre-defined hypothesis, since it was a compilation of data. For the second objective, our hypothesis was that in the future there will be an increase in Brachycephalus suitability values in regions of higher altitudes, where there will probably be little forest available for the maintenance of the populations of the species of this genus. The results of the compilation of the amphibian samples generated an overview of the diversity, composition, methods and sample effort of the communities of this taxon for the Atlantic Forest. Regarding the effects of climate change, our results indicated drastic changes in the suitability of the Brachycephalus genus in different areas of the biome. When we relate the values of suitability (current and future) with the altitude values, we observed that the higher altitude regions will have an increase in suitability values when compared to the current areas. However, these higher regions (generally above 1,500 m) will not have forest habitat, placing several species at serious risk of population size reduction and/or even local extinctions, especially species with restricted distribution. FAPESP: 2017/09676-8

Brachycephalus actaeus sp. nov. Figures 1, 2, 6, 7, and 8 Holotype. CFBH 39850, adult male, collected at Serra da Palha, Laranjeiras, Ilha de S��o Francisco do Sul, municipality of S��o Francisco do Sul, state of Santa Catarina, Brazil (26��17'50"S; 48 �� 40'28"W, Datum WGS 84, ca 60 meters above sea level), on 19 May 2015, by C.F.B. Haddad, J.P.C. Monteiro, and E.C. Nardin (Figures 1 and 2). Paratopotypes. CFBH 39851, adult female, collected with the holotype; CFBH 39872, 39873, and UFMG 18973, adult males, collected on 25 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; CFBH 39876, adult male, and CFBH 39877, adult female, collected on 0 2 December 2015, by J.P.C. Monteiro and E.C. Nardin. Paratypes. CFBH 39846, adult male, cleared and double-stained, collected at Fazenda Morro Grande, Morro Grande, Ilha de S��o Francisco do Sul, municipality of S��o Francisco do Sul, state of Santa Catarina, Brazil (26 �� 17'47"S; 48��37'10"W, Datum WGS 84, ca 60 meters above sea level), on 14 November 2014, by E.J. Comitti. UFMG 18970, adult female, collected on 15 November 2014, by J.P.C. Monteiro, T.H. Condez, and E.J. Comitti; CFBH 39849, sub-adult female, collected on 21 November 2014, by J.P.C. Monteiro and E.C. Nardin; CFBH 39848, adult male, cleared and double-stained, collected on 21 January 2015, by C.F.B. Haddad, J.P.C. Monteiro, T.H. Condez, and E.J. Comitti; UFMG 18971, adult male, collected on 0 1 August 2015, by J.P.C. Monteiro and E.C. Nardin; CFBH 39855���39858 and 39861, adult males, CFBH 39860, adult female, CFBH 39859, juvenile, collected on 23 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; all collected at Centro de Estudos e Pesquisas Ambientais da Univille (CEPA), Vila da Gl��ria, Distrito do Sa��, municipality of S��o Francisco do Sul, state of Santa Catarina, Brazil (26 �� 13'39"S; 48 �� 41'31"W, Datum WGS 84, ca 120 meters above sea level). CFBH 39853 and 39854, adult males, collected on 27 August 2015, by J.P.C. Monteiro and E.C. Nardin; CFBH 39862, adult female, collected on 23 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; at Estrada do Sa��, Distrito do Sa��, municipality of S��o Francisco do Sul, state of Santa Catarina, Brazil (26��12'06"S; 48 �� 41'37"W, Datum WGS 84, ca 80 meters above sea level). CFBH 39863, 39864, 39867, 39868, and UFMG 18972 adult females, CFBH 39865 and 39870, adult males, collected on 24 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; CFBH 39875, adult female, collected on 30 November 2015, by J.P.C. Monteiro and E.C. Nardin; at Bra��o do Norte, municipality of Itapo��, state of Santa Catarina, Brazil (26��07'29"S; 48 �� 43'48"W, Datum WGS 84, ca 220 meters above sea level). CFBH 42005���42008, adult females, collected on 17 September 2016, by J.P.C. Monteiro and E.C. Nardin; Fazenda Palmito Juriti, municipality of S��o Francisco do Sul, state of Santa Catarina, Brazil (26��08'09"S; 48 �� 43'54"W, Datum WGS 84, 125���170 meters above sea level). Diagnosis. Brachycephalus actaeus sp. nov. is a new species of the B. pernix group, distinguished from all its congeners by the following combination of characters: (1) ���bufoniform��� body; (2) general dorsal body color dark green with a dark brown vertebral stripe, and orange background more evident in ventral view; (3) absence of hyperossification of the skull and skeleton; (4) pectoral girdle arciferal and robust, with small ovoid fenestra, distant from the epicoracoid; (5) radius and ulna fused; (6) finger IV greatly reduced, almost not visible externally; (7) manus with two prepollical elements; (8) tips of terminal phalangeal elements of fingers I and IV pointed, II and III arrow-shaped; (9) tibiale and fibulare completely fused; (10) toes I and V present but externally indistinguishable, toe II greatly reduced, toe III short and distinct, and toe IV larger and robust; (11) pes with distal tarsal element I present; (12) pes with phalangeal formula 1���2���3���4���0; (13) tips of terminal phalangeal elements of toes I and II pointed, and of toes III and IV arrow-shaped; (14) arytenoid cartilages not mineralized; (15) body size (SVL of adults: 9.2���10.8 mm for males and 11.1���12.4 mm for females); (16) proportional measurements HL/SVL 18���24% and ED/HL 52���73%; (17) rounded snout in dorsal and lateral views; (18) protuberant nostrils; (19) skin texture rough; and (20) advertisement call short (0.02���0.03 seconds), composed of one high-frequency note (dominant frequency 6.6���7.3 kHz). Comparisons with other species. Brachycephalus actaeus sp. nov. exhibits a ���bufoniform��� body and an orange background color, both characteristics that clearly differentiate it from the ���leptodactyliform��� species: B. didactylus, B. hermogenesi, B. pulex, and B. sulfuratus (Izecksohn 1971; Giaretta & Sawaya 1998; Napoli et al. 2011; Condez et al. 2016). These species are generally smaller, exhibit ���leptodactyliform��� bodies, and always exhibit a brown background color (Napoli et al. 2011; Condez et al. 2016). The absence of hyperossification of the skull and skeleton distinguishes Brachycephalus actaeus sp. nov. from B. darkside, B. ephippium, B. garbeanus, and B. margaritatus, which exhibit the extreme condition of hyperossification within Brachycephalus, including a dorsal bony shield (Clemente-Carvalho et al. 2009; Guimar��es et al. 2017). The absence of hyperossification also distinguishes the new species from B. alipioi, B. atelopoide, B. bufonoides, B. crispus, B. guarani, B. nodoterga, B. pitanga, B. toby, and B. vertebralis, which exhibit the intermediate condition of hyperossification within Brachycephalus (Clemente-Carvalho et al. 2009; Haddad et al. 2010; Pombal 2010; Clemente-Carvalho et al. 2011; Condez et al. 2014; Condez et al. 2016). Brachycephalus actaeus sp. nov. differs from B. brunneus, B. coloratus, B. ferruginus, B. izecksohni, and B. pombali, which possess pectoral girdles with larger fenestra, disposed closer to the epicoracoid (small fenestra distant from the epicoracoid in the new species). Likewise, B. brunneus, B. ferruginus, B. izecksohni, and B. pombali differ in having the radius and ulna not fused, pes with distal tarsal element I absent, and toe V reduced (Ribeiro et al. 2005; Alves et al. 2006); the new species has the radius and ulna fused, pes with distal tarsal element I present, and toe V externally not visible. Also, B. albolineatus , B. coloratus, B. curupira, B. ferruginus, and B. pombali have just one prepollical element, and B. izecksohni has no prepollical element (Ribeiro et al. 2005; Alves et al. 2006; Bornschein et al. 2016b; Ribeiro et al. 2017); the new species has two prepollical elements. The phalangeal formula for the pes of B. brunneus , B. izecksohni, and B. pombali is 0���2���3���4���0 (Ribeiro et al. 2005; Alves et al. 2006), and for B. curupira it is 0���1���3���4���0 (Ribeiro et al. 2017); the phalangeal formula of the pes of the new species is 1���2���3���4���0. In B. albolineatus, B. coloratus, and B. curupira the fibulare and tibiale are not completely fused; tips of terminal phalangeal elements of toes II���IV are arrow-shaped (fibulare and tibiale completely fused; tips of terminal phalangeal elements of toes I and II pointed, III and IV arrow-shaped in the new species). Finally, the arytenoid cartilages are mineralized in B. albolineatus and B. coloratus (arytenoid cartilages not mineralized in the new species). Although the osteology of B. pernix was not studied in detail (Pombal et al. 1998), on the basis of the available information its osteology is quite similar to that of the new species. See Figure 3 for osteological details of the new species. Body size (males SVL = 9.2���10.8 mm; females SVL = 11.1���12.4 mm) distinguishes the new species from Brachycephalus ferruginus (males SVL = 11.6���12.5 mm; females SVL = 13.0��� 14.5 mm; Alves et al. 2006), B. pernix (males SVL = 12.0��� 13.3 mm; females SVL = 14.1���15.8 mm; Pombal et al. 1998), and B. pombali (males SVL = 12.6���13.9 mm; females SVL= 14.6���15.3 mm; Alves et al. 2006). Also, in B. coloratus (males SVL = 10.3��� 10.6 mm; females SVL = 12.2���13.3 mm; Ribeiro et al. 2017), B. izecksohni (males SVL = 10.3���12.1 mm; females SVL = 12.5���13.1 mm; Ribeiro et al. 2005), and B. tridactylus (males SVL = 10.6���11.6; females SVL = 13.5���13.8 mm; Garey et al. 2012) males and/or females are slightly larger than in the new species. Brachycephalus actaeus sp. nov. is distinguishable from B. albolineatus, B. auroguttatus, B. boticario, B. fuscolineatus, B. leopardus, B. mariaeterezae, B. olivaceus, B. quiririensis, and B. verrucosus by a proportionally shorter head relative to body length (HL/SVL) and by a proportionally larger eye diameter related to head length (ED/HL). In B. actaeus sp. nov., HL/ SVL is 18���24% (x��= 20, SD = 1) and ED/HL is 52���73% (x��= 63, SD = 4), for the 32 adult specimens of the type series, without sexual distinction. In B. albolineatus, HL/SVL is 28���34% (x��= 31, SD = 4) and ED/HL is 36���42% (x��= 38, SD = 4); in B. auroguttatus, HL/ SVL is 29���38% (x��= 33, SD = 2) and ED/HL is 30���44% (x��= 33, SD = 3); in B. boticario, HL/ SVL is 31���36% (x��= 34, SD = 2) and ED/HL is 30���34% (x��= 32, SD = 1); in B. fuscolineatus, HL/ SVL is 29���34% (x��= 31, SD = 1) and ED/HL is 36���41% (x��= 39, SD = 1); in B. leopardus, HL/ SVL is 31���35% (x��= 33, SD = 1) and ED/HL is 34���43% (x��= 38, SD = 3); in B. mariaeterezae, HL/ SVL is 29���36% (x��= 33, SD = 2) and ED/HL is 36���42% (x��= 39, SD = 2); in B. olivaceus, HL/ SVL is 32���36% (x��= 34, SD = 1) and ED/HL is 26���36% (x��= 32, SD = 3); in B. quiririensis, HL/ SVL is 31���36% (x��= 34, SD = 1) and ED/HL is 28���34% (x��= 32, SD = 2); and in B. verrucosus, HL/ SVL is 30���36% (x��= 33, SD = 2) and ED/HL is 30���40% (x��= 34, SD = 3) (Pie & Ribeiro 2015; Ribeiro et al. 2015; Bornschein et al. 2016b). Additionally, Brachycephalus actaeus sp. nov. exhibits a rounded snout in dorsal and lateral views, which distinguishes it from B. brunneus, which has a slightly mucronate snout in dorsal view (Ribeiro et al. 2005); from B. leopardus, which has a slightly truncate snout in dorsal and lateral views (Ribeiro et al. 2015); and from B. quiririensis, which has a mucronate snout in dorsal view (Pie & Ribeiro 2015). Nostrils are not protuberant in B. auroguttatus, B. pernix, and B. fuscolineatus (Pombal et al. 1998; Ribeiro et al. 2015), which differ from the protuberant nostrils of the new species. In Brachycephalus tridactylus, finger IV is not externally visible (Garey et al. 2012), while in the new species it is reduced but externally distinct. In B. actaeus sp. nov., fingertips I, II, and IV are rounded, differing from B. pernix in which these fingertips are pointed (Pombal et al. 1998) and from B. brunneus, B. izecksohni, and B. leopardus, which have the tip of finger II pointed (Ribeiro et al. 2005; Ribeiro et al. 2015). The texture of the skin on the dorsum of Brachycephalus actaeus sp. nov. is rough; this characteristic distinguishes the new species from B. albolineatus, B. brunneus, B. coloratus, B. curupira, B. ferruginus, B. izecksohni, B. leopardus, B. pernix, B. pombali, and B. tridactylus, which have a smooth dorsum (Pombal et al. 1998; Ribeiro et al. 2005; Alves et al. 2006; Garey et al. 2012; Ribeiro et al. 2015; Bornschein et al. 2016b; Ribeiro et al. 2017). The dark green general color of Brachycephalus actaeus sp. nov. in life is very distinct from B. boticario, B. coloratus, B. ferruginus, B. fuscolineatus, B. izecksohni, B. leopardus, B. mariaeterezae, B. quiririensis, B. pernix, B. pombali, B. tridactylus, and B. verrucosus, which exhibit a bright yellow or orange general dorsal body color (Pombal et al. 1998; Ribeiro et al. 2005; Alves et al. 2006; Garey et al. 2012; Pie & Ribeiro 2015; Ribeiro et al. 2015; Ribeiro et al. 2017). The body color of the new species is similar to B. albolineatus, which has a greenish general coloration (Bornschein et al. 2016b), and B. olivaceus, which is predominantly dark green to brown (Ribeiro et al. 2015). The advertisement call of Brachycephalus actaeus sp. nov. differs in structure and frequency from all known advertisement calls within Brachycephalus. It is clearly distinct from B. crispus, B. darkside , B. ephippium, and B. pitanga by having higher frequencies together with a different call structure (Pombal et al. 1994; Ara��jo et al. 2012; Condez et al. 2014; Guimar��es et al. 2017). In these species, the advertisement call is characterized by the regular repetition of one low-frequency note with several pulses, while in B. actaeus sp. nov. the high-frequency notes are composed of just two pulses. In B. crispus, the notes generally last 0.28 seconds and are composed of 10 pulses; the frequency range is 3.5���5.7 kHz, while the dominant frequency is 4.6 kHz (Condez et al. 2014). In B. darkside, the average note duration is 0.11 seconds and notes are composed of six pulses; the frequency range is 2.5���5.8 kHz [considered dominant frequency in Guimar��es et al. (2017)], while the dominant frequency is 3.4 kHz [considered peak frequency in Guimar��es et al. (2017)]. In B. ephippium, notes typically last 0.12 seconds and are also composed of 12 pulses; the minimum and maximum frequencies are 3.4���5.3 kHz (Pombal et al. 1994). In B. pitanga, notes last 0.17 seconds and are composed of 11 pulses; the dominant frequency is 4.9 kHz (Ara��jo et al. 2012). The high frequency of the advertisement call of the new species is comparable to that described for B. hermogenesi and B. sulfuratus, the latter having the highest dominant frequency known for the genus (Condez et al. 2016). Nevertheless, the advertisement calls of these species differ from the new species in general structure, which is long and composed of a set of high-frequency notes. In B. hermogenesi, the call is composed of 1���5 notes; call lasts 0.2 seconds, with 1���5 pulses (Verdade et al. 2008). The dominant frequency in B. hermogenesi is 6.8 kHz (Verdade et al. 2008). In B. sulfuratus, the call is composed of 4���7 notes, each one lasting 0.19 seconds, with 9 pulses (Condez et al. 2016). The frequency range is 4.9���9.3 kHz and the dominant frequency is 6.7 kHz (Condez et al. 2016). When compared to the advertisement calls of its most closely related species, B. pernix and B. tridactylus, the new species call has shorter notes and higher frequencies. In B. pernix, notes last 0.03���0.06 seconds and are composed of three pulses; the frequency range is 4.5 kHz���6.7 kHz (Wistuba 1998). In B. tridactylus, the frequency range is 3.2���6.4 kHz and dominant frequency is 4.8 kHz (Garey et al. 2012). Description of holotype. Body robust, bufoniform; head wider than long; head length 19% of SVL; snout short, rounded in lateral and dorsal views (Figures 1A and 1B); nostrils protuberant; canthus rostralis indistinct; loreal region slightly concave; eyes slightly protruding laterally and dorsally; eye diameter 72% of head length; tympanum absent; lips nearly sigmoid; vocal sac not expanded externally; vocal slits present; tongue longer than wide, with the posterior half not adherent to floor of mouth; vomerine teeth absent; choanae small and ovoid, anterior to eyes. Arm and forearm moderately slender; hands with fingers I and IV reduced; finger II short but distinct; finger III large and robust; fingertips I, II, and IV rounded, fingertip III pointed; finger lengths IV Measurements of holotype (in mm). SVL 9.4; HL 1.8; HW 3.0; ND 0.4; IND 1.1; ED 1.3; IOD 2.1; END 0.6; AL 2.0; FAL 2.1; HAL 1.4; THL 3.8; TBL 3.3; FL 4.6. Color in life. (Figure 6) Iris black. General body color orange, dorsal surface of body covered by dark green blotches; in dorsal view, a poorly defined dark brown stripe extends from the interorbital region to the posterior end of the vertebral column; arms, legs, fingers III and IV and toe IV dark green, other fing, Published as part of Monteiro, Juliane Petry De Carli, Condez, Thais Helena, Garcia, Paulo Christiano De Anchietta, Comitti, Estev��o Jasper, Amaral, Ivan Borel & Haddad, C��lio Fernando Baptista, 2018, A new species of Brachycephalus (Anura, Brachycephalidae) from the coast of Santa Catarina State, southern Atlantic Forest, Brazil, pp. 483-505 in Zootaxa 4407 (4) on pages 486-500, DOI: 10.11646/zootaxa.4407.4.2, http://zenodo.org/record/1221060, {"references":["Izecksohn, E. (1971) Novo genero e nova especie de Brachycephalidae do estado do Rio de Janeiro, Brasil. Boletim do Museu Nacional, Zoologia, 280, 1 - 12.","Giaretta, A. A. & Sawaya, R. J. 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(2005) Two new species of Brachycephalus Gunther, 1985 from the state of Parana, southern Brazil (Amphibia, Anura, Brachycephalidae). Boletim do Museu Nacional, Zoologia, 519, 1 - 18.","Alves, A. C. R., Ribeiro, L. F., Haddad, C. F. B. & Reis, S. F. (2006) Two new species of Brachycephalus (Anura: Brachycephalidae) from the Atlantic Forest in Parana state, southern Brazil. Herpetologica, 62 (2), 221 - 233. https: // doi. org / 10.1655 / 05 - 41.1","Bornschein, M. R., Ribeiro, L. F., Blackburn, D. C., Stanley, E. L. & Pie, M. R. (2016 b). A new species of Brachycephalus (Anura: Brachycephalidae) from Santa Catarina, southern Brazil. PeerJ, 4 (e 2629), 1 - 19. https: // doi. org / 10.7717 / peerj. 2629","Ribeiro, L. F., Blackburn, D. C., Stanley, E. L., Pie, M. R. & Bornschein M. R. (2017) Two new species of the Brachycephalus pernix group (Anura: Brachycephalidae) from the state of Parana, southern Brazil. PeerJ, 5 (e 3603), 1 - 28. https: // doi. org / 10.7717 / peerj. 3603","Pombal, J. P., Wistuba, E. M. & Bornschein, M. R. (1998) A new species of brachycephalid (Anura) from the Atlantic Rain Forest of Brazil. Journal of Herpetology, 32 (1), 70 - 74. https: // doi. org / 10.2307 / 1565481","Garey, M. V., Lima, A. M. X., Hartmann, M. T. & Haddad, C. F. B. (2012) A new species of miniaturized toadlet, genus Brachycephalus (Anura: Brachycephalidae), from southern Brazil. Herpetologica, 68 (2), 266 - 271. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00074.1.","Araujo, C. B., Guerra, T. J., Amatuzzi, M. C. O. & Campos, L. A. (2012) Advertisement and territorial calls of Brachycephalus pitanga (Anura: Brachycephalidae). Zootaxa, 3302, 66 - 67.","Verdade, V. K., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta and Sawaya, 1998) (Anura, Brachycephalidae). Journal of Herpetology, 42 (3), 542 - 549. https: // doi. org / 10.1670 / 07 - 287.1","Goutte, S., Mason, M. J., Christensen-Dalsgaard, J., Montealegre-Z, F., Chivers, B. D., Sarria-S, F. A., Antoniazzi, M. M., Jared, C., Sato, L. A. & Toledo, L. F. (2017) Evidence of auditory insensitivity to vocalization frequencies in two frogs. Scientific Reports, 7, 12121. https: // doi. org / 10.1038 / s 41598 - 017 - 12145 - 5","Pie, M. R., Meyer, A. L. S., Firkowski, C. R., Ribeiro, L. F. & Bornschein, M. R. (2013) Understanding the mechanisms underlying the distribution of microendemic montane frogs (Brachycephalus spp., Terrarana: Brachycephalidae) in the Brazilian Atlantic Rainforest. Ecological Modelling, 250, 165 - 176. https: // doi. org / 10.1016 / j. ecolmodel. 2012.10.019","Bornschein, M. R., Firkowski, C. R., Belmonte-Lopes, R., Correa, L., Ribeiro, L. F., Morato, S. A. A., Antoniazzi-Junior, R. L., Reinert, B. L., Meyer, A. L. S., Cini, F. A. & Pie, M. R. (2016 a). Geographical and altitudinal distribution of Brachycephalus (Anura: Brachycephalidae) endemic to the Brazilian Atlantic Rainforest. PeerJ, 4 (e 2490), 1 - 41. https: // doi. org / 10.7717 / peerj. 2490","Oliveira, J. C. F., Coco, L., Pagotto, R. V., Pralon, E., Vrcibradic, D., Pombal, J. P. & Rocha, C. F. D. (2012) Amphibia, Anura, Brachycephalus didactylus (Izecksohn, 1971) and Zachaenus parvulus (Girard, 1853): Distribution extension. Check List, 8 (2), 242 - 244. https: // doi. org / 10.15560 / 8.2.242","Clemente-Carvalho, R. B. G., Monteiro, L., Bonato, V., Rocha, H., Pereira, G., Oliveira, D., Lopes, R., Haddad, C. F. B., Martins, E. & Reis, S. F. (2008) Geographic variation in cranial shape in the pumpkin toadlet (Brachycephalus ephippium): a geometric analysis. Journal of Herpetology, 42 (1), 176 - 185. https: // doi. org / 10.1670 / 07 - 141 R 1.1","Pimenta, B. V. S., Bernils, R. S. & Pombal, J. P. (2007) Amphibia, Anura, Brachycephalidae, Brachycephalus hermogenesi: filling gap and geographic distribution map. Check List, 3 (3), 277 - 279. https: // doi. org / 10.15560 / 3.3.277","Pombal, J. P. & Izecksohn, E. (2011) Uma nova especie de Brachycephalus (Anura: Brachycephalidae) do estado do Rio de Janeiro. Papeis Avulsos de Zoologia, 51 (28), 443 - 451. https: // doi. org / 10.1590 / S 0031 - 10492011002800001","Clemente-Carvalho, R. B. G., Perez, S. I., Tonhati, C. H., Condez, T. H., Sawaya, R. J., Haddad, C. F. B. & Reis, S. F. (2015) Boundaries of morphological and molecular variation and the distribution range of a miniaturized froglet, Brachycephalus nodoterga (Anura: Brachycephalidae). Journal of Herpetology, 50 (1), 169 - 178. https: // doi. org / 10.1670 / 14 - 119","Abegg, A. D., Ortiz, F. R., Rocha, B. & Condez, T. H. (2015) A new record for Brachycephalus nodoterga (Amphibia, Anura, Brachycephalidae) in the state of Sao Paulo. Brazil. Check List, 11 (1769), 1 - 3. https: // doi. org / 10.15560 / 11.5.1769"]}

Brachycephalus darkside sp. nov. Figures 1–4 and 7. Brachycephalus ephippium —Pombal Jr. 2001 (part.) Brachycephalus ephippium — Dayrell et al. 2006 Brachycephalus ephippium —Pombal & Izecksohn 2011 (part.) Brachycephalus ephippium — Moura et al. 2012. Holotype. MZUFV 16636, adult male (17.9 mm SVL, Figure 1), collected at “Trilha do Cruzeiro” (20°52'40.7"S, 0 42°31'14.6"W; 1266 m a.s.l.), Parque Estadual da Serra do Brigadeiro (PESB), district of Careço, Municipality of Ervália, Minas Gerais State, Brazil, on 17 December 2015 by C.S. Guimarães, P.C. Rocha, C.L. Assis, R.N. Feio, and C. Novaes. Paratypes. Specimens collected at type locality: MZUFV 16634–35 (males) collected with holotype. MZUFV 16627, 16632–33 (males), 16628, 16631 (females), MNRJ 91327 and UFMG 19522 (males) collected on 20 November 2015 by C.S. Guimarães, P.C. Rocha and R.N. Feio. MZUFV 16491, 16579 (males), and 16780 (female) collected on 13 August 2015 by C.S. Guimarães, P.S. Hote and S. Luz. MZUFV 15716 (male), 15717, 15720–21 (adult specimens), and 15718–19 (juveniles) collected on 4 December 2014 by C.S. Guimarães, P.G. Taucce and B. Lisboa. MZUFV 15557–58, 15560–61, 15566–15571, 15717 (adult specimens), and 15559, 15565 (males), collected on 18 September 2014 by C.S. Guimarães, C.L. Assis and R.N. Feio. Specimens collected at “ Mata do Pai Inácio ”, PESB, Municipality of Miradouro, Minas Gerais State, Brazil (20°46'42" S, 42°29' W; 1340 m a.sl.): MZUFV 6658–60 (adults) collected on 5 December 2005 by R.N. Feio, E.F. Oliveira and J.S. Dayrell. MZUFV 2897 (adult) collected on 5 September 1996 by R. Harvey. Diagnosis. The new species is characterized by the (1) presence of black connective tissue covering all dorsal muscles; (2) body completely yellow-orange in life; (3) presence of skull and post-cranial plates; (4) presence of paravertebral plates; (5) lack of ossified warts; (6) parotic plates laterally projected; (7) convex-shaped paravertebral plates with rounded edges; (8) dorsal shield not projected up over the vertebral spine; (9) first spinal plate with trapezium shape; (10) second spinal plate with rectangular shape; (11) large size (SVL of adults: 14.8– 18.5 mm); (12) bufoniform body; (13) rounded snout; (14) absence of metacarpal and metatarsal tubercles; (15) absence of maxillae teeth; (16) large head; (17) presence of harmonic structures in the advertisement call. Comparison with other species. Brachycephalus darkside sp. nov. differs from the remaining species of the genus by having black connective tissue (which can be seen as background coloration through the skin of specimens), covering all sides of dorsal muscles (Fig. 4). There is no pigmentation in B. ephippium (Fig. 5), B. izecksohni, B. pernix, and B. pitanga; few scattered areas with pigmentation in the dorsolateral region adjacent to the dorsal musculature in some individuals of B. garbeanus and B. margaritatus; and there is little dark pigmentation internally around the line of the spinal vertebrae in B. vertebralis. The skin of Brachycephalus darkside sp. nov. is orange in life in all parts of the body, which distinguishes it from all other species of the genus with colored stripes and/or spots and/or blotches (as B. auroguttatus, B. boticario, B. crispus, B. ferruginus, B. fuscolineatus, B. guarani, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. pernix, B. pitanga, B. pombali), background greenish (as B. albolineatus, B. olivaceus, B. toby, B. verrucosus, B. tridactylus), or brownish (as B. brunneus, B. didactylus, B. nodoterga, B. pulex, B. quiririensis, B. sulfuratus). In preservative, specimens of the new species differ from those of B. alipioi, B. atelopoide, B. bufonoides, B. ephippium, B. garbeanus and B. margaritatus by the darkish background dorsum (pale cream in those species; see Fig. 1 and 5). The presence of skull and post-cranial plates of Brachycephalus darkside sp. nov. distinguishes it from B. albolineatus, B. auroguttatus, B. boticario, B. brunneus, B. didactylus, B. ferruginus, B. fuscolineatus, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. olivaceus, B. pernix, B. pombali, B. pulex, B. quiririensis, B. sulfuratus, B. tridactylus and B.verrucosus (absent in the referred species). The new species differs from the remaining species by the presence of paravertebral plates, except B. ephippium, B. garbeanus and B. margaritatus (paravertebral plates present in those species). The lack of osteoderms distinguishes B. darkside sp. nov. from B. atelopoide, B. crispus and B. margaritatus (present in those species). The presence of paravertebral plates in Brachycephalus darkside sp. nov. resembles the ones observed in B. ephippium, B. garbeanus and B. margaritatus. However, the new species differs from B. ephippium by the welldeveloped parotic plates, laterally projected (not projected in B. ephippium), and by the convex paravertebral plates (flat in B. ephippium). Brachycephalus darkside sp. nov. differs from B. garbeanus by having the dorsal shield not projected over the vertebral spine (projected over the borders of the vertebral spine in B. garbeanus), and by the convex central shape and rounded edges of paravertebral plates (paravertebral plates flat with square edges in B. garbeanus). It is distinguished from B. margaritatus by the first and second spinal plates with trapezium and rectangular shape, respectively (both triangular in B. margaritatus) and by the dorsal plate with rounded edges (nearly square and curved down in B. margaritatus). The new species differs by its larger SVL (14.8 – 18.5 mm) from Brachycephalus albolineatus, B. auroguttatus, B. boticario, B. brunneus, B. didactylus, B. fuscolineatus, B. guarani, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. nodoterga, B. olivaceus, B. pitanga, B. pulex, B. quiririensis and B. verrucosus (combined SVL in those species: 7.0–14.0 mm). Brachycephalus darkside sp. nov. differs from B. didactylus, B. hermogenesi and B. pulex by the bufoniform body and snout rounded in dorsal view (body leptodactyliform and snout pointed in those species). The absence of metacarpal and metatarsal tubercles distinguish the new species from Brachycephalus hermogenesi (metacarpal and out metatarsal present in this species), B. auroguttatus, B. boticario, B. brunneus, B. fuscolineatus, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. olivaceus, B. pitanga, B. pulex, B. quiririensis, B. toby and B. verrucosus (outer metatarsal present in those species). Brahycephalus darkside sp. nov. differs from B. brunneus, B. bufonoides, B. ferruginus , B. izecksohni and B. pombali by the absence of maxillae teeth (present in those species). The large head (wider than long) distinguishes B. darkside sp. nov. from B. ephippium and B. garbeanus (head longer than wide in those species). The advertisement call of Brachycephalus darkside sp. nov. distinguishes it from all its congeneric species by the presence of harmonic structures (absent in the other species of Brachycephalus; see Table 3 for complete comparison). It is distinguished from B. tridactylu s by the pulsed notes (not pulsed in this species), from B. pernix by having five to eight pulses per note (three in B. pernix), and from B. pitanga by having less pulses per note (mean of 11.1± 1.2 pulses per note in B. pitanga). It is distinguished from B. crispus, B. hermogenesi and B. pitanga by the shorter notes (ND = 83–163 ms in B. darkside sp. nov.; ND = 280 ± 20 ms in B. crispus; ND = 200 ms in B. hermogenesi; ND = 170 ± 13 ms in B. pitanga) and by the higher note rate (NR = 186.4–243.4 notes/s in B. darkside sp. nov.; NR = 1.67 ± 0.09 notes/s in B. crispus; NR = 1.09 notes/s in B. hermogenesi; NR = 2.65 ± 0.18 notes/s in B. pitanga). Brachycephalus darkside sp. nov. has the lowest peak frequency within the genus (PF = 2.8–3.8 kHz in B. darkside sp. nov.; combined dominant frequency in the other species = 4.6–6.8 in other species; Condez et al. 2014, Verdade et al. 2008, Araújo et al. 2012 and Garey et al. 2012). Description of holotype. Body robust and bufoniform; head wider than long, 32.9% (HL/SVL) and 34.4% (HW/SVL) (Fig. 1 A); snout short, rounded in dorsal and lateral views; nostrils slit-shaped, slightly protuberant, anterolaterally directed; canthus rostralis indistinct; loreal region weakly concave; lips sigmoid; eyes slightly protruding, anterolaterally directed, 8.7% (ED/HW) and 30% (ED/HL); tympanum indistinct; vocal sac not externally expanded; vocal slit presents; tongue longer than wide, posterior half not adhered to the mouth floor; choanae small and round; vomerine odontophores absent. Arm and forearm relatively slender; arm slightly shorter than forearm (AL 22.3% and FAL 22.3% of SVL); forearm slightly hypertrophied; hands shorter than arm. All fingers are distinct; fingers II and III robust; I and IV very small, vestigial; tip of finger I rounded and the others pointed; relative lengths of fingers IV Skin on head and dorsum rough, due to surface ornamentation of the skull and post-cranial plates; posterior region of the body with few scattered warts; skin of the body granular dorsolaterally and around cloacal opening; skin on belly and limbs smooth (Figs. 1 and 7). Measurements of holotype (in mm). SVL 17.9, HL 5.9, HW 6.2, ED 1.8, IOD 2.2, END 1.0, ND 0.4, IND 2.2, UAL 4.0, FAL 4.3, HAL 3.3, THL 7.1, SHL 6.7, TAL 4.1, FL 5.6. Osteology. (Figure 2) The cleared and double stained specimens revealed a well-developed parotic plate and ornamented dermal roofing bones in the skull of Brachycephalus darkside sp. nov. Dorsomedially, the parotic plate articulates with the frontoparietal. Laterally, the parotic plate is expanded, making the posterior region of head wide and, consequently, the head wider than long. Nasal, sphenethmoid, frontoparietals, prootics, and exoccipital fused, forming a dorsal cranial plate. Premaxillae broad, not fused medially; pars dentalis present, but odontoids absent. Alary process of premaxillae distinct, narrowly separated from the nasal and widely separated from each other, with length approximately twice of height. In ventral view, maxillae arched, odontoids absent. Quadratojugal absent. Pterygoid slender, anterior ramus long, articulating with maxillary arch; posterior ramus short, articulating with ventral ramus of squamosal; medial ramus short, articulating with the prootic. Dentigerous process of vomer absent, prechoanal and postchoanal ramus reduced but distinct. Palatine absent. Parasphenoid robust. Squamosal broad in lateral view; anterior zygmatic ramus extremely reduced, not ornamented; posterior otic ramus short, allocated below the parotic plate and may or not present ornamentations on the anterior portion not covered by the parotic plate. Anterior zygmatic ramus short and not ornamented. Tympanic annulus absent. Mandible edentate. Pectoral girdle arciferal and robust (Fig. 3 C). Procoracoid and epicoracoid fused and completely ossified. Procoracoid and epicoracoid synostotically united with clavicle, coracoid, and scapula. Suprascapula expanded, with anterior half ossified as cleithrum. Omosternum and sternum absent. Ventral column composed of eight nonimbricate vertebrae and hyperossified spinal process of vertebrae. Atlas (first pressacral vertebra) lacks transverse process. Presence of paravertebral and spinal plates well developed and ornamented. Spinal plates associated with the spinal process of all presacral and sacral vertebrae. First spinal plate (vertebra II) broad; trapezium convex-shaped; second spinal plate (vertebra III) thin, almost rectangle convex-shaped and nearly joint with principal group of dorsal plates. Dorsally, the paravertebral plate completely conceals the transverse process of vertebra IV-VII, and partially covers the posterior tip of transverse process of vertebra III. Lateral edge of the paravertebral plates almost rounded; anterior and posterior corners reach the level of transverse process of vertebra II and almost the sacral diapophysis, respectively. Paravertebral plates fused medially to the block consisting of the fusion of spinal plates IV–VII (VIII in some individuals). Ventrally, transverse process of vertebrae IV and V fused to the paravertebral plates. Dorsally, the central of each paravertebral plate has an elevation that provides a convex shape. Parotic, cranial, spinal and paravertebral plates ornamented with dermal roofing bones. Forearm slightly shorter than humerus. Radius and ulna fused and distinguishable. Manus with distal carpals (I–V) fused, with centrale, radiale and ulnare about the same size. Prepollex very reduced, with one element. Phalangeal formula 1-2-3-1 (Fig. 3 A). Tips of the terminal phalangeal elements of fingers arrow-shaped. Tibia and fibula fused, distinguishable, forming tibiafibula. Tibiafibula and femur of approximately the same length. Tibiale and fibulare fused at their distal and proximal ends, not fused medially. Hindlimbs with tarsal elements I, II, III present, and IV–V absent; centrale present. One very-reduced prehallical element. Phalangeal formula 1-2-3-4-1 (Fig. 3 B). Tips of terminal phalangeal elements of toes II, III and IV arrow shaped. Toes I and V reduced with terminal phalangeal element rounded in shape. Histology. Histological analysis showed a connective tissue covering the dorsal musculature (epimysium), and between muscle fibers (perimysium and endomysium) (Fig. 4 A-D). This tissue presented spots of extracellular matrix containing dark pigments, which were reflected in the macroscopy of fixed specimens as well (Fig. 1 A). Coloration of Holotype. In life, body bright yellow-orange. Venter yellow-orange, slightly pale compared to remaining of body. Dark background at central region of dorsum, from posterior region of skull to inguinal region, beneath the yellow-orange skin. Dorsal plates slightly dark. Eyes completely black (Fig. 7). Coloration in preservative. General body color cream. Dark background beneath the cream skin from central region of dorsum to inguinal region, including posterior region of skull. Ossified regions such as skull and dorsal plates grayish. Eyes completely black (Fig. 1). Variation. Measurements of adults and juveniles are given in Table 1. The density of warts along the posterior region of the body and granular regions vary between individuals (i.e., in some specimens the body is entirely smooth). Skull and post-cranial plates varies ontogenetically; juveniles may lack hyperossification or present skull and post-cranial skeleton less ornamented than adults, as previously described for Brachycephalus ephippium (Campos et al. 2010). Among adult individuals the paravertebral plates are proportionally different in size; they can be bigger or smaller in individuals of same size. The second spinal plate may contact spinal plates fused with paravertebral plates. The spinal plate of presacral VIII and sacral vertebra may be separated from the dorsal shield. The top of the squamosal may present ornamented dermal ossification. Some specimens presented 1/3 of tong adhered to floor of mouth, and oval choanae. Call description. We observed two distinct calls of Brachycephalus darkside sp. nov. herein referred as the advertisement call and an aggressive call (sensu Toledo et al. 2015). The advertisement call is the most common type of call (76% of all calls, n = 790 notes; Fig. 6 A, Table 2) and is characterized by pulsed notes emitted in extremely long sequences. The longest call recorded had more 250 notes and was not fully recorded (NN = 114 ± 97.1, 9–253 notes per call, n = 7; CD = 30.4 ± 25.3, 2.9– 66.2 s, n = 7 sequences). We were only able to record two intervals between calls, which ranged from 6.2 to 11.2 s, hampering the calculation of call rate. Each note had six pulses on average (PN = 6.3 ± 0.7, 5–8 pulses, n = 790 notes) emitted at constant rate (PR = 56.9 ± 4.9, 36.8–78.4 pulses/s). The first pulse is often the most energetic one (Fig. 6 A), followed by a decrease in amplitude in the second pulse and a new rise in the next one or two pulses, then the amplitude fades until the last pulse. Note duration was relatively stereotyped (ND = 111.5 ± 13.7, 83–163 ms, n = 790 notes), and so was the interval between notes (NI = 159.5 ± 14.5, 122–215 ms, n = 783 intervals), and the note rate (NR = 211.4 ± 25.6, 186.4– 243.4 notes/min, n = 5 recordings). Dominant frequency (DF) ranged from 2484.4 to 5765.6 Hz with peak of energy around 3.3 kHz (PF = 3382.1 ± 184.6, 2856.4–3796.9 Hz). Up to three harmonics could be observed in the advertisement call, with only two of them visible in all recordings. A second type of call was recorded whilst a male Brachycephalus darkside sp. nov. vocalized within 20 cm of a conspecific male in an aggressive social context (sensu Toledo et al. 2015). The aggressive call (23%, n = 245 calls; Fig. 6 B, Table 2) is also characterized by pulsed notes emitted in sequences shorter than the ones from the advertisement call (CD = 4.1 ± 1.3, 2.4– 6.9 s, n = 10 calls), with variable number of notes (NN = 24.5 ± 7.9, 15–41 notes/call, n = 10 calls) and emitted about eight times per minute (CR = 8.46 calls/min, n = 1) with brief intervals (CI = 2.8 ± 0.7, 2.0– 3.8 s, n = 9 intervals). Each note was shorter than the ones from the advertisement call (ND = 31.1 ± 5.7, 18–44 ms, n = 245 notes), emitted at higher rate (NR = 356.6 ± 6.5, 343.7–364.2 notes/min, n = 10 calls) and va

Brachycephalus darkside sp. nov. Figures 1���4 and 7. Brachycephalus ephippium ���Pombal Jr. 2001 (part.) Brachycephalus ephippium ��� Dayrell et al. 2006 Brachycephalus ephippium ���Pombal & Izecksohn 2011 (part.) Brachycephalus ephippium ��� Moura et al. 2012. Holotype. MZUFV 16636, adult male (17.9 mm SVL, Figure 1), collected at ���Trilha do Cruzeiro��� (20��52'40.7"S, 0 42��31'14.6"W; 1266 m a.s.l.), Parque Estadual da Serra do Brigadeiro (PESB), district of Care��o, Municipality of Erv��lia, Minas Gerais State, Brazil, on 17 December 2015 by C.S. Guimar��es, P.C. Rocha, C.L. Assis, R.N. Feio, and C. Novaes. Paratypes. Specimens collected at type locality: MZUFV 16634���35 (males) collected with holotype. MZUFV 16627, 16632���33 (males), 16628, 16631 (females), MNRJ 91327 and UFMG 19522 (males) collected on 20 November 2015 by C.S. Guimar��es, P.C. Rocha and R.N. Feio. MZUFV 16491, 16579 (males), and 16780 (female) collected on 13 August 2015 by C.S. Guimar��es, P.S. Hote and S. Luz. MZUFV 15716 (male), 15717, 15720���21 (adult specimens), and 15718���19 (juveniles) collected on 4 December 2014 by C.S. Guimar��es, P.G. Taucce and B. Lisboa. MZUFV 15557���58, 15560���61, 15566���15571, 15717 (adult specimens), and 15559, 15565 (males), collected on 18 September 2014 by C.S. Guimar��es, C.L. Assis and R.N. Feio. Specimens collected at ��� Mata do Pai In��cio ���, PESB, Municipality of Miradouro, Minas Gerais State, Brazil (20��46'42" S, 42��29' W; 1340 m a.sl.): MZUFV 6658���60 (adults) collected on 5 December 2005 by R.N. Feio, E.F. Oliveira and J.S. Dayrell. MZUFV 2897 (adult) collected on 5 September 1996 by R. Harvey. Diagnosis. The new species is characterized by the (1) presence of black connective tissue covering all dorsal muscles; (2) body completely yellow-orange in life; (3) presence of skull and post-cranial plates; (4) presence of paravertebral plates; (5) lack of ossified warts; (6) parotic plates laterally projected; (7) convex-shaped paravertebral plates with rounded edges; (8) dorsal shield not projected up over the vertebral spine; (9) first spinal plate with trapezium shape; (10) second spinal plate with rectangular shape; (11) large size (SVL of adults: 14.8��� 18.5 mm); (12) bufoniform body; (13) rounded snout; (14) absence of metacarpal and metatarsal tubercles; (15) absence of maxillae teeth; (16) large head; (17) presence of harmonic structures in the advertisement call. Comparison with other species. Brachycephalus darkside sp. nov. differs from the remaining species of the genus by having black connective tissue (which can be seen as background coloration through the skin of specimens), covering all sides of dorsal muscles (Fig. 4). There is no pigmentation in B. ephippium (Fig. 5), B. izecksohni, B. pernix, and B. pitanga; few scattered areas with pigmentation in the dorsolateral region adjacent to the dorsal musculature in some individuals of B. garbeanus and B. margaritatus; and there is little dark pigmentation internally around the line of the spinal vertebrae in B. vertebralis. The skin of Brachycephalus darkside sp. nov. is orange in life in all parts of the body, which distinguishes it from all other species of the genus with colored stripes and/or spots and/or blotches (as B. auroguttatus, B. boticario, B. crispus, B. ferruginus, B. fuscolineatus, B. guarani, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. pernix, B. pitanga, B. pombali), background greenish (as B. albolineatus, B. olivaceus, B. toby, B. verrucosus, B. tridactylus), or brownish (as B. brunneus, B. didactylus, B. nodoterga, B. pulex, B. quiririensis, B. sulfuratus). In preservative, specimens of the new species differ from those of B. alipioi, B. atelopoide, B. bufonoides, B. ephippium, B. garbeanus and B. margaritatus by the darkish background dorsum (pale cream in those species; see Fig. 1 and 5). The presence of skull and post-cranial plates of Brachycephalus darkside sp. nov. distinguishes it from B. albolineatus, B. auroguttatus, B. boticario, B. brunneus, B. didactylus, B. ferruginus, B. fuscolineatus, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. olivaceus, B. pernix, B. pombali, B. pulex, B. quiririensis, B. sulfuratus, B. tridactylus and B.verrucosus (absent in the referred species). The new species differs from the remaining species by the presence of paravertebral plates, except B. ephippium, B. garbeanus and B. margaritatus (paravertebral plates present in those species). The lack of osteoderms distinguishes B. darkside sp. nov. from B. atelopoide, B. crispus and B. margaritatus (present in those species). The presence of paravertebral plates in Brachycephalus darkside sp. nov. resembles the ones observed in B. ephippium, B. garbeanus and B. margaritatus. However, the new species differs from B. ephippium by the welldeveloped parotic plates, laterally projected (not projected in B. ephippium), and by the convex paravertebral plates (flat in B. ephippium). Brachycephalus darkside sp. nov. differs from B. garbeanus by having the dorsal shield not projected over the vertebral spine (projected over the borders of the vertebral spine in B. garbeanus), and by the convex central shape and rounded edges of paravertebral plates (paravertebral plates flat with square edges in B. garbeanus). It is distinguished from B. margaritatus by the first and second spinal plates with trapezium and rectangular shape, respectively (both triangular in B. margaritatus) and by the dorsal plate with rounded edges (nearly square and curved down in B. margaritatus). The new species differs by its larger SVL (14.8 ��� 18.5 mm) from Brachycephalus albolineatus, B. auroguttatus, B. boticario, B. brunneus, B. didactylus, B. fuscolineatus, B. guarani, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. nodoterga, B. olivaceus, B. pitanga, B. pulex, B. quiririensis and B. verrucosus (combined SVL in those species: 7.0���14.0 mm). Brachycephalus darkside sp. nov. differs from B. didactylus, B. hermogenesi and B. pulex by the bufoniform body and snout rounded in dorsal view (body leptodactyliform and snout pointed in those species). The absence of metacarpal and metatarsal tubercles distinguish the new species from Brachycephalus hermogenesi (metacarpal and out metatarsal present in this species), B. auroguttatus, B. boticario, B. brunneus, B. fuscolineatus, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaeterezae, B. olivaceus, B. pitanga, B. pulex, B. quiririensis, B. toby and B. verrucosus (outer metatarsal present in those species). Brahycephalus darkside sp. nov. differs from B. brunneus, B. bufonoides, B. ferruginus , B. izecksohni and B. pombali by the absence of maxillae teeth (present in those species). The large head (wider than long) distinguishes B. darkside sp. nov. from B. ephippium and B. garbeanus (head longer than wide in those species). The advertisement call of Brachycephalus darkside sp. nov. distinguishes it from all its congeneric species by the presence of harmonic structures (absent in the other species of Brachycephalus; see Table 3 for complete comparison). It is distinguished from B. tridactylu s by the pulsed notes (not pulsed in this species), from B. pernix by having five to eight pulses per note (three in B. pernix), and from B. pitanga by having less pulses per note (mean of 11.1�� 1.2 pulses per note in B. pitanga). It is distinguished from B. crispus, B. hermogenesi and B. pitanga by the shorter notes (ND = 83���163 ms in B. darkside sp. nov.; ND = 280 �� 20 ms in B. crispus; ND = 200 ms in B. hermogenesi; ND = 170 �� 13 ms in B. pitanga) and by the higher note rate (NR = 186.4���243.4 notes/s in B. darkside sp. nov.; NR = 1.67 �� 0.09 notes/s in B. crispus; NR = 1.09 notes/s in B. hermogenesi; NR = 2.65 �� 0.18 notes/s in B. pitanga). Brachycephalus darkside sp. nov. has the lowest peak frequency within the genus (PF = 2.8���3.8 kHz in B. darkside sp. nov.; combined dominant frequency in the other species = 4.6���6.8 in other species; Condez et al. 2014, Verdade et al. 2008, Ara��jo et al. 2012 and Garey et al. 2012). Description of holotype. Body robust and bufoniform; head wider than long, 32.9% (HL/SVL) and 34.4% (HW/SVL) (Fig. 1 A); snout short, rounded in dorsal and lateral views; nostrils slit-shaped, slightly protuberant, anterolaterally directed; canthus rostralis indistinct; loreal region weakly concave; lips sigmoid; eyes slightly protruding, anterolaterally directed, 8.7% (ED/HW) and 30% (ED/HL); tympanum indistinct; vocal sac not externally expanded; vocal slit presents; tongue longer than wide, posterior half not adhered to the mouth floor; choanae small and round; vomerine odontophores absent. Arm and forearm relatively slender; arm slightly shorter than forearm (AL 22.3% and FAL 22.3% of SVL); forearm slightly hypertrophied; hands shorter than arm. All fingers are distinct; fingers II and III robust; I and IV very small, vestigial; tip of finger I rounded and the others pointed; relative lengths of fingers IV Measurements of holotype (in mm). SVL 17.9, HL 5.9, HW 6.2, ED 1.8, IOD 2.2, END 1.0, ND 0.4, IND 2.2, UAL 4.0, FAL 4.3, HAL 3.3, THL 7.1, SHL 6.7, TAL 4.1, FL 5.6. Osteology. (Figure 2) The cleared and double stained specimens revealed a well-developed parotic plate and ornamented dermal roofing bones in the skull of Brachycephalus darkside sp. nov. Dorsomedially, the parotic plate articulates with the frontoparietal. Laterally, the parotic plate is expanded, making the posterior region of head wide and, consequently, the head wider than long. Nasal, sphenethmoid, frontoparietals, prootics, and exoccipital fused, forming a dorsal cranial plate. Premaxillae broad, not fused medially; pars dentalis present, but odontoids absent. Alary process of premaxillae distinct, narrowly separated from the nasal and widely separated from each other, with length approximately twice of height. In ventral view, maxillae arched, odontoids absent. Quadratojugal absent. Pterygoid slender, anterior ramus long, articulating with maxillary arch; posterior ramus short, articulating with ventral ramus of squamosal; medial ramus short, articulating with the prootic. Dentigerous process of vomer absent, prechoanal and postchoanal ramus reduced but distinct. Palatine absent. Parasphenoid robust. Squamosal broad in lateral view; anterior zygmatic ramus extremely reduced, not ornamented; posterior otic ramus short, allocated below the parotic plate and may or not present ornamentations on the anterior portion not covered by the parotic plate. Anterior zygmatic ramus short and not ornamented. Tympanic annulus absent. Mandible edentate. Pectoral girdle arciferal and robust (Fig. 3 C). Procoracoid and epicoracoid fused and completely ossified. Procoracoid and epicoracoid synostotically united with clavicle, coracoid, and scapula. Suprascapula expanded, with anterior half ossified as cleithrum. Omosternum and sternum absent. Ventral column composed of eight nonimbricate vertebrae and hyperossified spinal process of vertebrae. Atlas (first pressacral vertebra) lacks transverse process. Presence of paravertebral and spinal plates well developed and ornamented. Spinal plates associated with the spinal process of all presacral and sacral vertebrae. First spinal plate (vertebra II) broad; trapezium convex-shaped; second spinal plate (vertebra III) thin, almost rectangle convex-shaped and nearly joint with principal group of dorsal plates. Dorsally, the paravertebral plate completely conceals the transverse process of vertebra IV-VII, and partially covers the posterior tip of transverse process of vertebra III. Lateral edge of the paravertebral plates almost rounded; anterior and posterior corners reach the level of transverse process of vertebra II and almost the sacral diapophysis, respectively. Paravertebral plates fused medially to the block consisting of the fusion of spinal plates IV���VII (VIII in some individuals). Ventrally, transverse process of vertebrae IV and V fused to the paravertebral plates. Dorsally, the central of each paravertebral plate has an elevation that provides a convex shape. Parotic, cranial, spinal and paravertebral plates ornamented with dermal roofing bones. Forearm slightly shorter than humerus. Radius and ulna fused and distinguishable. Manus with distal carpals (I���V) fused, with centrale, radiale and ulnare about the same size. Prepollex very reduced, with one element. Phalangeal formula 1-2-3-1 (Fig. 3 A). Tips of the terminal phalangeal elements of fingers arrow-shaped. Tibia and fibula fused, distinguishable, forming tibiafibula. Tibiafibula and femur of approximately the same length. Tibiale and fibulare fused at their distal and proximal ends, not fused medially. Hindlimbs with tarsal elements I, II, III present, and IV���V absent; centrale present. One very-reduced prehallical element. Phalangeal formula 1-2-3-4-1 (Fig. 3 B). Tips of terminal phalangeal elements of toes II, III and IV arrow shaped. Toes I and V reduced with terminal phalangeal element rounded in shape. Histology. Histological analysis showed a connective tissue covering the dorsal musculature (epimysium), and between muscle fibers (perimysium and endomysium) (Fig. 4 A-D). This tissue presented spots of extracellular matrix containing dark pigments, which were reflected in the macroscopy of fixed specimens as well (Fig. 1 A). Coloration of Holotype. In life, body bright yellow-orange. Venter yellow-orange, slightly pale compared to remaining of body. Dark background at central region of dorsum, from posterior region of skull to inguinal region, beneath the yellow-orange skin. Dorsal plates slightly dark. Eyes completely black (Fig. 7). Coloration in preservative. General body color cream. Dark background beneath the cream skin from central region of dorsum to inguinal region, including posterior region of skull. Ossified regions such as skull and dorsal plates grayish. Eyes completely black (Fig. 1). Variation. Measurements of adults and juveniles are given in Table 1. The density of warts along the posterior region of the body and granular regions vary between individuals (i.e., in some specimens the body is entirely smooth). Skull and post-cranial plates varies ontogenetically; juveniles may lack hyperossification or present skull and post-cranial skeleton less ornamented than adults, as previously described for Brachycephalus ephippium (Campos et al. 2010). Among adult individuals the paravertebral plates are proportionally different in size; they can be bigger or smaller in individuals of same size. The second spinal plate may contact spinal plates fused with paravertebral plates. The spinal plate of presacral VIII and sacral vertebra may be separated from the dorsal shield. The top of the squamosal may present ornamented dermal ossification. Some specimens presented 1/3 of tong adhered to floor of mouth, and oval choanae. Call description. We observed two distinct calls of Brachycephalus darkside sp. nov. herein referred as the advertisement call and an aggressive call (sensu Toledo et al. 2015). The advertisement call is the most common type of call (76% of all calls, n = 790 notes; Fig. 6 A, Table 2) and is characterized by pulsed notes emitted in extremely long sequences. The longest call recorded had more 250 notes and was not fully recorded (NN = 114 �� 97.1, 9���253 notes per call, n = 7; CD = 30.4 �� 25.3, 2.9��� 66.2 s, n = 7 sequences). We were only able to record two intervals between calls, which ranged from 6.2 to 11.2 s, hampering the calculation of call rate. Each note had six pulses on average (PN = 6.3 �� 0.7, 5���8 pulses, n = 790 notes) emitted at constant rate (PR = 56.9 �� 4.9, 36.8���78.4 pulses/s). The first pulse is often the most energetic one (Fig. 6 A), followed by a decrease in amplitude in the second pulse and a new rise in the next one or two pulses, then the amplitude fades until the last pulse. Note duration was relatively stereotyped (ND = 111.5 �� 13.7, 83���163 ms, n = 790 notes), and so was the interval between notes (NI = 159.5 �� 14.5, 122���215 ms, n = 783 intervals), and the note rate (NR = 211.4 �� 25.6, 186.4��� 243.4 notes/min, n = 5 recordings). Dominant frequency (DF) ranged from 2484.4 to 5765.6 Hz with peak of energy around 3.3 kHz (PF = 3382.1 �� 184.6, 2856.4���3796.9 Hz). Up to three harmonics could be observed in the advertisement call, with only two of them visible in all recordings. A second type of call was recorded whilst a male Brachycephalus darkside sp. nov. vocalized within 20 cm of a conspecific male in an aggressive social context (sensu Toledo et al. 2015). The aggressive call (23%, n = 245 calls; Fig. 6 B, Table 2) is also characterized by pulsed notes emitted in sequences shorter than the ones from the advertisement call (CD = 4.1 �� 1.3, 2.4��� 6.9 s, n = 10 calls), with variable number of notes (NN = 24.5 �� 7.9, 15���41 notes/call, n = 10 calls) and emitted about eight times per minute (CR = 8.46 calls/min, n = 1) with brief intervals (CI = 2.8 �� 0.7, 2.0��� 3.8 s, n = 9 intervals). Each note was shorter than the ones from the advertisement call (ND = 31.1 �� 5.7, 18���44 ms, n = 245 notes), emitted at higher rate (NR = 356.6 �� 6.5, 343.7���364.2 notes/min, n = 10 calls) and va, Published as part of Guimar��es, Carla Silva, Luz, Sofia, Rocha, Pedro Carvalho & Feio, Renato Neves, 2017, The dark side of pumpkin toadlet: a new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Brigadeiro, southeastern Brazil, pp. 327-344 in Zootaxa 4258 (4) on pages 329-341, DOI: 10.11646/zootaxa.4258.4.2, http://zenodo.org/record/570094, {"references":["Dayrell, J. S., Oliveira, E. F., Cassini, C. S. & Feio, R. N. (2006) Brachycephalus ephippium (Pumpkin Toadlet): geographic distribution. Herpetological Review, 37, 357.","Moura, M. R., Motta, A. P., Fernandes, V. D. & Feio, R. N. (2012) Herpetofauna da Serra do Brigadeiro, um remanescente de Mata Atlantica em Minas Gerais, Brasil. Biota Neotrop, 12 (1), 209 - 235.","Condez, T. H., Clemente-Carvalho, R. B. G., Haddad, C. F. B. & dos Reis, S. F. (2014) A new species of Brachycephalus (Anura: Brachycephalidae) from the highlands of the Atlantic Forest, Southeastern Brazil. Herpetologica, 70, 89 - 99. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 13 - 00044","Verdade, K. V., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. C. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta and Sawaya, 1998) (Anura, Brachycephalidae). Journal of Herpetology, 42, 542 - 549.","Araujo, C. B., Guerra, T. J., Amatuzzi, M. C. O. & Campo, L. A. (2012) Advertisement and territorial calls of Brachycephalus pitanga (Anura: Brachycephalidae). Zootaxa, 3302, 66 - 67.","Garey, M. V., Lima, A. M. X., Hartmann, M. T. & Haddad, C. F. B. (2012) A new species of miniaturized toadlet genus Brachycephalus (Anura: Brachycephalidae), from southern Brazil. Herpetologica, 68, 266 - 271. https: // doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00074.1","Campos, L. A., da Silva, H. R. & Sebben, A. (2010) Morphology and development of additional bony elements in the genus Brachycephalus (Anura: Brachycephalidae). Biological Journal of the Linnean Society, 99, 752 - 767. https: // doi. org / 10.1111 / j. 1095 - 8312.2010.01375. x","Toledo, L. F., Martins, I. A., Bruschi, D. P., Passos, M. A., Alexandre, C. & Haddad, C. F. (2015) The anuran calling repertoire in the light of social context. Acta Ethologica, 18, 87 - 89.","Clemente-Carvalho, R. B. G., Klaczko, J., Perez, S. I., Alves, A. C. R., Haddad, C. F. B. & dos Reis, S. F. (2011) Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae). Molecular Phylogenetics and Evolution, 61, 79 - 89.","Bornschein, M. R., Firkowski, C. R., Belmonte-Lopes, L. C., Ribeiro, L. F., Morato, S. A. A., Antoniazzi-Jr., R., Reinert, B. L., Meyer, A. L. S., Cini, F. A. & Pie, M. R. (2016 a) Geographical and altitudinal distribution of Brachycephalus (Anura: Brachycephalidae) endemic to the Brazilian Atlantic Rainforest. PeerJ, 4, e 2490,","Alves, A. C. R., Sawaya, R., Reis, S. F. & Haddad, C. B. F. (2009) A new species of Brachycephalus (Anura: Brachycephalidae) from the Atlantic rain forest in Sao Paulo state, southeastern Brazil. Journal of Herpetology, 43, 212 - 219. https: // doi. org / 10.1670 / 0022 - 1511 - 43.2.212","Clemente-Carvalho, R. B. G., Giaretta, A. A., Condez, T. H., Haddad, C. F. B. & dos Reis, S. F. (2012) A new species of miniaturized toadlet, genus Brachycephalus (Anura: Brachycephalidae), from the Atlantic Forest of southeastern Brazil. Herpetologica, 68, 365 - 374.","Haddad, C. F. B., Alves, A. C. R., Clemente-Carvalho, R. B. G. & Reis, S. F. (2010) A new species of Brachycephalus from Atlantic Rain Forest in Sao Paulo State, Southeastern Brazil (Amphibia: Anura: Brachycephalidae). Copeia, 2010, 410 - 420. https: // doi. org / 10.1643 / CH- 09 - 102","Pie, M. R. & Ribeiro, L. F. (2015) A new species of Brachycephalus (Anura: Brachycephalidae) from the Quiriri mountain range of southern Brazil. PeerJ, 3, e 1179."]}

Made available in DSpace on 2020-12-10T16:58:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundacao Grupo Boticario de Protecao a Natureza Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The advent of next-generation sequencing allows researchers to use large-scale datasets for species delimitation analyses, yet one can envision an inflection point where the added accuracy of including more loci does not offset the increased computational burden. One alternative to including all loci could be to prioritize the analysis of loci for which there is an expectation of high informativeness. Here, we explore the issue of species delimitation and locus selection with montane species from two anuran genera that have been isolated in sky islands across the southern Brazilian Atlantic Forest: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). To delimit species, we obtained genetic data using target enrichment of ultraconserved elements from 32 populations (13 for Melanophryniscus and 19 for Brachycephalus), and we were able to create datasets that included over 800 loci with no missing data. We ranked loci according to their number of parsimony-informative sites, and we performed species delimitation analyses using BPP with the most informative 10, 20, 40, 80, 160, 320, and 640 loci. We identified three types of phylogenetic node: nodes with either consistently high or low support regardless of the number of loci or their informativeness and nodes that were initially poorly supported where support became stronger as we included more data. When viewed across all sensitivity analyses, our results suggest that the current species richness in both genera is likely underestimated. In addition, our results show the effects of different sampling strategies on species delimitation using phylogenomic datasets. Univ Fed Parana, Dept Zool, BR-81531980 Curitiba, Parana, Brazil Mater Nat Inst Estudos Ambientais, BR-80250020 Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No,Parque Bitaru, BR-11330900 Sao Paulo, Brazil Pontificia Univ Catolica Parana, Escola Ciencias Vida, BR-80215901 Curitiba, Parana, Brazil Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA Louisiana State Univ, Museum Nat Sci, Baton Rouge, LA 70803 USA Occidental Coll, Moore Lab Zool, 1600 Campus Rd, Los Angeles, CA 90041 USA Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No,Parque Bitaru, BR-11330900 Sao Paulo, Brazil Fundacao Grupo Boticario de Protecao a Natureza: 0895_20111 Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014 CNPq: 301636/2016-8

Brachycephalus sulfuratus sp. nov. Figures 1���3 Holotype. CFBH 39137, adult female, collected at Centro de Estudos e Pesquisas Ambientais da Univille, Vila da Gl��ria, Distrito do Sa�� (26 �� 13'39"S, 48 �� 41'31"W, 123 m above sea level, Datum WGS 84), munic��pio de S��o Francisco do Sul, State of Santa Catarina, Brazil, on 14 November 2014, by T.H. Condez, J.P.C. Monteiro, and E.J. Comitti. Paratypes. CFBH 39138, cleared and double-stained adult male, and CFBH 39139, adult male, both collected with the hotolype. CFBH 39140, adult female collected on 11 September 2014, CFBH 39329, adult female, and CFBH 39331, adult male, collected on 31 July 2015, CFBH 39330 and 39332, adult females, collected on 0 1 August 2015, at the same locality as the holotype, by J.P.C. Monteiro and E.C. Nardin. CFBH 39142, adult male, CFBH 39141 and 39144, adult females, CFBH 39143, cleared and double-stained adult male, collected on 0 4 December 2013, by T.H. Condez, J.P.C. Monteiro and E.J. Comitti, UFMG 17954 and 17955, adult females, CFBH 39407���39410 and UFMG 17956 and 17957, adult males, CFBH 39406, juvenile, collected on 28 August 2015, by J.P.C. Monteiro and E.C. Nardin, at Morro do Cantagalo, Vila da Gl��ria, Distrito do Sa�� (26 �� 10'31"S, 48 �� 42'44"W, 161 m above sea level, Datum WGS 84), munic��pio de S��o Francisco do Sul, State of Santa Catarina, Brazil. CFBH 39145���39147, adult females, collected at Castelo dos Bugres, ��rea de Prote����o Ambiental Dona Francisca (26 �� 13'43"S, 49 �� 03'27"W, 835 m above sea level, Datum WGS 84), munic��pio de Joinville, State of Santa Catarina, Brazil, on 29 November 2013, by T.H. Condez, J.P.C. Monteiro, E.J. Comitti, I. Borel, P.D. Pinheiro, and P.C.A. Garcia. CFBH 39148 and 39149, adult males, collected at Parque Estadual da Ilha do Cardoso (25 �� 06'53"S, 47 �� 55'40"W, 385 m above sea level, Datum WGS 84), munic��pio de Canan��ia, State of S��o Paulo, on 19 December 2013, by T.H. Condez, L.N. Bandeira, and S. Pinheiro. CFBH 39150, adult female, collected at Morro do Anhangava (25 �� 22'51"S, 49 �� 01'26"W, 915 m above sea level, Datum WGS 84), munic��pio de Quatro Barras, State of Paran��, on 0 2 February 2012, by T.H. Condez, T.B. Rocha, and P.F. Colas-Rosas. Referred specimens. MZUSP 129855, juvenile, collected at Ilha do Cardoso (non-georeferenced data), munic��pio de Canan��ia, State of S��o Paulo, on 22 January 1979, by an unknown collector. ZUEC 16602, juvenile, collected at ��rea de Prote����o Ambiental de Guaratuba (25��47��� S, 48��54��� W, 291 m above sea level), munic��pio de S��o Jos�� dos Pinhais, State of Paran��, on 18 January 2008, by A.K. Cunha and I. Soares. Diagnosis. Brachycephalus sulfuratus sp. nov. is a new species of flea-toad, distinguished from all its congeners by the following combination of characters: (1) small body size (SVL of adults: 7.4���8.5 mm for males and 9.0��� 10.8 mm for females); (2) ���leptodactyliform��� body; (3) pectoral girdle arciferal and less robust compared to the Brachycephalus species with ���bufoniform��� body; (4) procoracoid and epicoracoid fused with coracoid but separated from the clavicle by a large fenestrae; (5) toe I externally absent; toes II, III, IV, and V distinct; phalanges of toes II and V reduced; (6) skin smooth with no dermal ossifications; (7) in life, general background color brown with small dark-brown spots; skin of throat, chest, arms, and forearms with irregular yellow blotches; in ventral view, cloacal region of alive and preserved specimens surrounded by a dark-brown inverted v-shaped mark outlined with white; (8) advertisement call long, composed of a set of 4���7 high-frequency notes (6.2���7.2 kHz) repeated regularly. Description of holotype. The holotype of Brachycephalus sulfuratus sp. nov. has a ���leptodactyliform��� body (Figure 1 and 2); head wider than long, narrower than body; head length approximately 26% of SVL; snout long, with length slightly short than the eye diameter, rounded in lateral and dorsal views (Figure 3A and 3B); nostrils protuberant, directed anterolaterally; canthus rostralis distinct and straight; loreal region weakly concave; mouth nearly sigmoid; eye slightly protruding in dorsal and lateral views, eye diameter 48% of HL; tympanum absent; tongue longer than wide, posterior half not adherent to floor of mouth; choanae relatively small and round; vomerine odontophores absent. Upper arm slightly shorter than forearm; length of upper arm plus forearm 43% of SVL; hands approximately as long as upper arm; fingers II and III thin and distinct; finger I and IV very small, vestigial; tip of fingers II and III slightly pointed; relative lengths of fingers II Measurements of holotype (in mm). SVL 9.8; HL 2.5; HW 2.7; ND 0.4; IND 1.0; ED 1.2; IOD 1.9; END 0.8; THL 4.3; TBL 4.1; FL 6.3; AL 2.0; FAL 2.3; HAL 1.8. Color of holotype in life. Dorsal coloration grey to brown covered with dark brown and red spots (Figure 1A). The dark brown spots are concentrated on the head and medial portion of the dorsum, in which a large irregular mark is distinguishable. The interorbital area exhibits a dark brown nearly v-shaped mark. Dorsal surfaces of arms and legs exhibit dark brown blotches, which on the thigh and tibia resemble incomplete stripes. Above the cloacal region, in dorsal view, there is a dark brown inverted m-shaped mark. A dark brown stripe extends laterally from the tip of the snout to the flanks and the surface of thigh (Figure 1A). In lateral view, the tip of the snout and posterior regions of maxilla and ocular globe present undefined yellow spots. The maxilla is dark brown with distinct white spots. Ventral surface of body pale-brown, slightly transparent, with small dark brown spots and white blotches (Figure 1B). Some yellow blotches are also spread among the gular region, arms, forearms, and disposed in an inverted v-shape on the chest. In ventral view, the cloacal region is surrounded by an inverted vshaped dark brown mark with white borders. The pupil is black and the iris is golden. Color of holotype in preservative. General background color is pale-brown covered with small dark brown dots (Figure 2); dorsum with dark brown marks on the ocular region, knees, and cloacal region; dark brown stripes on dorsal surfaces of thigh, tibia, and foot; ventral surfaces of hands and feet with dark brown blotches; in ventral view, cloacal region surrounded by an inverted v-shaped dark-brown mark. Osteology. The cleared and double-stained material revealed the following characters in Brachycephalus sulfuratus sp. nov. No hyperossification of the skull or skeleton. Nasals, sphenethmoid, frontoparietals, prootics, and exoccipitals fused. Premaxillae broad, not fused medially; odontoids absent; alary process of premaxillae distinct and separated from the nasal. Maxillae arched in ventral view; odontoids present. Quadratojugal and pterygoid present. Vomer not fused medially, vomerine odontophores absent. Palatine absent. Parasphenoid and sphenethmoid fused and robust. Squamosal elongated in lateral view, zygomatic ramus short and not ornamented. Tympanic annulus absent. Mandible edentate. Pectoral girdle arciferal and less robust than in other Brachycephalus; clavicle, coracoid, and scapula fused and completely ossified; procoracoid and epicoracoid fused with coracoid, but separated from the clavicle by a large fenestrae; suprascapula expanded, anterior half ossified as cleithrum; omosternum present and cartilaginous; sternum absent. Vertebral column composed of eight presacral, non-imbricate vertebrae; hyperossification absent in the spinal processes of vertebrae; all presacral vertebrae with transverse processes; transverse process of sixth presacral vertebra elongated but not ornamented. Humerus slightly shorter than forearm; radius and ulna fused but distinguishable. Manus with distal carpals (I���IV) fused with centrale; radiale and ulnare about the same size; one prepollical element; phalangeal formula 1���2���3���1; tips of terminal phalangeal element of fingers I and IV falciform and tip of finger II and III pointed. Hindlimbs with tibia and fibula fused but distinguishable, forming the tibiafibula; tibiafibula slightly shorter than femur; tibiale and fibulare fused at their distal and proximal ends (medially not fused). Pes with distal tarsal element I, II, III present and IV���V absent; centrale present. One very reduced prehallical element present; phalangeal formula 1���2���3���4���1; tips of terminal phalangeal elements of toes I and V rounded; tips of the terminal phalangeal elements of toes II, III, and IV arrow-shaped. Advertisement call. The advertisement call of the new species is long, composed by the regular repetition of five or six high-frequency pulsed notes (Figure 4). The call lasts 1.5���2.3 seconds (x��=1.8��0.2) and the interval between calls is 3.1���7.4 seconds (x��=5.1��1.4). The call is composed of 4���7 notes (x��=5.3��0.9), repeated in a rate of 0.1���0.3 notes/second (x��=0.2��0.0). Notes last 131���233 milliseconds (x��=195��13) and present 7���11 pulses (x��=8.8��1.3). Pulses last 20���30 milliseconds (x��=23.6��4.8) and are repeated at a rate of 6.1���12.3 pulses/second (x��=9.3��1.8). The minimum frequency is 4.5���5.5 kHz (x��=4.9��0.3), the maximum frequency is 8.2���10.3 kHz (x��=9.3��0.3), and the dominant frequency is 6.2���7.2 kHz (x��=6.7��0.3). Variation. Morphometric variation is given in Table 1. In our sample, females are larger than males (SVL of females x��= 9.9 mm ��0.4; SVL of males x��=8.0 mm��0.4, Welch���s t-test t = 10.2, df = 9.6, p p F = 12.86, TukeyHSD tests p = 0.02 and p p F = 31, TukeyHSD tests p B. sulfuratus sp. nov. is preserved and the range of divergent call parameters overlapped among the analyzed populations (Table 2). Call (s) Interval (s) Notes/call Notes/s Note (ms) Pulses/ note A 1.7��0.1 4.5��1.7 4.9��0.4 0.2��0.1 177��12 8.25��0.8 (1.5���1.8) (3.1���7.4) (4���6) (0.2���0.3) (131���205) (7���9) B 2.2��0.1 5.9��0.8 5.6��0.6 0.2��0.1 202��14 8.5��1.5 (2.1���2.3) (5.2���6.7) (5���7) (0.1���0.2) (180���233) (7���10) C 1.8��0.1 5.2��1.2 5.1��0.5 0.2��0.1 206��10 9.4��1.2 (1.7���2.1) (3.7���7.0) (4���6) (0.1���0.2) (171���228) (8���11) continued. Pulses/s Pulse (ms) Min Freq (Hz) Max Freq (Hz) Dom Freq (Hz) A 9.1��0.9 25��5 4.9��0.3 9.1��0.2 6.6��0.1 (8.5���10.7) (20���30) (4.5���5.2) (9.0���9.4) (6.5���6.7) B 7.6��1.4 20��0 5.1��0.4 8.2��0.0 6.4��0.2 (6.1���9.0) (20���20) (4.7���5.5) (8.2���8.2) (6.2���6.6) C 10.1��1.9 24��5 5.0��0.3 9.9��0.3 6.9��0.2 (7.7���12.3) (20���30) (4.5���5.4) (9.4���10.3) (6.7���7.2) Comparisons with other species. Brachycephalus sulfuratus sp. nov. shares the diminutive size and reduced number of functional toes with other species of Brachycephalus. The texture of the skin of the head and dorsum of B. sulfuratus sp. nov. is smooth, and without dermal ossification. This characteristic distinguishes the new species from B. ephippium, B. garbeanus, and B. margaritatus, all of which have the largest SVLs in the genus (pers. observation) and the most extreme condition of hyperossification, as revealed by the presence of a dorsal bony shield (Clemente-Carvalho et al. 2009). Brachycephalus sulfuratus sp. nov. also differs from B. alipioi, B. atelopoide, B. bufonoides, B. crispus, B. guarani, B. nodoterga, B. pitanga, B. vertebralis and B. toby, species with intermediate condition of hyperossification of the skull and skeleton (Clemente-Carvalho et al. 2009; Haddad et al. 2010; Pombal 2010; Clemente-Carvalho et al. 2012; Condez et al. 2014). The lack of hyperossification is shared among the new species and B. auroguttatus, B. boticario, B. brunneus, B. didactylus, B. ferruginus, B. fuscolineatus, B. hermogenesi, B. izecksohni, B. leopardus, B. mariaterezae, B. olivaceus, B. pernix, B. pombali, B. pulex, B. quiririensis, B. tridactylus, and B. verrucosus (Clemente-Carvalho et al. 2009; Napoli et al. 2011; Ribeiro et al. 2015; Pie & Ribeiro 2015). Except for B. didactylus, B. hermogenesi, B. pulex, and B. sulfuratus sp. nov., all of these species have a ���bufoniform��� body and in most of them the body background color is orange. The fleatoads, B. didactylus, B. hermogenesi, B. pulex, and B. sulfuratus sp. nov., have the smallest SVLs within the genus, a ���leptodactyliform��� body, and a brown general background color (pers. observation). The main morphological differences among these species are related to the loss of phalangeal elements and the reduced number of toes. Brachycephalus sulfuratus sp. nov. has the toe I externally absent and toes II, III, IV, and V distinct and functional. This condition is very distinct from B. pulex, which presents toes I, II, and V absent, and toes III and IV distinct and functional (Napoli et al. 2011). Brachycephalus didactylus is easily distinguished from B. sulfuratus sp. nov. by having toe I absent, toes II, III, and IV distinct and functional, and toe V vestigial (Izecksohn 1971). Toes of B. hermogenesi have the same configuration as B. sulfuratus sp. nov. (see Giaretta & Sawaya 1998). According to the original description of B. hermogenesi the tip of toe II is pointed, which could be considered distinct from the new species. However, after analyzing several individuals of both species we conclude that this character is variable (being rounded or slightly pointed) and not informative. The general color of Brachycephalus sulfuratus sp. nov. in life and preservative is very distinct from all currently known species of Brachycephalus, except for the flea-toads B. didactylus, B. hermogenesi, and B. pulex. These species exhibit a brown general body color with variable dorsal and ventral patterns of dark ornamentation (Izecksohn 1971; Giaretta & Sawaya 1998; Napoli et al. 2011). The m-shaped mark around the cloacal opening in dorsal view, the ventral inverted v-shaped mark in the chest, and variable patterns of stripes on the legs are shared among the four species. The new species differs from B. didactylus, B. hermogenesi, and B. pulex by having (in life) yellow blotches on the ventral surfaces of the throat, chest, arms, and forearms. Another small differences among the flea-toads relate to the x-shaped dorsal mark, which is diagnostic for B. pulex (Napoli et al. 2011) and can be slightly visible in B. didactylus (Izecksohn 1971) and some B. hermogenesi specimens (Giaretta & Sawaya 1998). In the specimens of B. sulfuratus sp. nov. the irregular mark on the middle of the dorsum and the two circular dark blotches on the dorsal view of pelvic girdle are coincident with the x-shaped dorsal mark, though in this species this mark is not clearly distinguished. Another remarkable feature of the new species is the singular inverted v-shaped mark around the cloacal region in ventral view (Figure 6A), which is not clearly distinguishable in B. pulex (Napoli et al. 2011) and generally rounded and not ornamented in B. didactylus (Izecksohn 1971) and B. hermogenesi (Giaretta & Sawaya 1998; Figure 6C). The ornamented marks around the cloacal region of B. sulfuratus sp. nov. are usually sharper when compared to B. hermogenesi (Figure 6). The m-shaped mark around the cloacal opening, which is dark and defined in B. sulfuratus sp. nov. (Figure 6B), is present but not clearly defined in B. hermogenesi (Figure 6D). The main structure of the advertisement call of B. sulfuratus sp. nov. is exceptional within Brachycephalus. It differs greatly from B. ephippium, B. pitanga, and B. crispus because it is composed of a set of 4���7 high-fr, Published as part of Condez, Thais Helena, Monteiro, Juliane Petry De Carli, Comitti, Estev��o Jasper, Garcia, Paulo Christiano De Anchietta, Amaral, Ivan Borel & Haddad, C��lio Fernando Baptista, 2016, A new species of flea-toad (Anura: Brachycephalidae) from southern Atlantic Forest, Brazil, pp. 40-56 in Zootaxa 4083 (1) on pages 42-52, DOI: 10.11646/zootaxa.4083.1.2, http://zenodo.org/record/1050887, {"references":["Clemente-Carvalho, R. B. G., Antoniazzi, M. M., Jared, C., Haddad, C. F. B, Alves, A. C. R., Rocha, H. S., Pereira, G. R., Oliveira, D. F., Lopes, R. T. & Reis, S. F. (2009) Hyperossification in miniaturized toadlets of the genus Brachycephalus (Amphibia: Anura: Brachycephalidae): microscopic structure and macroscopic patterns of variation. Journal of Morphology, 270 (11), 1285 - 1295. https: // dx. doi. org / 10.1002 / jmor. 10755","Haddad, C. F. B., Alves, A. C. R., Clemente-Carvalho, R. B. G. & Reis, S. F. (2010) A new species of Brachycephalus from the Atlantic Rain Forest in Sao Paulo state, southeastern Brazil (Amphibia: Anura: Brachycephalidae). Copeia, 2010, 410 - 420. https: // dx. doi. org / 10.1643 / CH- 09 - 102","Pombal, J. P. Jr. (2010) A posicao taxonomica das \" variedades \" de Brachycephalus ephippium (Spix, 1824) descritas por Miranda-Ribeiro, 1920 (Amphibia, Anura, Brachycephalidae). Boletim do Museu Nacional, Nova Serie, Zoologia, 526, 1 - 12.","Clemente-Carvalho, R. B. G., Giaretta, A. A., Condez, T. H., Haddad, C. F. B. & Reis, S. F. (2012) A new species of miniaturized toadlet, genus Brachycephalus (Anura: Brachycephalidae), from the Atlantic Forest of southeastern Brazil. Herpetologica, 68, 365 - 374. http: // dx. doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00085.1","Condez, T. H., Clemente-Carvalho, R. B. G., Haddad, C. F. B. & Reis, S. F. (2014) A new species of Brachycephalus (Anura: Brachycephalidae) from the highlands of the Atlantic Forest, southeastern Brazil. Herpetologica, 70 (1), 89 - 99. https: // dx. doi. org / 10.1655 / HERPETOLOGICA-D- 13 - 00044","Napoli, M. F., Caramaschi, U., Cruz, C. A. G. & Dias, I. R. (2011) A new species of flea-toad, genus Brachycephalus Fitzinger (Amphibia: Anura: Brachycephalidae), from the Atlantic rainforest of southern Bahia, Brazil. Zootaxa, 2739, 33 - 40.","Izecksohn, E. (1971) Novo genero e nova especie de Brachycephalidae do estado do Rio de Janeiro, Brasil. Boletim do Museu Nacional, Rio de Janeiro, nova serie, Zoologia, 280, 1 - 12.","Giaretta, A. A. & Sawaya, R. J. (1998) Second species of Psyllophryne (Anura: Brachycephalidae). Copeia, 1998, 985 - 987. https: // dx. doi. org / 10.2307 / 1447345","Pombal, J. P. Jr., Sazima, I. & Haddad, C. F. B. (1994) Breeding behavior of the pumpkin toadlet, Brachycephalus ephippium (Brachycephalidae). Journal of Herpetology, 28, 516 - 519. https: // dx. doi. org / 10.2307 / 1564972","Araujo, C. B., Guerra, T. J., Amatuzzi, M. C. O. & Campos, L. A. (2012) Advertisement and territorial calls of Brachycephalus pitanga (Anura: Brachycephalidae). Zootaxa, 3302, 66 - 67.","Garey, M. V., Lima, A. M. X., Hartmann, M. T. & Haddad, C. F. B. (2012) A new species of miniaturized toadlet, genus Brachycephalus (Anura: Brachycephalidae), from southern Brazil. Herpetologica, 68 (2), 266 - 271. https: // dx. doi. org / 10.1655 / HERPETOLOGICA-D- 11 - 00074.1","Verdade, V. K., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta & Sawaya, 1998) (Anura, Brachycephalidae). Journal of Herpetology, 42 (3), 542 - 549. http: // dx. doi. org / 10.1670 / 07 - 287.1","Clemente-Carvalho, R. G. B., Klaczo, J., Perez, I., Alves, A. C. R, Haddad, C. F. B. & Reis, S. F. (2011) Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae). Molecular Phylogenetics and Evolution, 61, 79 - 89. https: // dx. doi. org / 10.1016 / j. ympev. 2011.05.017","Padial, J. M., Grant, T. & Frost, D. R. (2014) Molecular systematics of terraranas (Anura: Brachycephaloidea) with an assessment of the effects of alignment and optimality criteria. Zootaxa, 3825 (1), 1 - 132. https: // dx. doi. org / 10.11646 / zootaxa. 3825.1.1","Pimenta, B. V. S, Bernils, R. S. & Pombal, J. P. Jr. (2007) Amphibia, Anura, Brachycephalidae, Brachycephalus hermogenesi: Filling gap and geographic distribution map. Notes on Geographic Distribution. Checklist, 3 (3), 277 - 279.","Oliveira, J. C. F., Coco, L., Pagotto, R. V., Pralon, E., Vrcibradic, D., Pombal, J. P. Jr. & Rocha, C. F. D. (2012) Amphibia, Anura, Brachycephalus didactylus (Izecksohn, 1971) and Zachaenus parvulus (Girard, 1853): Distribution extension. Checklist, 8 (2), 242 - 244.","Clemente-Carvalho, R. B. G., Monteiro, L. R., Bonato, V., Rocha, H. S., Pereira, G. R., Oliveira, D. F., Lopes, R. T., Haddad, C. F. B., Martins, E. G. & Reis, S. F. (2008) Geographic variation in cranial shape in the pumpkin toadlet (Brachycephalus ephippium): a geometric analysis. Journal of Herpetology, 42 (1), 176 - 185.","Clemente-Carvalho, R. B. G., Perez, S. I., Tonhati, C. H., Condez, T. H., Sawaya, R. J., Haddad, C. F. B. & Reis, S. F. (2015) Boundaries of morphological and molecular variation and the distribution range of a miniaturized froglet, Brachycephalus nodoterga (Anura: Brachycephalidae). Journal of Herpetology. [in press]"]}

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18. mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate., Facultad de Ciencias Naturales y Museo

The Pumpkin toadlet, Brachycephalus ephippium (Spix, 1824), is an orangish daily anuran, which inhabits the litter of the Atlantic Forest of Southeastem Brazil. During a survey of this species' natural history in the vicinities of Campinas (22º52'S, 46º49'W), São Paulo State, field observations were made on the oviposition behavior, in which after the male left the oviposition site, the female covered the surface of ali eggs with soil particles. She pressed and rolled each egg against the ground using her hindfeet, until the eggs were indistinguishable from the litter background. A clutch was obtained in laboratory and two others were found in the field. After 25 days of development the embryo possess a big yolk sac, differentiated mouth and a small tail with caudal membranes poorly developed. After 41 days, fingers and toes are fully formed, the tail is relatively smaller than the previous state, the abdômen is large due to the yolk within, and the mouth possess two egg tooth. After 54 days, the tail remains as a small appendix, there is only one egg teeth, and body is densely pigmented. The hatehling oceurred after 64 days of development, the young possess reddish brown coloration, vestigial tail, and no egg teeth.

Brachycephalus pulex sp. nov. Figures 1���3 Holotype. MNRJ 69646 (ex-UESC 8357), adult specimen, collected at the Serra Bonita mountain (15 o 23 'S, 39 o 33 'W, 830 m above sea level), Municipality of Camacan, State of Bahia, Brazil, on 0 4 July 2010, by I.R. Dias. Paratopotypes. MZUESC 8352, adult specimen, collected on 25 March 2010, by I.R. Dias. MZUESC 8604��� 8605, adult specimens, collected on 26 July 2010, by I.R. Dias. UFBA 10415���10416, juveniles, collected on 16 April 2009, by M.F Napoli and A. Y.C. Vargas. UFBA 10455, sub-adult, collected on 11 September 2010, by I.R. Dias. Diagnosis. The new species is promptly included in the genus Brachycephalus by the small body size (snout��� vent length less than 18 mm), reduced number of toes, terminal digits not expanded, toes without circumferential grooves, and digits apically pointed. Brachycephalus pulex is diagnosed from all its congeners by the combination of the following character states: (1) toe II externally absent; (2) toe V vestigial; (3) fingers I and IV externally absent; (4) body leptodactyliform; (5) small body size (adult specimens SVL 8.0��� 8.4 mm); (6) dorsal ground color maculated with variegated browns without yellow or orange vivid colors; (7) dorsum with a x-shaped mark from posterior corners of eyes to sacral region; (8) an inverted depigmented v-shaped mark on chest, bordered above by a dark brown stripe on each side; (9) smooth skin texture with no dermal co-ossification on top of the head and central part of the back body. Comparisons with other species. Brachycephalus pulex is closely allied to B. didactylus and B. hermogenesi by the small body size (combined SVL of B. didactylus and B. hermogenesi 8.6���10.2 mm; B. pulex 8.0��� 8.4 mm), body leptodactyliform, dorsal ground color maculated with variegated browns without yellow or orange vivid colors, a dark brown x-shaped mark on dorsum, and short and pointed snout with a rounded tip in dorsal view. These features promptly diagnose B. pulex from B. alipioi, B. atelopoide, B. brunneus, B. bufonoides, B. ephippium, B. ferruginus, B. garbeana, B. izecksohni, B. nodoterga, B. pernix, B. pitanga, B. pombali, and B. vertebralis, which have body size larger (combined SVL 9.3���19.7 mm), body bufoniform, general body color vivid yellow to orange in life and pale cream in preservative (dorsal background color in B. brunneus brown, but not variegated), and rounded or mucronate snout shape in dorsal view. Brachycephalus pulex has smooth skin texture with no dermal co-ossification on top of the head and central part of the back body, whereas B. ephippium, B. nodoterga, and B. vertebralis have such characters. The toe II externally absent diagnoses B. pulex from all congeners, in which toe II is developed. Also, an only vestigial toe V diagnoses B. pulex from B. hermogenesi, which presents a small, but developed, toe V. The external absence of fingers I and IV diagnoses B. pulex from B. didactylus and B. hermogenesi, in which such fingers are vestigial. An inverted depigmented v-shaped mark on chest bordered above by a dark brown stripe on each side diagnoses B. pulex from B. didactylus, which has a dark brown trapezoidal mark on chest, and from B. hermogenesi (without dark brown marks on chest). Description of holotype. Body slender, leptodactyliform (Fig. 1). Head wider than long, its length 38 % of snout���vent length; snout long with length almost equal to eye diameter, rounded in lateral and dorsal views (Fig. 2 A���B); nostrils directed anterolateraly, elliptical, elongated, almost perpendicular to the canthus rostralis; canthus rostralis rounded; loreal region weakly concave; lips nearly sigmoid; eye slightly protruding in dorsal and lateral views, eye diameter 42 % of head length; tympanum indistinct; vocal sac indistinct and vocal slits absent; tongue longer than wide, free behind; choanae relatively small, rounded; vomerine odontophores absent. Upper arm and forearm relatively slender, upper arm approximately as long as forearm; fingers I and IV absent; finger II distinct, short; finger III long, robust; tip of finger II nearly rounded, tip of finger III pointed; relative lengths of fingers II Color of holotype. In preservative (70 % ethyl alcohol), dorsal surfaces light brown, darkened by numerous brown pin dots; eyeball blackish brown; dorsum maculated by dark brown marks: a thin and medially interrupted interorbital stripe, a x-shaped mark from posterior corners of eyes to sacral region, somewhat dimmed in its ends, and an inverted v-shaped mark from sacrum to vent, continuous with the x-shaped mark. Forearms with scattered dark brown blotches somewhat anastomosed, absent on upper arms; outer edges of fingers II and III dark brown. Groin with a dark brown blotch, continuous with a dark brown diagonal transverse large stripe on thigh; knee dark brown; posterior surfaces of thighs with a continuous and curved dark brown stripe just below the vent; tibia with two dark brown stripes cranially, and a third one over the heel; foot with five small dark brown blotches from tarsus to fourth toe. Background color of ventral surfaces pale cream, darkened by numerous brown pin dots; chest with an inverted depigmented v-shaped mark bordered above by two dark brown stripes on each side; edges of throat and belly delimited by distinct depigmented circular areas, also scattered on the inferior surfaces of thighs and tarsus. Palm of hand and sole of foot widely depigmented. Color in life. Description based in the paratype MZUESC 8604. Dorsal ground color maculated with variegated browns looking like dead leaves (Fig. 3). A dark brown x-shaped mark is discernible over the dorsum, anastomosed caudally with a dark brown v-shaped mark over the sacrum, which in turn is continuous with dark brown stripes over the thighs, tibia and tarsus. Dorsal ground color surfaces anterior and posterior to the x-shaped mark, reddish brown. Loreal region, flanks, concealed surfaces of thighs, and anterior surface of tibia, faded reddish brown. A light brown v-shaped interorbital area is delimited on the head cranially and caudally by two dark brown stripes, the former narrower than second. A dark brown mid-dorsal stripe extends perpendicularly from the cranial v-shaped stripe to near the tip of snout, which in turn presents the same background color of the v-shaped interorbital area. Tarsus and posterior surface of tibia light brown. Ventral surfaces dark brown (Fig. 3), with an inverted blackish brown v-shaped mark on chest, and white blotches bordering the throat, belly, thighs, and tibia. An xshaped mark is outlined from the groin to the posterior undersurface of thighs by a row of white botches bordered externally by a blackish stripe. From the groin, these white blotches extend cranially to the posterior margins of chest, bordering the belly. Pupil black, iris orange. Variation. Specimens are congruent with respect to the external morphologic characters and color pattern. Snout-vent lengths of juveniles are 5.5 mm (UFBA 10415) and 6.8 mm (UFBA 10416); sub-adult UFBA 10455 is 7.4 mm. Measurements of adult paratopotypes are in Table 1. Etymology. The specific name (pulex), a noun in apposition, refers to the flea, Pulex irritans, and is allusive to the small size and to the striking capacity for large leaps presented by the new species. Geographic distribution and Natural History. Brachycephalus pulex is only known from the type locality, a forest remnant located in a mountain range regionally named Serra Bonita, with elevations ranging from 200 to 950 m above sea level, which leads to altitudinal changes in the vegetation, from evergreen forest with elements of moist lowland semi-deciduous forest, to moist submontane forest near the summits. The Serra Bonita mountain range lies in the municipalities of Camacan and Pau Brasil, in the State of Bahia, Brazil, and is one of the last remnants of moist submontane forest in the region. It covers an area of approximately 7,500 hectares, 130 km from the city of Ilh��us on the Atlantic coast and 526 km from the State capital of Salvador. Approximately 50 % of the land cover is primary forest, and the remaining is a mosaic of forests in advanced stages of recuperation mixed with ���cabruca��� (cocoa trees planted under natural forest canopy) and small areas of pasture. In the Serra Bonita there are several springs that provide fresh water for the inhabitants of Camacan and Pau Brasil. Brachycephalus pulex was captured within the Serra Bonita Reserve Complex (SBR), a Private Natural Heritage Reserve (RPPN), which comprises ca. 1,800 hectares of a ���consortium��� of privately owned properties (Instituto Uira��u 2009). This is the first record of a Brachycephalus for the State of Bahia and the northernmost record for the coastal Atlantic Forest (but see below). The new species was captured in a primary forest remnant provided with streams near the top of the mountain (800���930 m a.s.l.), within an area of dense leaf litter and several epiphytic bromeliads. At the time of specimens capture (December 2009 to July 2010), the minimum mean air temperature was 16.9 ��C (12���20 ��C), the maximum 23.9 ��C (20���28 ��C), and the average monthly precipitation was 188.6 mm (22.5���315 mm). The specimens were captured beneath the leaf litter, except by one individual that was on a tree trunk 30 cm above the ground. One specimen when disturbed during the survey of anurans jumped away many times quickly and stopped over a dry leaf, remaining immovable (deaf feigning) even after we raise the leaf. This behavior was also observed for both juvenile paratypes. No vocalization was heard, although the advertisement call of the closely allied B. hermogenesi has been recorded and described by Verdade et al. (2008), and also for B. ephippium (Pombal et al. 1994). Remarks. The northern geographic distribution limit of the genus Brachycephalus deserves some considerations. Hedges et al. (2008) stated that the genus Brachycephalus is restricted to the Atlantic Coastal Forest in the states of Rio de Janeiro, S��o Paulo, and Paran�� in southeastern Brazil. In the same article, the authors provided a map with the geographic distribution of the genera Brachycephalus and Ischnocnema, in which the range distribution of the former extends to near the Brazilian State of Alagoas (ca. 10 o S). This mapped geographic distribution is contradictory to the information presented in the article itself and is not congruent with the literature, as the northernmost geographic distribution limit known for the genus Brachycephalus is that of B. alipioi in the municipalities of Vargem Alta, Forno Grande and Santa Teresa (ca. 20 o S), all in the State of Esp��rito Santo, Brazil (Pombal & Gasparini 2006), and that of B. ephippium in the Serra do Brigadeiro (ca. 20 o S) (Pombal 2001; Dayrell et al. 2006), a regional designation of the Serra da Mantiqueira mountain range in the State of Minas Gerais, Brazil. Dayrell et al. (2006) and Frost (2010) included in the geographic distribution range of B. ephippium the State of Bahia, Brazil. Indeed, the type locality of B. ephippium lies in the Municipality of Ilh��us, State of Bahia (Bokermann 1966), but Pombal et al. (1998) and Pombal and Gasparini (2006) already contested this information pointing out that possibly Spix���s material was incorrectly labeled and the type locality is in error, as B. ephippium is common in the mountains of Rio de Janeiro city, where Spix also collected. In assuming this error in the label of Spix���s material, Pombal and Gasparini (2006) considered B. alipioi as the northernmost species of the genus. Therefore, the presence of B. pulex in the Municipality of Camacan (ca. 15 o S), southern Bahia State, currently corresponds to the northernmost distribution limit of the genus Brachycephalus., Published as part of Napoli, Marcelo Felgueiras, Caramaschi, Ulisses, Cruz, Carlos Alberto Gon��alves & Dias, Iuri Ribeiro, 2011, A new species of flea-toad, genus Brachycephalus Fitzinger (Amphibia: Anura: Brachycephalidae), from the Atlantic rainforest of southern Bahia, Brazil, pp. 33-40 in Zootaxa 2739 on pages 34-39, DOI: 10.5281/zenodo.276578, {"references":["Verdade, V. K., Rodrigues, M. T., Cassimiro, J., Pavan, D., Liou, N. & Lange, M. C. (2008) Advertisement call, vocal activity, and geographic distribution of Brachycephalus hermogenesi (Giaretta & Sawaya, 1998) (Anura, Brachycephalidae). Journal of Herpetology, 42, 542 - 549.","Pombal, Jr., J. P., Sazima, I. & Haddad, C. F. B. (1994) Breeding behavior of the pumpkin toadlet, Brachycephalus ephippium (Brachycephalidae). Journal of Herpetology, 28, 516 - 519.","Hedges, B., Duellman, W. E. & Heinicke, M. P. (2008) New World direct-developing frogs (Anura: Terrarana): molecular phylogeny, classification, biogeography, and conservation. Zootaxa, 1737, 1 - 182.","Pombal, Jr., J. P. & Gasparini, J. L. (2006) A new Brachycephalus (Anura: Brachycephalidae) from the Atlantic Rainforest of Espirito Santo, southeastern Brazil. South American Journal Herpetology, 1, 87 - 93.","Pombal, Jr., J. P. (2001) A new species of Brachycephalus (Anura: Brachycephalidae) from Atlantic Rain Forest of southeastern Brazil. Amphibia-Reptilia, 22, 179 - 185.","Frost, D. R. (2010) Amphibian Species of the World: an Online Reference. Version 5.4 (08 April, 2010). Electronic Database available from http: // research. amnh. org / vz / herpetology / amphibia / American Museum of Natural History, New York, USA (accessed 24 August 2010).","Pombal, Jr., J. P., Wistuba, E. M. & Bornschein, M. R. (1998) A new species of Brachycephalid (Anura) from the Atlantic Rain Forest of Brazil. Journal of Herpetology, 32, 70 - 74."]}

Orientador: Sergio Furtado dos Reis Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O gênero Brachycephalus compreende atualmente seis espécies, viz., B. ephippium (SPIX, 1824), B. nodoterga MIRANDA-RIBEIRO, 1920, B. pernix POMBAL-JR, WISTUBA & BORNSCHEN, 1998, B. vertebralis POMBAL-JR, 2001, B. didactyla (IZECKSOHN, 1971) e B. hermogenesi (GIARETTA & SAWAYA, 1998), que são endêmicas da Mata Atlântica. Brachycephalus didactyla e B. hermogenesi, originalmente descritas no gênero Psyllophryne, ocorrem desde o nível do mar até cerca de 700 m de altitude, ao passo que B. ephippium, B. nodoterga, B. pernix e B. vertebralis, ocorrem na faixa de altitude de 700 a 1600 m (IZECKSOHN, 1971; GIARETTA & SAWAYA, 1998; POMBAL-JR, 2001). Estas quatro espécies (B. ephippium, B. nodoterga, B. pernix e B. vertebralis) têm distribuição disjunta nas Serras do Mar e da Mantiqueira nos Estados de Espírito Santo, Rio de Janeiro, São Paulo e Paraná e este padrão de distribuição sugere a existência de populações diferenciadas ainda não caracterizadas e descritas formalmente. Em particular, para o Estado do Paraná, somente uma espécie, B. pernix, foi descrita até o momento. O presente estudo envolveu a amostragem de populações de Brachycephalus na Serra do Mar do Estado do Paraná das localidades de Pico Caratuva na Serra Ibitiraquire, Pico Marumbi na Serra do Marumbi, Torre da Prata na Serra da Prata e Pico da Igreja na Serra da Igreja, além de B. pernix do Pico Anhangava, que foram caracterizadas na coloração, morfologia e seqüências do gene mitocondrial citocromo b. As populações de Brachycephalus do Pico Caratuva, Pico Marumbi, Torre da Prata e Pico da Igreja, apresentam padrões de coloração exclusivos e podem ser unicamente diagnosticadas com base na cor e também diferem no padrão de coloração de B. pernix. Estas populações diferem também entre si e de B. pernix na sua morfologia externa e óssea. Com base nos padrões únicos de coloração e atributos morfológicos, as populações do Pico Caratuva, Pico Marumbi, Torre da Prata e Pico da Igreja são reconhecidas neste estudo como novas espécies, sendo que as populações do Pico Caratuva e Torre da Prata foram formalmente descritas neste estudo. A filogenia inferida com base nas seqüências de um fragmento do gene mitocondrial citocromo b mostrou que os taxa do Estado do Paraná formam um grupo monofilético com relação ao grupo externo, representado por B. ephippium. Os dois taxa representados pelas populações das localidades de Torre da Prata e Pico da Igreja formam uma linhagem cujo grupo-irmão é composto por B. pernix e os taxa das localidades de Pico Marumbi e Pico Caratuva. Nesta última linhagem, B. pernix e o taxon do Pico Marumbi compartilham um ancestral comum mais recente Abstract: The genus Brachycephalus currently includes six species, viz., B. ephippium (SPIX, 1824), B. nodoterga MIRANDA-RIBEIRO, 1920, B. pernix POMBAL-JR, WISTUBA & BORNSCHEN, 1998, B. vertebralis POMBAL-JR, 2001, B. didactyla (IZECKSOHN, 1971) and B. hermogenesi (GIARETTA & SAWAYA, 1998), which are endemic to the Atlantic Forest of eastern Brazil. Brachycephalus didactyla e B. hermogenesi, originally described in the genus Psyllophryne, occur from sea level to near 700 m of altitude, whereas B. ephippium, B. nodoterga, B. pernix, and B. vertebralis, occur from 700 to 1600 m of altitude (IZECKSOHN, 1971; GIARETTA & SAWAYA, 1998; POMBAL-JR, 2001). Brachycephalus ephippium, B. nodoterga, B. pernix, and B. vertebralis have disjunct distributions in the Serra do Mar and Serra da Mantiqueira in the states of Espírito Santo, Rio de Janeiro, São Paulo, and Paraná and this pattern of distribution suggests the existence of differentiated populations still not characterized and formally described. In particular, for the state of Paraná, only one species, B. pernix, has been described so far. The present study involved the sampling of populations of Brachycephalus in the Serra do Mar in the state of Paraná from the localities of Pico Caratuva in the Serra Ibitiraquire, Pico Marumbi in the Serra do Marumbi, Torre da Prata in the Serra da Prata, and Pico da Igreja in the Serra da Igreja, in addition to B. pernix from Pico Anhangava. All samples were characterized from the perspectives of variation in color, morphology, and sequences derived from the mitochondrial cytochrome b gene. Populations of Brachycephalus from Pico Caratuva, Pico Marumbi, Torre da Prata e Pico da Igreja, display unique color patterns and can be uniquely diagnosed on the basis of color and they also differ in color pattern from B. pernix. These populations also differ among themselves and from B. pernix in their morphology. Based on unique color patterns and morphological traits, the populations from Pico Caratuva, Pico Marumbi, Torre da Prata, and Pico da Igreja are recognized here as new species, and the populations from Pico Caratuva and Torre da Prata are formally described. The phylogeny inferred from the mitochondrial cytochrome b gene indicates that all taxa in the state of Paraná comprise a monophyletic group, having B. ephippium as an outgroup. The two taxa from the localities of Torre da Prata and Pico da Igreja are joined in a lineage whose sister-group is composed of B. pernix and the taxa from Pico Marumbi and Pico Caratuva. In this latter lineage, B. pernix and the taxon from Pico Marumbi share a most recent common ancestor Doutorado Genética Animal e Evolução Doutor em Genética e Biologia Molecular

The Pumpkin toadlet, Brachycephalus ephippium (Spix, 1824), is an orangish daily anuran, which inhabits the litter of the Atlantic Forest of Southeastem Brazil. During a survey of this species' natural history in the vicinities of Campinas (22º52'S, 46º49'W), São Paulo State, field observations were made on the oviposition behavior, in which after the male left the oviposition site, the female covered the surface of ali eggs with soil particles. She pressed and rolled each egg against the ground using her hindfeet, until the eggs were indistinguishable from the litter background. A clutch was obtained in laboratory and two others were found in the field. After 25 days of development the embryo possess a big yolk sac, differentiated mouth and a small tail with caudal membranes poorly developed. After 41 days, fingers and toes are fully formed, the tail is relatively smaller than the previous state, the abdômen is large due to the yolk within, and the mouth possess two egg tooth. After 54 days, the tail remains as a small appendix, there is only one egg teeth, and body is densely pigmented. The hatehling oceurred after 64 days of development, the young possess reddish brown coloration, vestigial tail, and no egg teeth.

The Pumpkin toadlet, Brachycephalus ephippium (Spix, 1824), is an orangish daily anuran, which inhabits the litter of the Atlantic Forest of Southeastem Brazil. During a survey of this species' natural history in the vicinities of Campinas (22º52'S, 46º49'W), São Paulo State, field observations were made on the oviposition behavior, in which after the male left the oviposition site, the female covered the surface of ali eggs with soil particles. She pressed and rolled each egg against the ground using her hindfeet, until the eggs were indistinguishable from the litter background. A clutch was obtained in laboratory and two others were found in the field. After 25 days of development the embryo possess a big yolk sac, differentiated mouth and a small tail with caudal membranes poorly developed. After 41 days, fingers and toes are fully formed, the tail is relatively smaller than the previous state, the abdômen is large due to the yolk within, and the mouth possess two egg tooth. After 54 days, the tail remains as a small appendix, there is only one egg teeth, and body is densely pigmented. The hatehling oceurred after 64 days of development, the young possess reddish brown coloration, vestigial tail, and no egg teeth.

It was sampled the litter frogs at altitudinal sites of a semideciduous mesophytic forest located at Sena do Japi (Jundiaí, São Paulo, 23º17'S, 47º00'W, 800-1300m altitude, 1500mm rain/year). Forty two plots of 8x8m were sampled in the dry/cold season of 1995: 21 at a Higher Site (HS) (1000m) and 21 at a Lower Site (LS) (850m). The plots were evenly located near, mid-distance, and far from the stream-bed. It was found a total of five species, three at each site. Eleutherodactylus guentheri comprised 83% of the individuals at HS and E. juipoca 67% at LS. The density of individuals was higher at HS and near the stream. The greater density at HS may be due to greater humidity there, provided by mist. The frogs may aggregate in wetter places (stream edges) in the dry/cold season. The studied sites represent harsh and early successional environments and both these factors may be acting together to produce the low observed diversity.